.“HE..W.M ¢
We: "Mummy

 

 

          

32.x...r.
31“.. vi
. :

             

    

{
V .14IL.
.S t

‘ .. {mfg
brunt?!“ ‘3. ufrnv .

‘2’”...me 9 Kr

 

 

2 . 3:}?
.3 3:...

         

 

 
      

     

 

.,.o\ y . 1.
wiuhsn.
; 4 . may.
Kr . . .
. . V
4 ‘.., , .
o . v 1

    

       
   

  

r3;

  

x . aria?

£3 :3...” m».

 

25-, .2. : . Firm.

   
   

 

oh ‘ .,
«was

 

 

 

' It... t ‘ I,

“flaw

N “ llllllllllllHIHUHHIHHLIlllllll’lllllllllllll

02.54)?) 01801 7404

LIBRARY
Michigan State
University

I“,

 

 

 

 

This is to certify that the

dissertation entitled

31P-NMR SPECTROSCOPY OF PHOSPHOCREATINE

RECOVERY TRANSIENTS IN RAT SKELETAL MUSCLE
presentedby

Anthony Toby Paganini

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Physiology

 

Meal/9R

Major professor \

Date I Z///al 72

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

'___. -'-_.4 _._ -

 

 

_ _..——-.-- "—‘——q-___ 4 _ 4
'- v—— w. ___r“sr44 w.—

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

DATE DUE

 

 

 

. Jflfléogfifl .‘

‘——_

 

 

 

 

 

 

 

 

 

 

 

 

 

ma W14

 

31P—NMR SPECTROSCOPY OF PHOSPHOCREATINE
RECOVERY TRANSIENTS IN RAT SKELETAL MUSCLE

By

Anthony Toby Paganini

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Physiology
1998

3ll’-l\'.\IR .
RECOVER}

I

' v.

”I Y". “‘1'.
he t‘yJ'Q‘gO

“r?“ -115 I]

37“..
=pr ii,

L“t

ABSTRACT

31P-NMR SPECTROSCOPY OF PHOSPHOCREATINE
RECOVERY TRANSIENTS IN RAT SKELETAL MUSCLE

By

Anthony Toby Paganini

The first part of this dissertation examines the effect of perturbations in skeletal
muscle oxidative capacity on phosphocreatine (PCr) recovery kinetics after
submaximal workloads (8 minutes of isometric twitch stimulation) in addition to
the influence of intracellular acidosis on the PCr recovery rate constant. In vivo
31—phosphorous nuclear magnetic resonance spectroscopy (31P-NMRS) is used to
quantify the temporal changes of phosphagens and intracellular pH in the rat
superficial gastrocnemius (approximately 85% fast—twitch—glycolytic fibers) after
mitochondrial content is either decreased by chronic hypothyroidism via chemical
thyroidectomy [IO—weeks of 0.025% w/v methimazole (MMI) in drinking water] or
increased by progressive, interval, aerobic training [IO—weeks on a running wheel
t0 a final regimen of 37m/minute, 60 minutes/day, five days/week (z 85% VOZmaxH.
After these treatments, citrate synthase activity (an index of mitochondrial content)
0f the superficial gastrocnemius was 29% and 179%, respectively, of that in the
corresponding control groups. The results indicate a significant linear correlation (r
= 0-84, P<0.0l) between the PCr recovery rate constant after submaximal
stimulation and a 6—fold range of citrate synthase activity. Additionally, within the

control groups, there was a significant correlation (r=0-73, P4101) between the

g

   

. -... ,
l'Tfinrl'i ram

..
_,,,... 4.0"- A»
I

,4
_‘.:-: “In JLt.o

vv-"I‘ rct‘ ' .
fa‘:¢ DSUC '4).-

, . .
,.,.. ...\_.,.[4
.4

i- .‘a: x\‘ ’ci

0
D
o

-0-‘v..~ A. .’r
. .
nun-”no; Jt:bn .
. U

" - A v. 1,.
$541.29 uLSs'

“"';L
2- .4 1' ‘1‘ <..
..‘I....‘ U" :MI‘.‘..

33%,”; l i
.' Y‘.‘
‘10 .mk.ud‘ C
I
l'v‘ .' _
”uni" Fri .
‘ ‘ . 'Ju.

l"'~r”' ‘
~..,_' u... lv‘wor,
V‘_..~L u:.d\

.Pv..'
w, _
-. :1 r.

r :r ‘
detht‘d‘

s,

¢.v.;"’4 .
4 -
““ka 3U:

.‘ .
.' f H.
t . I‘
“:31 u‘ :C'E’i
\\.

"ew.. I .
\~:‘ h‘MrJF .
Van-Jud
2..
\‘ "'

i...
Q- - P

". A I

«.d“. u‘. trlegv
""h. a

“ wt . 9 .'

Nest L‘JM

‘.

I.‘
.‘t-s: ' n

W! = i

. 00:1“, ..

"‘3
\1‘ -

\ .P‘o

“ Y",

N‘ Nttabi

l
\.
I.

\

 

 

PCr recovery rate constant and the intracellular pH at the end of stimulation. This
suggests intracellular acidification complicates the correlation between PCr
recovery rate constants and muscle oxidative capacity.

In the second part of this dissertation, an attempt was made to identify the
underlying distribution of distinct muscle fiber type populations as differentiated
by relative oxidative capacity from decomposition of global PCr recovery
transients. High signal-to—noise 31P—NMRS data from the posterior rat leg were
obtained by summing across eight contiguous, stimulation—recovery cycles. The
novel Principal Component Analysis (PCA) method for spectral processing was
used prior to Fourier transformation to correct for frequency drifting and other
equipment instabilities. Experimental results indicates the apparent
monoexponential PCr recovery can be resolved into multiple components using
the unbiased Non-negative Least Squares (NNLS) analysis method. This suggests
multiple, discrete, fiber type populations may exist. Modelling studies, however,
illustrate applying the NNLS method to two disparate fiber type distributions
(discrete bimodal versus Gaussian) gives similar results. Therefore unambiguous
identification of the distribution of fiber type populations in mammalian mixed
muscle by non—invasive, high—resolution, NMR spectroscopy remains elusive.
Secondarily, these results document significant differences in end—stimulation pH
over the first three cycles of an intermittent, constant, supramaximal workload.
This may confound the interpretation of gated—NMR experiments which assume
invariant metabolic responses from cycle to cycle and point to the importance of a

“warm—up” period prior to engaging in short bursts of high—intensity workloads.

r ‘ ‘
vfi°:'fi flfi’g'
g... Mu r.’ .

.. “"11"

s r u.

culvu‘shufll L
\.

..-.
at,

v 'fP'F'r 0
““It nibblt‘. \

r
“4...... .. O
‘ ‘ “I.

. 4
"“t- div-JUL .u‘

‘ 4...." .5 _'

I 9 z

‘. "“4. theta

-
w ' ' ' \
‘5' y. 4“

i .

m.“‘ .ea‘: D

n

k

U

9... -- ‘, l-
“t. $2.1M 1';

\. .

- I

.P 3’... "
“”9HWUSW

 

‘15 33"6 been '

5.
"1”; ;~"‘v~
‘
““330: s
I ' ‘
gr .

3.»,

n!

.5,"

>4
KP
' J
0’:
r—o
T

5.
‘3.
~' than I ‘
, ‘fi‘v’ O a" v
9 i
is. ‘1 .
\ ~"‘ s.
.WQPEQ
cg ‘
\
L‘\
'5
u, ‘.‘
{2‘ ’h ‘
“‘3: ‘- h
¥ lkiy (
5‘.“
“‘C‘ "n .
«id

ACKNOWLEDGMENTS

First and foremost, I genuinely thank my research mentor, Dr. Ron Meyer, for
his intellectual guidance, impeccable scientific integrity, and treating me like a
colleague rather than just another minion. You have shown me how to think
deeply about nature, balance skepticism with open—mindedness, wear many hats
while “doing science”, and have fun doing it all. You are one of the very few people
who truly leads by example rather than decree. I am also deeply indebted to my
former senior lab colleague, Dr. Jeanne Foley. I’ve always been able to count on
you for professional guidance, lively scientific discourse, friendship, and help with
the lab protocols when I was wet behind the auricles. I loathe to think where I
would have been without your detailed lab notes and patience showing me how to
run the assays, spin the dials on the Bruker spectrometer, and perform the rat
surgery. Many thanks go to my teaching mentor, Dr. Tom Adams, for the countless
hours I have spent in his office discussing physiology pedagogy, class management
skills, and the finer points of heat transfer in Australian Brush Turkey nest
mounds. Tom, you are a genuine Renaissance man. Thanks also go to Drs. Rudy
Bernard and Jim Pivarnik for their valuable perspectives and thorough review of
my dissertation. I also want to thank Drs. Dick Heisey, Burnell Selleck, Bob
Pitman and especially the late Jack Krier for their tutelage and sage advice over
the years. Jack, I’ll never forget your passion for research and willingness to spend
as much time as I needed discussing neurophysiology. Without the spectral
processing help of Dr. Truman Brown and Radka Stoyanova at Fox Chase Cancer
Center my data would seem much less interesting.

I am extremely grateful to my friends and current and former colleagues while
I’VE been at Michigan State (Marya Liimatta, Roop J ayaraman, Barry Prior, Marco

Cabrera, Bob Wiseman, Rick Kustasz) and Ohio State (Ken—”Kenny H”—Helal,

'0' hit. fiEY“
. .

high bit \

IE... \'.F "e“
., 14
.-.'. .t-hcho‘. BA!

 

. . ‘

,.....,...

.32....;\ JC‘.‘
U.

.17‘1'9'-M '

- “no. 4 .1.15. 'J

n,.,’.‘_ '

6-5-«,.tI.

.
A;
q “'”!-0
“ ‘at‘~dun
i'I ‘
i_ i I

 

Jim-”Jimmy R”—Raeburn) for the many thought provoking discussions, help with
homework or lab techniques, and general inspiration.

Last, but certainly not least I’d like to thank the Physiology (Amylou, Jan, Barb,
Nancy, Marilyn, Sharon, Shirley, Bobby, Greg, Vance), Anatomy (Judy, Jill), and
Kinesiology (Jo Ann, Jan, Verna, Darcie) department support staff; Dan from
Physical Plant, Jim from the Physics machine shop, and Ron’s former lab
technicians (Tom, Mary, Rob). All of you have kept the gears well greased and
made life much easier for me over the years. To all who have help me, thanks a
googolplex!

This dissertation was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-38972. A portion of my graduate

program was financed by a Recruiting Fellowship from the College of Human
Medicine at Michigan State University.

 

 

7.1"? 0? TABLE)
Eomcmi

Cupierl NH

.fi". 9 vur.
7'. \'-1 F I
vs- --'u~ AEAL
F ~~"""\'H
II. A v.4 I J |
UAlvudnfiyL-

A Y
"" ‘ 'Ir
V v- .,F Tap
""" '3 AnsLa ..
'p,,.. .
W- - l}\\l -

u...n..i.

Tia??? . 2 L111»
skeletal mus.

".4 P - ' " - . .
~‘.-_: Cl_3.\i E.”
:"._D-~v- :-

”l"
~...~ ‘U i A“

.l
N V '; rl'v
h...” “‘- Ab~|r
I
a v 'F' ‘_ ,-
"“ .5»): {rd
~~
' ‘ t
l: a, .
.A’ .‘AA—I.“d‘ 3'
f‘ . . .
I.” ‘1 . .._
.i “'1! 1“‘\‘
_ . . ck

.3
' I-
et: .Jnni. 5‘“ r
'i~ ‘ 4
' i
“‘ h,
‘ 1.... “p; P“,

 

‘ "v- . v‘

.‘ r "71: .\x

“QR." Fri
h a‘l“ ‘

 

 

 

TABLE OF CONTENTS
LIST OF TABLES ...................... - --- v---v-:v.00...OOOOOOOOOOOOOOOOOOOOOOOOOix
LIST OF FIGURES - - - x
Chapter 1 INTRODUCTION - -- -- ........ ............ -- -- 1
GENERAL ORIENTATION .................................................................................................. 1
BACKGROUND OF THE PROBLEM AND SPECIFIC AIMS .................................................. 2
SCOPE OF THE DISSERTATION ........................................................................................ 4
LIMITATIONS OF THE DISSERTATION ............................................................................. 4

CHAPTER 2 LITERATURE REVIEW The role and utility of phosphocreatine in
skeletal muscle energy metabolism - - -- - - -- - -- - -- -- 5

 

 

EARLY CONCEPTS (18808—19408) ................................................................................... 5
Energy for muscle contraction was viewed as a heat transfer phenomenon ...................... 5
Energy for muscle contraction was related to biochemical phenomena ............................. 6
Energy for muscle contraction was explained by PCr hydrolysis ....................................... 9

THE ROLE REVERSAL (194OS—EARLY197OS) ................................................................ 12
ATP emerges as the primary energetic determinant of contraction ................................ 12
Myothermal techniques refine the bioenergetic role and experimental utility of PCr ...... 14
PCr hydrolysis helps estimate muscle efficiency ............................................................. 20

CONTEMPORARY VIEWS (EARLY 197OS—PRESENT) ....................................................... 23
The connection between PCr kinetics and oxidative capacity and its implications ......... 23
PCr and the chemical energy balance method ................................................................ 29
Oxidative capacity exhibits plasticity in skeletal muscle fibers ...................................... 3 1
Phosphorus N MR spectroscopy becomes an important tool in bioenergetics ................... 32
Transient and steady-state analysis of PCr kinetics .................................................... 38

CHAPTER 3 Linear dependence of phosphocreatine kinetics on skeletal
muscle oxidative capacity - - -- 46

INTRODUCTION ................................................................................................................ 46
METHODS ........................................................................................................................ 46
Animal care and feeding ................................................................................................ 46
Treatment groups .......................................................................................................... 47
Muscle surgical protocol ................................................................................................. 48
Spectral acquisition parameters and muscle stimulation protocol ................................. 5O
Spectral analysis ........................................................................................................... 50
Statistical analysis ........................................................................................................ 5 1
RESULTS .................................. . ....................................................................................... 51
Treatment effects .......................................................................................................... 5 1
Effect of submaximal stimulation .................................................................................. 55
Effect of 2.0Hz Stimulation ............................................................................................ 6 1
DISCUSSION .................................................................................................................... 64

MIR 4 £51

I
phosphocre.
371-31177. {1N

”no-'~I\~
gf h

v

 

”.5; ...n--.

latpa‘ n. .— -- v
3......‘:.. (if: .1.
I

,... . -..’,-
x '4 \ . ~
no)": -U..,1\ .~

Ellis-1:5. L- T.

"w - .
A
Lynn“; ' id.'
lob- .g...,." A
manna. any,

‘l“*~ .
r.‘ 1:
""'Ul~.n

an.”

w .
|v.,., 1 __ ‘ _
l' '4? 3,4‘
' ““ ooun.¢....

" D
T W" 1‘ PET
~~sl Lu ‘ hi,

. .
if: 22",?” Ir'
.

c155

“no“

u .. ’. .
|~~..r"“ . 4.,
I" A: J. A‘

.ii'jSSZaN

K-Zp'éltlgn

;- 9 ‘ m o
..T?"‘14 .11":

 

.4
-u
T‘;.
“9"“ r' ..
“it It
..... anI

.
\"im' ,,

ON“. I I... .
.

REDICES

‘00....

$7 of REFER};

CHAPTER 4 Estimation of skeletal muscle fiber distributions by

 

 

 

 

phosphocreatine transient analysis ------------------- -- - ...... - - 69
INTRODUCTION ................................................................................................................ 69
METHODS ........................................................................................................................ 71

Animal care and feeding ................................................................................................ 7 1
Muscle surgical protocol ................................................................................................. 7 1
Stimulation—Recovery protocol ....................................................................................... 72
Spectral acquisition parameters .................................................................................... 73
Spectral processing ........................................................................................................ 74
Spectral analysis ........................................................................................................... 7 6
Statistical analysis ........................................................................................................ 7 7
RESULTS .......................................................................................................................... 77

Cyclic changes in metabolites and force ......................................................................... 77

Utility of Principal Component Analysis ...................................................................... 1 10

Decomposition of global PCr time constants ................................................................ 1 12

Modelling of fiber type distributions ............................................................................ 1 16
DISCUSSION .................................................................................................................. 120

Decomposition of PCr recovery transients .................................................................... 1 20

Temporal metabolite patterns during prolonged intermittent exercise ......................... 124

Viability of in vivo fiber typing by 31P—NMRS ............................................................. 126

CHAPTER 5 SUMMARY AND RECOMMENDATIONS -- ------ - - -------------- 130
SUMMARY ...................................................................................................................... 130
RECOMMENDATIONS FOR FUTURE STUDIES ............................................................... 131

APPENDICES- - - - - - -- - 134

LIST OF REFERENCES - ------------ - ------------- ------- 143

INDEX - ...... - ...-.--l70

 

 

.
'1‘. -.
an... .‘4 T-t'ul‘wi'

_ a
.;h

1’ ‘. n
".1- ..

CO?

.8

r
.1

l

118 f'

47‘
e .4

I
3...

O. .
O

LIST OF TABLES

Table 1: Evolution of fiber type classification schemes ............................................... 28
Table 2: Treatment groups and effects ........................................................................... 52
Table 3: Mechanical characteristics—Bench experiment ............................................... 54
Table 4: Metabolites in resting superficial gastrocnemius .......................................... 55
Table 5: PCr kinetics and pH during submax. stimulation ........................................ 58
Table 6: Treatment groups .............................................................................................. 73
Table 7: NMRS Acquisition parameters ......................................................................... 74
Table 8: Time constants from NNLS output ............................................................... 116
Appendix B Figure 28 & Table 9: Leg cross-section ................................................. 136

' O
o- w .
' U

.3...

9";

..'...

g .
‘I‘Mr '
. sun-“r.

3 lme ;

 

 

1.3.1319 (

F) “N
.5 b.”"
Gk“)
all: \.\.Lf
a ‘ ‘
\

Z'= Q

I

A k‘\."'
v";

LIST OF FIGURES

Figure 1: Sample 31P—NMR spectra stackplots ........................................................... 56
Figure 2: 'Iime course of PCr changes (submaximal stim.) ......................................... 57
Figure 3: PCr recovery rate constant v. oxidative capacity .......................................... 60
Figure 4: 2.0Hz Stimulation time courses ...................................................................... 62
Figure 5: 2.0Hz Recovery time courses .......................................................................... 63
Figure 6: Rate constant v. pH (supramaximal stim.) ................................................... 64
Figure 7: Sample 31P—NMR spectra stackplots ........................................................... 79
Figure 8: Time course of PCr changes (0.7 5H2) ............................................................ 80
Figure 9: Time course of PCr changes (2.0Hz) .............................................................. 82
Figure 10: Time course of PCr changes (5.0Hz) ............................................................ 84
Figure 11: Steady—state PCr characteristics vrs. cycle # .............................................. 87
Figure 12: Time course of ICF pH changes (0.7 5Hz) .................................................... 89
Figure 13: Time course of ICF pH changes (2.0Hz) ...................................................... 91
Figure 14: Time course of ICF pH changes (5.0Hz) ...................................................... 93
Figure 15: End—stimulation ICF pH vrs. cycle # ........................................................... 95
Figure 16: Time course of isometric twitch force (0.7 5H2) ........................................... 97
Figure 17 : Time course of isometric twitch force (2.0Hz) ........................................... 100
Figure 18: Time course of isometric twitch force (5.0Hz) ........................................... 103
Figure 19: Initial and end—stimulation peak twitch force .......................................... 108
Figure 20 Force kinetics of single twitches vrs. cycle # ............................................. 109
Figure 21: Spectral correction by PCA ........................................................................ 111
Figure 22: Sample high signal—to—noise 31P—NMR spectra ...................................... 113
Figure 23: NNLS applied to a PCr recovery transient ............................................... 114
Figure 24: Hypothetical fiber type distributions ........................................................ 117
Figure 25: Synthesized global PCr recovery time—courses ....................................... 118
Figure 26: NNLS output of hypothetical distributions .............................................. 119

 

 

 

Appendix A Figure 27: 31P—N MR Probe Photographs ............................................. 135
Appendix B Figure 28 & Table 9: Leg cross—section ................................................. 136
Appendix C Figure 29: Temperature Homeostasis ................................................... 137
Appendix D Figure 30: N versus Cycle Number ....................................................... 138
Appendix E Figure 31: Methimazole Inhibition ......................................................... 139
Appendix F Figure 32: Intensity-Duration Protocol ................................................. 140
Appendix G Figure 33: Training Compliance ............................................................ 141
Appendix H Figure 34: Methimazole Effects ............................................................. 142

 

 

 

Senetal 0r

i'Z‘PP 'r‘r'

“m Ut vita u‘
0

‘9'.

: ‘ "ICF"
'W “A shunt...
‘ i

‘9 __ . .
. :43": ‘r 0'
“a“ “‘ 5.

~
. . :
.‘-"~on_.l

\‘l. .utxoukd‘ Cf.

V
3., F

, '- "Wt,-
~.-... ‘tittltr‘:

‘2'.- A? r ‘
~ . I v v'. . .
u. ~l e. “ ‘lt‘~

 

in. 2' ‘

\fl .,
.‘Fr- \ \
u~1x1¥ \

'5." v

\ci ‘- i.‘
a": 1x .3 l

_',:"-..

1., .
4‘...“ ‘ l'-v~..
AJA 3 tl‘g‘
an
i.“
5 \~
\:

*2
'6 LP. ~
“km or
M
‘|
\.,‘-
em a
‘ a
:4.
I
“nil:- Enzv"
a“
5,;
“ A
"inl‘e ‘Li y
‘<a‘ ?}
\-
Us"
\Z. “’i
‘J
r: {'5'-
é‘iJIr‘P‘Uh
H

s“,
I‘h-“~
"b: :r
*u "rFr
E c-
”A.
'\- .‘
ii‘hfii ‘
.

 

 

 

 

Chapter 1
INTRODUCTION

Gene ral Orientatio n

A major thrust of this dissertation explores the viability of non-invasive fiber
typing in mammalian skeletal muscle using 31—phosphorus nuclear magnetic
resonance spectroscopy (31P-N MRS). Fiber typing has been of perennial interest
to clinicians in the rehabilitative medicine fields who wish to catalog the
histochemical changes from muscle tissue biopsies as a result of acute trauma or
chronic degenerative processes. More recently, in the applied exercise sciences, the
debate over whether central cardiovascular factors or peripheral muscle factors
limit maximal whole—body oxygen consumption (VOZmax) has been re—ignited.
Since V021,”x is synonymous with a person’s aerobic capacity, determining what
limits V02max is of profound interest to cardiologists, exercise scientists, and
coaches. Support for limitation by peripheral muscle factors comes from
observations that over 80% of the slow V02 component reflects the oxygen
consumption (Q02) kinetics of the exercising muscle mass (for reviews see Refs.
246, 309). This phenomenon necessarily reflects muscle fiber Q02 kinetics which
are dependent on the communication between cytosolic ATP demand and
mitochondrial ATP supply. Invasive fiber typing protocols based upon staining for
oxidative enzyme contents and isoenzymes of myosin ATPase have led to the
Conjecture that the fast V02 component is due to Type I fiber recruitment and the
Slow V02 component is due to Type II fiber recruitment (18). This explanation is
derived from observations that Type I fibers generally have higher mitochondrial
contents and greater capillary densities resulting in greater oxidative capacities

compared to Type II fibers (261). The greater the oxidative capacity, the greater the

 

.,o o» “What
' '..| .9 AM..-

- r: ..'..
2".” hi‘ “it
I

“ch

iackgroun,

...._’ V .-l -o
\. {,W'Ip‘

-'--bvm\u‘ : ,-

H‘" “NF,“ 3 '
1‘5" “Mttgdr
Tran; :- n 7‘ ~
a. km?! a goru u

.3 . ., ‘
.F‘V 0'. . .
. "' r.
"“‘~'~ u.“ ...
.

23.71% FG \v-

 

70; ‘
‘ I
"4.; n

5!;
v?

v,
C
.
b
{a}
(-O

‘f-‘3 muscle '1

:ffl- ' i ‘
i.~.§.:;h‘c lab-4‘ ‘

Mgr

: ., ,
\Mt. “‘C're‘ m i;

. .4“. 4"
‘qu‘u ‘1'}00mf‘rjl

4‘3“. l,
.~~“Ll\ '
7 K
n

 

 

 

 

extent to which fibers rely on oxygen consumption to meet the instantaneous ATP

demand by the contractile apparatus.

Background of the Problem and Specific Aims

Consequently, it is incumbent to systematically classify fiber type distributions
in working skeletal muscle to provide a basis for mechanistic interpretations of
V02max' Traditional fiber typing methods over the past 40 years have employed
histochemical stains, immunocytochemical markers, and/or gel electrophoresis to
assay numerous enzyme activities and protein constituents. These methods have
provided a cornucopia of information unattainable through other techniques (261,
242), and they have led to the commonly reported bipartite (Type I v. Type II) or
tripartite (FG v. FOG v. SO) fiber type taxonomy.

Unfortunately, these traditional methods require in vitro assays of freeze;
clamped muscle biopsies, and several biopsies are usually needed with respect to
geographic location within the muscle and/or time during the experiment.
Furthermore, multiple biopsies necessitate halting the experiment and may cause
patient discomfort once the local anesthetic has worn off.

Because of these limitations, measuring oxidative capacity via in vivo 31P—NMRS
has recently been proposed as a non—invasive alternative for estimating muscle
fiber type distributions. The promise of 31P-NMRS is it offers real—time tracking of
energetically important intracellular metabolites (PCr, ATP, Pi, pH) within the
Same muscle. Not only does this greatly increase time resolution, it also increases
Statistical power over the one-muscle—one—time—point (and/or one—geographic—
location) limitation of the muscle biopsy method.

Consequently, a necessary prerequisite would be to correlate a 31P—NMRS
derived parameter to an energy supply or energy demand fiber type parameter.

Previous theoretical and empirical studies have shown that the steady—state PCr

 

. ‘rn" -.'n.

n
:7" ‘fl‘g‘ :L“:-
U

.3»; - pr‘f‘ 9
00 .0 i
.q-hav $~thu . O

' - v a
u-. .:4- Op Our
#:4‘: 5U but -

IL \ .4 ,
a..- but: v

I, -

.‘V‘m—o...
4

'~umu..| I l

Specifji

l».
4.1.. uu.

a n ‘ .
Unuatlb ' '

I
;"v .. '
*nu‘ FPF" "

7 \toole ,
r-w‘ .
m. .u 13‘ er a

1-
| .‘h ‘ '
-” | :1" F 9

{Wu gne I»;

M.

.1‘\ " . ‘
l~“‘ c‘.

l.~l

'l

{V . r

 

 

 

 

 

level during stimulation scales with oxygen consumption, and the time constant of
PCr recovery after submaximal stimulation should be directly proportional to total
creatine content and inversely proportional to oxidative capacity (192, 203, 208).

This leads to the first specific aim of this dissertation:

Specific Aim #1: Does skeletal muscle oxidative capacity in a
relatively homogeneous fiber population scale with an in vivo
31P—NMRS derived parameter such as the PCr recovery time
constant?

If affirmative, this leads to the second specific aim of this dissertation.

Specific Aim #2: Can 31P-NMRS be used to elucidate the
distribution of discrete fiber type populations based upon relative
orddative capacity in a known heterogenous fiber population?

In answering Specific Aim #1, skeletal muscle mitochondrial content will be
perturbed over a six—fold range, a range significantly larger than in any human
study, and the time constant of PCr recovery will be measured with 31P—NMRS in
the rat superficial white gastrocnemius (mostly fast—glycolytic fibers, (8). This is
based on previous studies which suggest the time constant of PCr recovery is
inversely proportional to oxidative capacity when normalized to total creatine
content (203).

Specific Aim #2 will be addressed by first constructing a high signal-to—noise
31P—-NMR spectral time course from repeated, contiguous, stimulation—recovery
Cycles with phase and frequency correction performed by the novel Principal
Component Analysis (PCA) method. An intentional effort will be made to
interrogate a deeper volume of muscle in the rat hindlimb which is known to

Contain a heterogenous population of fibers with respect to oxidative capacity (8).
Next, the global, NMR—derived, PCr recovery time course will be decomposed

using the unbiased Non—Negative Least Squares (NNLS) algorithm. This is done to

"h . .,-
P1 It CUAs~
.5“.

'
“0.? "‘7‘ 0;. H
3.....th A ~4-

I

.,. ~01 "- e
:.:n:- mi:'~o -

Empe oith

' l
F~v~ r v 0
,‘r'r.
on". LAW“. A
I I

.53.”. :LSCTElE i

 

1:»; 2569 to :

fo.‘ _."-
“1.: J.’ Ind!

.mitations
l T315»

' I
For r
r" 1|" 0;

“due ..I

dl‘rfln 0.

““‘K s.
\—

~O VII

animal: '
0: ‘Li" --oi
a he \ \

... l I
mate I.

'2: lite

‘9-0
“elsen '

 

T101 onlr

'1‘
I

aphiar-

 

extract time—constant amplitudes and durations in an effort to elucidate the

distribution of discrete fiber type populations within heterogeneous mammalian

skeletal muscle.

Scope of the Dissertatio n

The primary focus of this study is to clarify the potential utility of 31P—N MRS to
discern discrete fiber type populations in mixed mammalian muscle. No attempt
will be made to directly contribute to the ongoing, high—profile debates of muscle

fatigue or metabolic control theory.

Limitations of the Dissertation

1) This is an in vivo study using genotypically and phenotypically
similar rodents. These rodents spend nearly all of their time
within the confines of a small cage, are under general anesthetic
during the experiment, and undergo supramaximal sciatic nerve
stimulation simultaneously activating all motor units. The results
of this study, therefore, may not be directly applicable to human
muscle biology and routine locomotion patterns.

2) The in vitro enzymatic assay used to assess mitochondrial
content may not be representative of in vivo cellular activities.

3) Oxidative capacity in the working skeletal muscle is dependent
not only on mitochondrial content but also on capillary density.
Capillary density was not measured.

The following Literature Review places the investigation of phosphocreatine
and its kinetics in energy metabolism in a historical context as a preface for the

Specific Aims mentioned above.

EwyConc

Ergy for mu
momenon

. ‘ b
u. , . ‘
£3.”th Q“

mekuu.“ __

\~. 'Di; : I"
:“M\ ‘n p“‘ \

l".:_1 ‘
“Lawn ,,

.,

.i-‘agrfifl‘ D21:

3» ,

 

 

CHAPTER 2
LITERATURE REVIEW
The role and utility of phosphocreatine
in skeletal muscle energy metabolism

Early Co ncepts (18803-19403)

Energy for muscle contraction was viewed as a heat transfer
phenomenon

Since the late 1920s phosphocreatine (PCr) has been recognized as an
important metabolite in mammalian skeletal muscle energy metabolism. Prior to
this time, a paradigm shift occurred in muscle bioenergetics that allowed this
recognition to take place. The contemporary archetype of muscle as a biological
chemo—mechanical energy transducer was not the prevailing theory during most of
the 18003 (225). During that century, muscle energy conversion was believed to
behave like a heat engine. A heat engine is a device, such as a steam engine,
performs mechanical work when heat is transferred from a high temperature
reservoir to a low temperature reservoir. The explanation of a muscle’s mechanical
Output based on heat engine principles was undoubtedly linked to the excitement
Surrounding the birth of classical (a.k.a. macroscopic) thermodynamics as a new
discipline in physics during the nineteenth century (88). This was accomplished by
a confederation of physicists, chemists, and engineers such as Carnot, Clausius,
Clayeperon, Dalton, Gibbs, Helmholtz, Joule, Kelvin, and Mayer who all lived
approximame during the same time and were all instrumental in formalizing and
extending the laws of classical thermodynamics (196).

Not surprisingly this excitement in physics over thermodynamics infected other
disciplines such as physiology. As an illustration of the muscle—heat engine
paradigm, Engelmann in 1895 (88) attempted to apply the second law of
thermodynamics to muscle by postulating the existence of numerous intracellular

high temperature foci. These foci were to provide the muscle with the necessary

‘9' 1‘ 'P. .'
I‘yl Ill. '4
uh‘ .ov‘s «Act LA

~~~;- x" .
c....:: Ilu......

 

1:41:13 occur
“We. If :.

0.4...4‘.
c

”In.

‘--: 3331! act.

..

‘5 .

._-_'-.3'; ‘7“

.""f Au 1;.“
\-

Elfiavor at :2

5"ill for mu

 

.57» ._~
" ‘ '\
w \ ~.
S‘Edtfin t
A
a.“

intracellular temperature gradients for sufficient heat transfer in order to perform
mechanical work in a thermodynamically reversible fashion. Unfortunately, the
heat engine theory was seriously flawed on two accounts. First, it required
numerous intracellular high temperature foci be maintained at more than three to
ten times normal physiological temperatures (314). Second, Hill (130) showed heat
production occurs during the recovery period from contraction in an oxygen
atmosphere. If muscle were a heat engine, then heat should be produced only
during contraction and not during recovery as Hill showed. Consequently, the heat
engine paradigm as an explanation for the energy of muscular contraction was
losing favor at the beginning of this century.
Energy for muscle contraction was related to biochemical phenomena
Muscle physiologists sought to explain muscle contraction based upon
biochemical mechanisms as a way to supplant the heat engine paradigm. By the
early 19308 PCr hydrolysis emerged as the leading process most closely associated
with muscle force development; however, as discussed next, PCr arrived at this
central role in the early history of muscle biochemistry through a circuitous route.
It was well known before the discovery of PCr in 1927 by Fiske and Subbarow
(94) that the temporal change in the concentrations of PCr hydrolysis products
(creatine, Cr, and inorganic phosphate, Pi) was strongly correlated with muscle
contraction. Uyeno noticed a greater creatine content in tonically contracting
pectoralis muscles of mating male frogs engaged in clasping females during the
breeding season than non-mating male frogs (301). Thompson showed ischemic
contractions of cat hindlimb increased the creatine released by the muscle into the
plasma (297) and Embden was able to show increased urine inorganic phosphate
in humans after exhaustive exercise (86).
Prior to the late 19203 creatine was considered a waste product of generalized

protein catabolic processes occurring during muscle contraction (238). Moreover,

. ' 9- l
‘0‘- F
v'. i
.‘ , “H: “a“.
u

.
.e “’ ‘
“c.1536 '31 "
e- f‘.‘

'4

. ‘
.r‘ ‘. A M. '
h-‘L‘LJC uektlk‘a .

. y I

M- ,. ‘0'. PP '
:3... WI.“ id“—
'I. z" 1

.~w""'"
.3 .A n. ult‘ “1..

12:: WOW“

2:; the resuli

l v
“Lg the 351
331'." ’l “ "
hstéEblc: “ Ci
:‘Meli 1
. 3“;an 1016 I

33“»;
“01's. hm &"

agnmh 'll '
‘

l

 

.~::..:.‘¢c intern.-

i‘xi EIRCKlE-ll: I

R in the re

I‘.
‘39... '

«date the

'Efizn F
«A Inseam
is" F
~..__ 4 ‘
.1. Was 13‘.
L;
\‘l _' isrl
a l‘ll'r ‘
niGiE‘C
1‘59?“ {'5’ .
,mtlon ’
5th."-
“Hg“ L

 

 

during this time frame, most muscle biochemists investigating creatine did so for
the purpose of understanding the factors that caused increased creatinuria under
pathological conditions such as myasthenia gravis, kidney disease, starvation, or
diabetes mellitus (20, 147, 202, 237). Interestingly, it was recognized early on,
creatine had its greatest concentration in tissues with capacity for large changes in
metabolic demand (e.g. skeletal muscle, brain, heart), but not necessarily in
tissues with large constant metabolic demand, per se, such as kidney or liver (20,
147). Furthermore, as early as 1911, Thunberg showed adding creatine to muscle
extracts proportionally increased 02 consumption and C02 production (298).
Unfortunately, these observations did not resonate in the muscle bioenergetic
community prior to the 19308 since increased creatine during contraction was not
viewed as being directly tied to the energy supply of the contractile apparatus but
rather the result of muscle protein catabolism.

During the same time frame the emerging role of inorganic phosphate in muscle
bioenergetics was far more captivating to bioenergeticists than the putative waste
by—product role of creatine. The metabolic significance of inorganic phosphate
evolved from being considered as an obscure anion known to promote plant and
yeast growth (117) into an essential ionic species covalently bonded to key
glycolytic intermediates involved with lactic acid formation. Much of the motivation
bebind Embden’s ground breaking work on the glycolytic pathway during the
1920s was the result of tenacious attempts to isolate a carbohydrate—based
intermediate that liberated phosphate during muscle contraction, resulting in the
Observed increased urine phosphate and increased intramuscular lactic acid. Of
Course, it was later determined the phosphorylated C3 and C6 intermediates
Embden isolated were directly or indirectly involved in substrate level
phosphorylation of adenosine diphosphate (ADP) and were not intimately linked to

Providing the immediate energy for the contractile machinery.

. .

“... gr. - a. »

‘-..“AAJH‘ -
—

. u p
0'. ‘3"! 9r""
. u

‘i‘ nib-6U ALIA as.

-.~ 1”" It i.

I... a». J .

~ .
.._.....,.,,.5. 1..
o . , A“

bum“. ‘4‘

- . ‘ ‘
D 91 'h.‘ P,_,
Ltd White: at:

"—“I‘n ‘ ~
._ ‘5’" .
x“-~J:tJCu..,

I
l
|
|

- .
lrL"‘r-.~

I.“ B. .7-
M-5 JC..

"."vtl. r‘ ,
“'r~3~-Ul1: .j~.

11“.“
car Emmi-e:
I ‘b‘.

. .
5‘
"up \ ‘,\

enlisted {an

 

 

Throughout most of the first three decades of the 20th century the process of
lactic acid formation was seen as the energy source for muscular contraction. Why
lactic acid? It has been well appreciated by hunters since the beginning of time
that animals killed after a pursuit will have more sour tasting flesh than animals
killed while resting. According to N eedham (225) in the middle of the 19th century
Berzelius identified the ultimate source of this sourness in hunted stags as lactic
acid. The early interest in the biochemical process of lactic acid formation was
largely motivated by developments in the alcoholic fermentation process by yeast.
Extending Buchner’s earlier work for the German beer industry on intact yeast
preparations (34), Harden and Young where able to show yeast-juice was also able
to ferment sugars, thereby producing ethanol and C02 (105, 117). They later
postulated hexoses (of unknown structure at the time) plus inorganic phosphates
were directly converted in a single reaction step into C02, ethanol, and hexose
monophosphates. These insights encouraged Fletcher and Hopkins (99, 100) to
suggest the process by which lactic acid is anaerobically formed from an unknown
precursor in skeletal muscle could provide the energy for contraction—the so called
‘lactate hypothesis’. During recovery, this unknown precursor would be
reconstituted from intramuscular lactic acid. This instigated an intensive search for
the ‘unknown precursor’ and the catabolic pathway that resulted in lactic acid
formation during muscle contraction. Toward this end, the collective seminal
Efforts of Embden, Meyerhof, Parnas, and the Cori husband and wife team
elucidated the glycogenolytic/glycolytic pathways and identified glycogen and
hexose monophosphates as putative candidates for the role of the ‘unknown
precursor’ (162, 215).

As the glycogenolytic/glycolytic metabolic pathway began to materialize it
became necessary to explain two experimental observations during muscle

Contraction: 1) why does muscle Pi increase above resting levels 2) how does

”i l”?— v-
:.\n: :l-t:otr in

r» vh- up}; 1‘
on. .us A6‘Ll LA»

.
.u-«Op-v-
'4

. ‘3
u.» gun's: J‘Ai .
M

2.31??? were

 

‘5.

Q
.u

’u ""-n
. ' A

w, .L, A'M‘
....§... “0361' I!

9‘,

#2) " .
.~,fl. fl
. J bl

‘

“I"

’\

 

F‘l
II on.- 7r. ‘
‘ “40 19"..
‘4
k‘
"am;
'i‘“ ”Eats
l

. .
"3:33;.
““6 tr? 1
*P
.‘.. k
‘..l '.
\‘L‘

 

muscle buffer the lactic acidosis that occurs under anaerobic conditions. At the end
of the 19203 Fisk and Subbarow (95, 96) proposed PCr hydrolysis would explain
both the increased Pi during contraction and the buffering of lactic acid. At the
time, although lactate formation was seen as necessary for muscle contraction,
excessive lactic acidosis was thought to be the cause of muscle fatigue. Fisk and
Subbarow were able to dovetail their findings into the prevailing ‘lactate
hypothesis’ doctrine by suggesting the buffering action of PCr hydrolysis was the
essential factor allowing the muscle to contract without fatigue. Retrospectively,
the significance of their studies was that PCr, Pi, and Cr were simultaneously
brought closer to the energetic determinants of muscle contraction thereby

beginning to turn attention away from carbohydrate catabolism as articulated by

the lactate hypothesis. .
Energy for muscle contraction was explained by PCr hydrolysis

At the start of the 19303, the sovereignty of carbohydrate catabolism as the
direct energy source in muscle contraction was being seriously called into question
(131). Lundsgaard used iodoacetate to block glycolysis and showed contractions
Could still occur (190). If the lactate hypothesis were operative, then blocking
glycolytic flux should have resulted in no contractile response upon stimulation.
Furthermore, he also showed PCr breakdown, assessed by increasing Pi, was
directly proportional to the external work (force x distance) done by the muscle.
Taken together these observations suggested to Lundsgaard that the “contraction
energy is supplied directly and exclusively by phosphagen breakdown” (190).

By the mid 19303, Lohmann had discovered the enzyme responsible for PCr
hydrolysis, creatine kinase, and the existence of an indispensable coenzyme he
identified as a ‘pyrophosphate of adenylic acid’ (AMP-PPi) more commonly known
as adenosine triphosphate (ATP) (189). More importantly, he suggested PCr

breakdown was energetically coupled to the immediate resynthesis of ATP via the

I
h- ‘F‘F (‘9
.131..er J_

‘ w
-"—"11-w gm ‘. .
;._'-‘ V g‘uA‘u

-

I I
..,. ”"nnI-u
‘r'v'_.. ‘ ‘ .
"""-\~-Luu
y C

 

I

{I “a _ 11 ‘
...ce.u13r b

~55.

\ .
{Fir N,

.u HiJLent. r 1
|~~

 

following reaction catalyzed by creatine kinase:

#1 ATP —> AMP+ 2Pi
#2 AMP+ 2PCr <—> ATP + 2Cr
Net reaction: 2PCr ——> 2Pi + ZCr

These observations broke new ground on two different fronts. First, from a general
thermodynamic perspective, Lohmann provided the first experimental evidence of
an energetically coupled chemical process. Prior to this time the idea one chemical
reaction could energetically drive another chemical reaction was only theoretically
entertained (225). Since then it has been repeatedly shown that most of
intermediary metabolism occurs due to energetic coupling of highly exergonic
reactions to less exergonic reactions or to endergonic reactions (14, 105). Second,
and more specific to muscle bioenergetics, Lohmann suggested muscle contraction
will first hydrolyze ATP which is then quickly resynthesized at the expense of PCr
breakdown. From Lohmann’s perspective, PCr was the direct energy source for
contraction and the near constant level of the ATP provided the necessary

inorganic phosphate donor pool to sustain glycogenolytic/glycolytic flux resulting
in lactic acid (189). He viewed ATP as only a “coenzyme” and not the central
metabolite responsible for muscle contraction.

During the beginning of the 19403 Lipmann published a seminal paper in the
field of cellular bioenergetics in which he suggested the central purpose of
intermediary metabolism was to generate compounds of high phosphate—group
transfer potential (188). In other words, the chemical potential energy of a large
number of carbohydrate intermediates (lipids were acknowledged later) is
harnessed via orddation—reduction reactions and stored in a relatively small
number of phosphate based molecules. The major mammalian molecules with
high phosphate—group transfer potential are (in order of decreasing transfer

POtential): phosphoenolpyruvate (PEP) > 1,3—bisphosphoglycerate > PCr > ATP.

10

.n- VFW. '
....( u‘vmy"‘

 

' I
i
""“‘H" '7‘
‘ \ ‘5 '
I-cuuu..'t I “1‘ A
. o l

.Q. on: 1‘--
1".
n.

l
P-‘I
t '§ "
A— un- ‘1 db»
5
. . . I
'0 V’FFI‘V‘F - O -

f"

' I l‘ 4.
.- I'M-~psaub.
n l I

a u .
a . ,

"Pl 3" h: ‘7‘

-~d‘v0\ t‘“ x..-

"V'N‘ha

32'..2':..:..L PEI

t

l "__
«T 3“th “-er’

I.. .L ~
0 ’ Y‘H"~'
...... «if ‘Uu'lth,

WW I ' I
t_|~' F ~‘v- .
sun . :‘der

\—

 

~.,_‘.
‘ky‘ifie Till‘
‘3

H ' \
6",-

“1&6 'currer

2325‘
“U“ and 1
if?“ t‘
“a ' .
m6 lacte

_‘

 

‘———————

 

These compounds would transfer their phosphate group (technically, the
phosphoryl moiety, (91)) to molecules of lower phosphate group transfer potential
such as glucose—6—phosphate (G6P) > glycerol—B—phosphate. In most cases the
high phosphat<+group transfer potential of a molecule arises from its hydrolysis
products having greater resonance stabilization plus lower intramolecular
electrostatic repulsions plus a greater ability to be solvated in the aqueous cytosol
(14). There were two important insights Lipmann provided in this paper (188).
First, the phosphate—group transfer potential of ATP was viewed as intermediate
between higher transfer potential groups such as PEP and PCr and lower transfer
potential groups such as G6P. This realization is tantamount to the characterization
of ATP as the single universal energy “currency” of the cell whereby all metabolic
networks are ultimately linked to the production and utilization of ATP. Second, he
viewed PCr as a metabolic “capacitor” that could store “charge” in the form of
phosphate. This phosphate charge would flow through the cell forming a
metabolic “current” that is utilized to do work. It is worthwhile to note Lipmann’s
view of the role of PCr as metabolic capacitor storing phosphate charge was
conceptually similar to the view that A.V. Hill held in 1913 of lactate as a metabolic
capacitor storing redox charge (128). Lipmann also suggested the role of ATP was
to accept phosphates as a mechanism that prevents phosphates from clogging up
the redox reactions of the triose phase of glycolysis. As significant as the. above
insights were, Lipmann did not waver from the view held by Lundsgaard and
LOhmann whereby PCr hydrolysis was the direct energetic determinant of muscle
contraction and ATP was simply a coenzyme. But compared to nearly two decades
wherein the lactate hypothesis was the solitary working theory, the unrivaled

Primacy of PCr hydrolysis as the direct energy supply was relatively shorted lived.

11

 

me Role

ll? emerge.

.-,Fo , ,-
I‘ -
Alb. “tube“?

—_=-_

. . ‘
~..--r.r~-c.
. .-- at .
IIu~~PAO¢I

. I

Q .
"-y'A, 'y...
.lft‘ i

, \
be . salt»

 

...~o
~~o€qui L‘.F.'
-

 

 

The Ha le Reve rsal (19403—ea rly1970s)
A TP emerges as the primary energetic determinant of contraction
As mentioned above, the initial function assigned to ATP was a coenzyme role as

a phosphate donor to the glycolytic/glycogenolytic cascade and to creatine;
however, this subordinate role was slowly phased out by the early 19603.
Throughout the 19403 there was mounting, persuasive, circumstantial evidence
that turned attention away from the phosphoguanidine bonds of PCr toward the
phosphoanhydride bonds of ATP as the source of chemical potential energy for
muscle contraction. Although myosin had been considered the major structural
protein of muscle since the middle of the 18003 (225), the ATPase enzymatic activity
of myosin was not firmly established until 1939 (87). This discovery of myosin’s
ATPase activity had a profound effect on the muscle bioenergetic community.
Forcing the question: is the functional connection between the role of myosin as
the major intracellular structural protein of muscle and myosin’s ability to
hydrolyze ATP more than just coincidence? To make matters even more intriguing,
by the mid 19403, Szent-Gyorgyi and his Hungarian colleagues had discovered
filamentous actin, another intracellular muscle protein found in high
concentration, and showed under optimal Mgz+ and K+ concentrations, ATP was
required for dissociation of myosin from actin (286). Dainty et a1. (64) honed these
key results by showing only adenosine triphosphate—not simply any generic
tI'iphosphate such as various purines, pyrimidines, or inorganic triphosphates—will
dissociate myosin fiom actin. Consequently, by the end of the 19403 two general
tonets were well appreciated: (#1) based upon Lipmann, all metabolic networks
are ultimately linked to the production and utilization of ATP; and (#2) ATP is the
only molecule that dissociates the two structural proteins coincidentally having the
highest collective concentration of any chemical species in skeletal muscle. The

inference from these tenets was inescapable to the muscle physiology community—

12

 

m: age:

‘r
.
“a

f-oo p A‘ n .
25-. ...E::

"' :rrw-r Al‘ ' .
31“... JdLlat.

"" ‘ Arno ,a . .
‘cht [Ll-lundr‘

I
‘;II :9.
~~-uug L:
In. .

-' DP Fr ~ .
5.3.1: U-Euldr_i,
‘ A

c. .L
t. “ . u , ,
{a “.5“ ml_, ‘
’9'»...

P . -
i“ Lise; al Cl}:

3373336 to 5

 

Fo_.__..-. n :A
‘hotukrdtlin C0
72...; _
“Wilt 0i lhE

“-l n
QA’LH.

agent'nge \\.

:l

ii.“ F o

1“. : P 1
«EL aha

‘v.

I

i; _
p
‘\_ lhy.
u“e:‘1n,‘
\LI“ J
ya
\_~
‘n‘i;f\ ,
.~, ‘
Chg, Cl)"
(1
‘u
‘4...
K. 1..
a": In ‘h
L E]
..-
‘sfifu' ‘
"RAH.

ATP is the direct source of energy in muscle contraction.

Unfortunately, as seductive as the above hypothesis was at the time, there was
no direct experimental evidence ATP actually broke down during contraction to the
extent necessary to drive the contractile apparatus. Sensing a need for infusion of
new approaches to directly determine if ATP was significantly hydrolyzed during
muscle contraction, the esteemed Nobel laureate A.V. Hill issued a “Challenge to
Biochemists” (134). He made several suggestions in his challenge: use in vivo
muscle preparations rather than in vitro muscle extracts, use muscles from
species that move slower (i.e. tortoise rather than a frog), use limb muscles 30 a
contralateral control muscle can easily be obtained, and decrease the operating
temperature to slow the reactions. The overall objective of this challenge was to
slow down the reaction velocity such that statistically significant changes in ATP
concentration could be observed during contraction within the frequency response
limitations of the instrumentation available at the time. A year later in response to
this challenge Weber and Weber showed the tension developed by single
glycerinated muscle fibers depends on the ATP concentration (310), and
Mommarets and Rupp showed ATP is broken down during contraction in frog limb
muscle compared to contralateral resting controls (220). Both of these experiments
were consistent with the view that ATP serves as the direct energetic determinant,
but neither result could also exclude the chemical potential energy provided by
PCr hydrolysis as a direct energetic determinant of muscle contraction.

Over the next ten years additional incidental evidence of the central role of ATP
emerged from other domains of muscle physiology and bioenergetics. Based on
earlier investigators’ work with cytochromes, flavoproteins, and pyridine
nucleotides, Chance and Williams elucidated the precise sequence of redox
reactions in the mitochondrial respiratory chain responsible for oxidative

phosphorylation (52). They also suggested mitochondrial oxygen consumption

13

,,.,. ordsl‘ I

Dunn-0‘ t .I-Sh —

.-.--b~op any“
\ a I‘ -

~J~g¢urc Suki
A

-....... 4..
t'l
. .-

. “Ci: «it .

I-I ‘Wl'f.~ O.
- r ‘
“a-smhdn so

.

1" q
x. r M‘
«nutaJJl‘.

 

 

“.‘
I“ h ‘

‘3 “n-Llal‘tir
p

"9.11

“ill in 19.
the.
"‘VL Of :ht‘

ngj rl.

RAE cre

:3..me ‘
. HQ: abdor

~.‘.
Qab...
‘n Mm
bu,

L Ieil‘f

during twitch contractions of frog sartorius muscle is limited by the amount of
phosphate acceptor (i.e. ADP) within the matrix (53). Taken together their results
supported the idea of oxidative phosphorylation as a third ATP supply system that
complements the other two ATP supply systems based upon substrate level
phosphorylations, i.e. the creatine kinase reaction and glycolysis. This is consistent
with the “linked hydraulic reservoir model” of interdependent ATP supply systems
Lundsgaard postulated in 1932 (190)-well before the tricarboxylic acid cycle and
the mitochondrial respiratory chain were discovered! Chance’s results also neatly
tied ADP, a hydrolysis product of increased ATP demanded by the contractile
apparatus, to increased ATP supply, thereby supporting Lipmann’s
characterization of ATP as the cell’s energy currency. At the same time during the
mid 19503, H. E. Huxley and Hanson articulated the Sliding Filament Theory as a
mechanism explaining the changes in the cross—striation patterns occurring during
contraction of rabbit psoas myofibrils (148). Although they entertained other
possibilities in their paper, they strongly felt the interdigitation of actin and myosin
during contraction was driven by the enzymatic splitting of ATP.

Finally, in 1962, direct experimental evidence ATP actually broke down during
activation of the cross—bridge machinery was obtained when Cain and Davies
inhibited the creatine kinase reaction with fluorodinitrobenzene (FDN B) in isolated
frog rectus abdominus (40). The results were unequivocal: FDNB treated muscle
underwent ten—fold fewer contractions and consumed significantly more ATP while
Sustaining no change in PCr as compared to controls. A.V Hill’s challenge had been

conclusively answered—ATP was the indisputable primary energetic determinant of

muscle contraction.

14

Edi of PCr

. ‘ l ‘
.uoe F‘
L“ we on.

 

. ".ogl")
:25. Jew"
«1

.

u—‘V Vim - I

!:L1¢“D:
O

-._>«-\" . 'i‘ r
. r “ ii I
r+-..vd . u.

_ u
‘- QFH'FI“ I r.
I "l -¢.Jt“ .

' |
..,..;~" F ""
‘. Lt. Lat/(\‘t'i

r. '
L :25. PCT dOIl
genius .i. v

.13 . then it

 

 

union will

M othermal techniques refine the bioenergetic role and experimental
ut lity of PCr ~

With the basic bioenergetic interrelationship between ATP as the primary
energy metabolite and PCr as its phosphagen capacitor now clearly delineated,
several camps within the muscle energy metabolism community turned their
attention toward more precise characterization of the thermochemistry and the
time dependent behavior of PCr changes during and after stimulation. In general,
the thermochemical lines of investigation stemmed from motivation to quantify
changes in enthalpy, free energy, and work as a way to assess the thermodynamic
efficiency of muscle as a chemomechanical energy transducer (219, 314, 316).
Interest in the timwependent behavior of PCr was based upon exploring the
ramifications of a corollary to Cain and Davie’s unequivocal answer to A.V. Hill’s
challenge. That is, since ATP is temporally buffered by PCr during contraction (40,
45) and PCr donates its phosphoryl group to ATP through a single, biomolecular,
equilibrium, phosphoryl—transfer reaction catalyzed by creatine kinase (91, 137,
171, 230), then it is reasonable to infer measuring PCr changes during and afier
contraction will provide a direct measure of the energy cost of the underlying
metabolic processes. As will be discussed later, tracking phosphocreatine’s
behavior became, and remains today, an important experimental technique to
discern the fundamental bioenergetic properties of muscle efficiency, metabolic
energy costs, and oxidative metabolism. What follows next is a brief survey of
Pragmatic reasons as well as methodological and technological advancements that
facilitated understanding of the thermochemistry and kinetic behavior of PCr.

The pragmatic reason was simply experimental convenience—that is, the “when
Nature hands you lemons you make lemonade” maxim. Unlike all of the other
imwrtant energy metabolites (AMP, ADP, ATP, NAD, FAD) in skeletal muscle,
phosphocreatine is found at much higher concentrations (53, 91, 146, 230) and

undergoes readily detectable changes even during mild stimulations. Without

15

.
"

ol .

........,.

.a...‘faJ':uLt‘ . A
C .

'l
rww-r...‘ ’.‘
‘ tswtetbdii '

U
’ ‘er a Q
D' '
“-5- u-o. . .
hr

“‘9'. A'df' I -"..’V

oi--...r‘dvsu‘. .

. . I
~""‘ Pr“.-.

.n
""‘I ‘ LuaatdsI
. I

.iI Eliza a: f

‘V\
”‘9'" (“NHL ‘|
-‘-. .JlJrJls“ ‘
. l
I H

t {-
II:"I 5",." «O
.v“~~‘ d‘ Li‘d‘u.

‘9.,,ul A ~ ~
-’ ..-.. may

Ii‘iI‘iI't- U
I~~~~L\I 2) A“

:J’cd to the

 

w‘~_ M
(““iiiples u

5;; a h _
c LCittraCtl‘
\erh

“3 -. ,
uddon
." .
fr
.\ y.
"M:

FDNB, the creatine kinase inhibitor, only the most severe stimulation protocols,
which may require blocking blood flow, will significantly change the [ATP].
Consequently, PCr was a prime candidate for investigation since it was the most
experimentally accessible energy metabolite and was intimately linked to
buffering ATP, the primary energy molecule.

Methodological and technological advancements between the 1940-1970s also
greatly facilitated the growing interest in PCr kinetics that continues to this day.
Two examples relevant to myothermal techniques include: 1) The development of
more thorough rapid—freezing procedures for excised muscle tissue, which greatly
reduced artifactual hydrolysis of labile phosphate groups prior to extraction and
chemical assay (170), resulted in more accurate determination of [PCr] with less
variability. 2) Measurement of muscle heat production was substantially improved
compared to the pre—W.W.II era due to construction of thermopiles from individual
thermocouples which greatly improved temperature sensor gain (135). Moreover,
the temperature sensor’s response time was significantly reduced to = 10msec by
the newly available commercialized engineered plastics (e.g. Teflon) and epoxy
resins which abated nuisance heat capacity (101, 321).

Given PCr’s experimental accessibility, its role as ATP’s phosphagen capacitor,
and the improvements in temperature measurements previously noted,
bioenergeticists in the 19603 exploited the behavior of PCr during and after
stimulation as a way to understand the efficiency and energy costs involved with
muscle contraction and relaxation. Selected highlights are discussed below, for
comprehensive reviews see (63, 219, 320). In an effort to simplify the metabolic
systems under study, all of the myothermic experiments mentioned below used
isolated frog muscle at 0°C with glycolysis blocked by iodoacetate and oxidative
phosphorylation inhibited by a 100% nitrogen atmosphere.

Carlson showed there was a linear relationship between the extent of PCr

16

‘ ”MI; 3L6 '

”Sui-l

. «AL,
o‘.v.‘au D “k‘l

:cezgcal per

93:: 3f the:

 

”0...; ' ...~.

aux. UL“;

'25: ELSE in}
' v

.,_‘_ 'uo.‘ u, ,
*“mitlt‘ 0V

 

kt: ,
Q"

‘46 [hEV
‘:"\‘_I k

,4" ‘ TP

. CG
‘5
a.“
\."p g‘

‘4. \

K.

utilization and the number of isometric twitches (45) or external work done during
isotonic twitches (44). Coincidentally, he also confirmed the Fenn Effect (93) from a
biochemical perspective by showing the extra energy liberated during shortening
in excess of the isometric condition can be accounted for by PCr hydrolysis and
associated buffer reactions. In Fenn’s particular frog muscle preparation it was
shown that muscles undergoing isotonic contractions, thereby performing external
work on the environment, liberate an extra amount of heat beyond that
accountable by the work performed. Carlson also argued in his two papers that
PCr dephosphorylation and its associated buffer reactions are the major net energy
yielding reactions taking place during a complete contraction and recovery cycle in
either isometric, lightly—loaded isotonic, or heavily-loaded isotonic contractions.
This was based upon the observation that thermopile—measured global enthalpy
production in isolated frog muscle was not significantly different than the predicted
enthalpy production of PCr hydrolysis from thermochemical tables.

A few years later in 1966, Sandberg analyzed the Sliding Filament Theory from
a bioenergeticist’s perspective (264). He found ATP consumption, as estimated by
PCr hydrolysis, had both a length—dependent fraction and a length—independent
fraction during isometric contractions. The length—dependent fraction of ATP
consumption had the same qualitative dependence upon sarcomere length as
isometric tension. That is, both ATP consumption and isometric tension reached a
maximum around the optimal length, Lo, (0.26umole ATP per gram muscle per
second at Lo 2 2.25um) and both dropped off precipitously below 70%Lo or above
140%Lo. When they stretched the sarcomeres to various fixed lengths greater than
140%Lo—minimizing myosin’s ability to form crossbridges with actin—and stimulated
the muscle they found ATP consumption was approximately 25—50% of the
maximal ATP consumption at Lo. They termed this the length-independent

fraction. Sandberg astutely suggested this length—independent ATP consumption

17

 

‘. r1

.‘l-r"'| ' "

'2 ..;-'Jul\ at
I .

1

’ ”use i.

.lll U

E.

I l ‘ ‘
f" .p - “o‘ch
‘5‘]

a-.&t

 

”‘v *: '
a “F ~
mm 03.»

"O ‘ r‘
"" - Cd.

~.
I”“"‘ P ;

9 .
N b‘vw‘tc L6 !
.

 

\-"‘~
R.

Trr- '
“31111ch
\.D

‘li'C‘PA ‘
“a 1n the

'..-- .
\ fire.

:0 to UK

was probably due to calcium pumping and represented a significant fraction of the
total ATP use during a complete stimulation and recovery cycle. This result
conflicted with Carlson’s two papers a few year’s earlier which suggested net PCr
hydrolysis and its associated buffer reactions are the only significant ATP
consuming process. Furthermore, this indirect energetic evidence of an ATP
consuming calcium pump nicely complemented the 1960s surge of interest in
calcium handling by muscle cells as summarized in the next paragraph.

A decade earlier Sandow (265) formulated the “Calcium Theory” of muscle
contraction based upon electrophysiological measurements and light microsc0py
studies. His Calcium Theory pr0posed that calcium is stored in an intricate
interconnected network of internal membranes (the sarcoplasmic reticulum), is
released in response to sarcolemmal depolarization that requires a transverse—
tubular system, binds to the contractile apparatus and is taken back up into the
sarcoplasmic reticulum. Unfortunately, most muscle physiologists in the 1950s
dismissed the Calcium Theory largely on the grounds that they saw the internal
membrane network as simply an optical artifact from the fixation process required
to view muscle tissue slices under a light microscope. It required the development
of the ultramicrotome, freeze—fracture techniques, and heavy—metal shadowing
processes in the 19503 (275) that allowed thin—slice transmission electron
microscopy to unveil the structural detail required to pique the curiosity of
audiences other than histologists and cell biologists. Persuasive functional evidence
for the Calcium Theory came after Ebashi discovered calcium uptake required ATP
in muscle microsomes (79), Padykula localized an ATPase in the SR of rat
diaphragm muscle (233), and Winegrad (317) elucidated transient intracellular
fluxes of radiolabeled calcium during and after tetanic contractions. To
bioenergeticists these studies suggested muscle cells must budget at least some of

their total ATP currency toward handling calcium during and after contractions. As

18

,....-.-.4l FL

.3...‘...‘:u u»—
' .‘l ..o
v--:“ i"

.;z,_.+.\it 8:.

[tag at L.

l I
I. - - O-
, 7'
-‘i: Adhbt‘k

Q
Hunt. x"...
_v 0- ., t
'v~v.‘u A

rp-v

.‘r0- .
'1'»th l‘t.‘
.
.;~"‘Y'rnw - .
.~L...._!‘I“d“A.L

16.1mm. c1

1.
Err; ‘rlq.'_ '9-
I~.M“.‘;=‘
\—

 

\-
J33? cl
M [‘56 0L
1 n

mentioned above, Sanberg’s discovery of length—independent ATP consumption in
frog muscle estimated this allocation at 25—50% of the maximal ATP consumption
occurring at Lo.

In the latter half of the 1960s a fresh experimental paradigm, called the
“myothermal energy balance method”, emerged in which measuring PCr changes
played a vital role. The basic premise behind all myothermal energy balance
experiments rested upon a conspicuous application of the First Law of
Thermodynamics and assumed isolated skeletal muscle behaves as an isothermal,
isovolumetric, closed system-able to exchange heat but not matter with its
surroundings. The principle is that one should be able to correlate all of the
“biochemically explained” enthalpy production of known reactions (e.g. PCr
hydrolysis, lactate production) to the observed enthalpy production during, and
after isometric or isotonic contractions (63, 141, 219). The biochemically explained
enthalpy (AH) production requires knowing in vitro AH values plus measuring the
extents of reactions via direct chemical assay of metabolites extracted from freeze-
clamped muscles. The extent of reaction is evaluated by comparing metabolite
concentrations in the stimulated limb muscle versus its unstimulated contralateral
control limb muscle. The contribution of heat to the total observed enthalpy
production is measured by thermopiles; the contribution of external work (isotonic
contractions only) to the total observed enthalpy production is determined by
measuring force acting through a distance. So, within experimental error, the
myothermal energy balance method dictates it should be possible to quantitatively
fully account for the observed enthalpy production by comparing it to the
measured extent of the biochemical reactions multiplied by in vitro AH values
from thermochemical tables. If there was a statistically significant difference
between the observed enthalpy production and the biochemically explained

enthalpy production then it would be concluded some unknown reaction(s) was or

19

.C'lv.".‘ V‘“:~
.

“L «.er1.“
A

‘D"v~r N
4 The

.5... ‘ no; hi.
i. - ~

‘
wv- A- 0,.
and: dull L

I i ‘ ‘
Crux q p...
-. . r‘
I-vii' a (“u‘ ‘.‘

I
~.

I
“any .
~--.u..¢¢4

‘c

nr’u.

'W ,
0.. khutFDUu

‘; .. ‘. =
h P 'l “n -
N»X2¢.1“‘lti

\“
\lh ~I.
.uLé‘ [hf
x.
{at}
4‘ 'v-
' ti . v-
.9. En
_\‘
.\ ~_.
“I“:‘h. H“. l
- “Lnr‘
,u
\‘3'1, ‘

were taking place. The enthalpy of the unknown reaction(s) would comprise the
total “unexplained enthalpy”.

Applying the myothermal energy balance principle, Woledge (320) using frog
sartorius and Gower using rat soleus (111) found the enthalpy (heat + work)
produced during contraction and relaxation is greater than can be accounted for by
the biochemically explained enthalpy of PCr hydrolysis and lactic acid formation as
measured calorimetrically. But over an entire contraction—relaxation—oxidative
recovery cycle the total energy measured as heat plus work was not significantly
different than the enthalpy produced by the known metabolic reactions occurring.
After numerous investigations (cf. (63, 141, 321)) the major observations were that
the unexplained enthalpy is dependent upon the stimulus duration, exhibits a Q10
effect, is greater if a muscle rapidly shortens than if it isometrically contracts, and
is quantitatively and qualitatively different than the isometric maintenance heat.
(The Q10 effect is a generic term describing the increase in reaction rate seen when
temperature increases by 10°C.) It is believed the unexplained enthalpy is
primarily due to the heats of binding, ionization, and solvation associated with
calcium movements during contraction and relaxation. This includes release from
the terminal cisternae of the sarcoplasmic reticulum (SR), binding to troponin,
binding to parvalbumin (in fast twitch fibers only) binding to the longitudinal

segments of the SR and binding to calsequestrin within the SR.

PCr hydrolysis helps estimate muscle efficiency

In addition to quantifying the energetic costs of contraction and calcium
movements, the simultaneous measurement of enthalpy production and PCr
breakdown provided necessary data to attempt to estimate skeletal muscle
efficiency during shortening. Without requiring a derivation from first principles,
an intuitive notion of machine efficiency became obvious when the first steam

engine, a heat engine, was developed by James Watt in 1769 (196). Simply put, a

20

 

Iii?"

...v . -r ‘
. D _
n. -m-‘"‘

_ ...,.-,,-
'1’ .- -
~vo..‘.u‘.n-

~¢l --> a n
. . C .
nuguusl \-
u

C 'l
.5"!" K,

n —
c-‘uiah‘ u!
.

 

.ns-u .4;

 

 

 

 

 

heat engine’s efficiency is the ratio of its work output to heat input. Unfortunately
for bioenergeticists, this straightforward interpretation of efficiency is not
applicable since muscles are isothermal energy converters. Up until the 1960s, the
efficiency definition most widely used by muscle physiologists was the one
suggested by A.V. Hill. He defined the mechanical efficiency (emech) of a muscle as
the ratio of total work done to enthalpy produced (129, 133), emech = W/(W+AH).
Total work done includes both external work of the muscle exerting a force acting
through a distance plus internal work against an ill—defined elastic component in
series with the contractile component of muscle. Enthalpy is the sum of heat
produced plus total work. Using this definition, Hill found mechanical efficiency
during an isotonic contraction was dependent upon shortening velocity and had a
maximum of almost 40% at 88% of the maximal velocity of shortening (132). In his
1960 paper (314), Wilkie suggested Hill’s mechanical efficiency concept is imprecise
because the thermopile—measured AH term is actually the sum of the free energy
available to do work, AG, plus an entropy creation term, TAS (i.e. AH=AG+TAS).
He argued it is only the free energy term, exclusive of entropy creation, driving the
cyclic changes in the myosin kinetic states during contraction. Wilkie suggested a
thermodynamic efficiency, etherm’ be used in place of emech, where etherm =

AH ATP/AG ATp(emech). AH ATP and AG ATP were to be obtained from in vivo
measurements of ATP hydrolysis. Toward this goal, Wilkie’s laboratory measured
phosphocreatine hydrolysis as a way to estimate the in vivo value of AH ATP during
isometric and isotonic contractions (43, 315). Using frog muscle at 0°C with
glycolysis and oxidative phosphorylation inhibited they found that for either
isometric twitch or tetanic contractions the in vivo AHpCr was z10.6kca1/mol. By
taking the ratio of heat produced before inhibiting creatine kinase to heat produced
after inhibiting creatine kinase they estimated the in vivo AHATP value to be

11.0kcal/mole based on knowing AHpCr (315) . AGATP was not directly measured in

21

 

 

  
 

2:": 3339! Hill

 

.-vv‘ r.
3...)..ayf :

"'v 4. .
r-no

“,~," .
it 3"”: vi .‘JL

‘ I
b“. ,* . ’ ‘
- ’0‘“! n

Inlay-y“ .“ ‘
c

U
N

3;!» ‘
x» w, con‘?.

~LI

lib

,-..;
.‘. fine _
"“‘dusc

I" 4

. u

. ‘ y'{J‘i-«l

“L"‘is;
L

 

their paper and so they did not calculate etherm' Instead, they inferred the upper
limit of ethem would be approximately 80%. This was based upon previous studies
in FDNB poisoned frog muscle that showed AGATP z mechanical work/mole of ATP
split and was at least 5.5 kcal/mole (numerically: etherm = AHATp/AGATP(emech) =
11.0/5.5(40%) = 80%).

A somewhat more direct approach toward estimating muscle efficiency was
attempted the following year by Kushmerick and Davies (173) with the same
animal model Wilkie’s group used. Kushmerick and Davies suggested the
thermodynamic efficiency of chemo—mechanical energy conversion during
contraction is the ratio between work done and in vivo AGATP. (Note: their
efficiency definition, ethem = W/AGATP, is intuitively appealing since it is analogous
to Watt’s straightforward definition of heat engine efficiency, e = W/AQ.). Different
values of work done were obtained by integrating the force versus time record for
different constant distances shortened. In vivo AGATP at different values of work
was estimated by a formula requiring educated assumptions about intracellular
ionic strength based on Debye—Hiickel theory and activities of all of the various
ionic species of the adenine nucleotides based on chemically assayed
concentrations. A unique aspect of their experimental design was the use of an
isovelocity ergometer which, in contrast to an isotonic work measurement,
measures the work done by the muscle exclusive of the passive series elastic
components. In conjunction with results from their companion paper in the same
journal (174), this permitted them to directly measure the work done by the
myosin ATPase or the SR Ca2+ ATPase or both during shortening. The key result of
their experiment was they found etherm is not constant, but exhibited a complex
high—order polynomial dependence on shortening velocity. More specifically, they
found the etherm of the myosin ATPase, per se, reached a maximum of 97% at

approximately 30% of the maximum shortening velocity. The etherm of the myosin

22

 

|
... nu w 0"
l...L".' um: tut
A

a

. ..,.

I'V'f"‘"1‘r‘.!

...'. .Jmpfio. wAh
.

r. umfh: pf,
a nouvu~ a

- .
‘
fine -..'l, ‘5'
‘ . . ,
a... xicAulhnn.
' 'v

u

I .‘F' :
.QTI‘.

1;; .ur

“NZH'
“AA; .A‘ ‘
o

la.

'1‘

..._s

. 2 1 .
"W‘ r n..-
r ‘ \
~Ԥ

A. .‘_I

‘m... -
u“ 1?? ‘v :9.

.mn...‘; ~\f
v

"5" «u ‘ ‘
‘5- a. “1er, E:

h‘ h

"v 'n (N M-
. ~¢th lilg
1.“ no.“

‘-..

\ ~| .4 Lit-m, r
| us,‘ “if:

u

v2;

“Vii ’ 1
. -
a
.0
I"
‘.l 'r.'
\ut.‘:s a“
‘.
“a
\f‘is.
“Ni-K ,-
“15 'J 9
L
’a.‘
"-.'I~.

b
»iu.'
\ A. ‘
mg"; a
a, r.
NIJL
‘.
V.”
"4 if“.
‘u l??-
c"

ATPase plus the SR Ca2+ ATPase reached a maximum of 66% at the same %Vmax.
For comparison, the thermal efficiency of a typical four-stroke gasoline engine
reaches a maximum of about 28% at approximately 50% of its maximum
revolutions per minute (227) and the photosynthetic efficiency of C3 plants in
strong sunlight is around 30% (249). (C3 plants are so-named since they initially fix
002 in the form of three—carbon acids.)

In summary, simultaneous measurement of the enthalpy and chemical extent of
PCr hydrolysis before and after contraction provided bioenergeticists of the 19603 a
“measuring stick” to routinely estimate energetic costs of isometric or isotonic
contractions, estimate the energetic costs of the newly discovered calcium—ATPase
required for muscle fiber relaxation, and quantify the thermodynamic efficiency of
contraction.

Contempo ra ry Views (early1970s—pre se nt)

The connection between PCr kinetics and oxidative capacity
and its implications

By the mid to late 19703 most of interesting experimental permutations using
the myothermal energy balance method had been done: “unexplained” enthalpy
was explained, global thermodynamic efficiency of contraction was estimated, and
more accurate assessments of the value and temporal distribution of activation
heat, shortening heat, and maintenance heat evolved during contraction were
obtained (172, 321). During this same time period, attention in the bioenergetic
community began to be directed toward assessing the interrelationship between
PCr kinetics and oxidative metabolism. The purpose was to gather a functional
assessment of the extent and rate of ATP demand during a contraction—relaxation—
recovery cycle and the capacity of the ATP supply systems to meet these demands.
This rebirth of interest in this interrelationship was based upon three historical

lines of inquiry that converged in the early 19703.

23

 

n I I
. .9 o . - '
I: U. .err
11:): {KL-\-

- \
I» prev-’1‘ .‘n
Lt..:...cu 55"

‘ "N‘ w- .
I .v . \
- M nu I.

77"? M‘F:j1n
"yl-u ...§. 4‘.

tr A ~lhv '
f" ' 5 :51) if C

'5'...“ \
'. - ' r
‘M‘hh-dr

'30.

, I.

%~_fl';."‘, r”
I ‘”‘ l

Iii“ 1

~ ,

t"): "r

A ‘H’HAE;
Q.
t ‘.

b

\ A

a. l
$14 rr“
».

 

One of these lines was the discovery of the temporal association between
oxidative recovery and PCr resynthesis. Almost fifty years earlier, A.V. Hill
documented that recovery heat is only produced in an oxygen, not pure nitrogen,
atmosphere (130). This showed recovery processes, of unspecified mechanisms at
the time, must consume oxygen. Building upon this, Margaria showed recovery
oxygen consumption after an intense workload was composed of three exponential
components (193): a fast component (t [/2 z 30 seconds) necessary for regenerating
the ATP and PCr used during contraction which he coined the “alactacid oxygen
debt”, a slow component (t 1/2 z 15 minutes) necessary for regenerating
intramuscular glycogen from lactic acid—the “lactacid oxygen debt”, and a very slow
component (t 1/2 > 60 minutes) due to a thermogenic effect raising the basal
metabolic rate. In 1940, A.V. Hill’s son, D.K Hill, showed recovery oxygen
consumption and recovery heat production followed qualitatively similar
monoexponential time courses over a thirty minute period (136). He also showed it
Was possible to titrate the recovery rate of oxygen consumption with the degree of
inhibition of cytochrome oxidase by sodium azide. This latter observation was the
first study using whole skeletal muscle to show the rate of oxidative recovery is
Strongly dependent on the oxidative enzyme content. By 1970, Hultman and then
Piiper showed the above correlations between PCr kinetics and oxidative
metabolism were not limited to isolated in vitro amphibian muscles at low
temperatures. Hultman was the first to illustrate using human subjects that PCr
declines rapidly and reaches a steady—state that is a function of workload intensity
( 146). The higher the workload, the lower the steady—state value of [PCr] during
exercise. Upon cessation of exercise, PCr immediately begins to exponentially
return to resting levels. Using an in situ dog preparation, Piiper showed the
eliponential time course of recovery oxygen consumption after moderate

workloads had two components mechanistically corresponding to the fast and very

24

 

4..
-.- g.»
r.

W"? ram

- |
.2”; :r (“KEG

“1.. .Anv

”I; 7‘." FT
“"huJu u
i .

T‘”' ,I‘ ' fi‘é »
L2: .111an

. . 3 1
-I."‘."‘"h.
.J '3

........j “‘1

|__ I.
I "fihd .‘
"tad-l ui

’ ‘
m». ,
v.\.:rlq."
h....~~uié“u.i
.

\‘JTS “Lt
»._ u. h
"“4: CH

2.- . ,

4, l
.1». .
\‘y (1”,,1'“
5“ ‘3 Ltd
‘5'
v“ ‘r
.. J,‘l' n1”
3... _
‘35:" A
hu‘c ‘ra

slow components that Margaria elucidated (244, 245). Because of the moderate
workload Piiper used, he was able to unambiguously correlate the fast component
of oxygen recovery with the regeneration of PCr and ATP. Lastly, Sahlin and
Harris showed neither PCr nor lactate returned to their resting levels after intense
exercise in humans as long as blood flow was restricted with a pressure cuff during
recovery (120, 258). Taken together these studies empirically linked ATP
production by oxidative recovery processes to PCr resynthesis via the creatine
kinase reaction and established the rate of oxidative recovery is dependent on
mitochondrial content.

A second line of investigation coming to fruition by the early 19703 involved
understanding the mechanistic link between oxygen consumption and the adenine
nucleotides. In a brilliant piece of deductive reasoning based on mitochondrial
inhibitors and uncouplers, Peter Mitchell concluded that the reduction of oxygen to
Water was directly coupled to the phosphorylation of ADP to ATP by a proton
electrochemical potential difference across the inner mitochondrial membrane—the
Chemiosmotic hypothesis (216). Furthermore, it was recognized and verified that all
adenine nucleotides (ATP, ADP, AMP), both pyridine nucleotides (NAD, NADP),
PCr and Pi were interlinked by a network of near—equilibrium reactions even
though oxidative metabolism is a non—equilibrium process in toto (42, 157, 158,
169, 306). The profound significance of these insights is that tracking the steady—
State and transient behavior of oxygen consumption kinetics not only provides
information about PCr kinetics (and vice versa), but it also indirectly allows one to
monitor the kinetics of the major metabolic networks responsible for providing
Dhosphorylation chemical potential and redox chemical potential in the muscle
fiber. The phosphorylation chemical potential provides free energy to drive the
myosin kinetic state changes during contraction, the SR calcium pump during

contraction and relaxation, and the sarcolemmal Na+/K+ pump at all times. The

25

I35

 

. . q
. urH-w‘n‘. _
?:1 wiruubau

F .- I;
p- 'y' 7'.
no: a.“ h ail

(a. . -
.'._P "

l. .:
mad-c.1511 u

F’u

- i
-' "Ir-nus J.
l

'; filth-“MTV ,‘

.,‘.. ...,,~ , a.
" ht .AuitiiJ

:‘r

. ~-.. f
“‘- «rte '
'5'.“

"maul ext

if» A, ,
\\.-:~£.P “.1,
L

flieoj.

., . ‘

3%....3.‘ ‘ ar
‘

“$3.6 6112‘

V

‘1
.|5~._
\»‘\

mzlica

redox chemical potential provides free energy to drive the first reaction of the
triose phase of glycolysis in the cytosol and several reactions of Krebs cycle and
fatty acid B—oxidation in the mitochondrial matrix.

The third and final line of inquiry arose from recognition that muscle tissue was
a heterogeneous population of cells with respect to the rate of ATP demand and
the capacity of its ATP supply systems. As summarized in Table 1 below, it was
first documented in the late 19th century that the commonly observed color
differences of poultry, fish, and rabbit meat had physiological manifestations.
Visibly red rabbit skeletal muscle tissue (“dark meat”) generally developed tension
more slowly-i.e. took longer to reach peak tension—than white muscle (251). Due
to the time required for the discovery and cataloging of most of the key enzymes
and intermediates found in skeletal muscle, a period of almost 90 years elapsed
before muscle fiber classification was systematically revisited in the 19603.
Dubowitz extended Ranvier’s basic result by correlating an ATP demand
parameter with an ATP supply parameter. He suggested there were two fiber type
populations arbitrarily designated as Type I, that had low ATPase activity and high
Oxidative enzyme content, and Type II fibers, which had the opposite profile (76).
This classification was expanded by Brooke who exploited the differential pH
Sensitivity of the myosin ATPase staining process and suggested a total of four fiber
type populations could be discerned: Type I plus three subtypes of Type II—IIA,
IIB, IIC (32). Focusing on the ATP supply capacity, Stein characterized three fiber
type populations as White—A, Red—B, or Red—C based upon the relative intensity of
Staining of succinate dehydrogenase (SDH) (280). Burke showed the muscle fibers
lShaking up a single motor unit are functionally identical with respect to rate of
force development and fatigue characteristics (36, 38). He classified three motor
unit populations as Fast Fatigable, Fast Fatigue Resistant, or Slow Fatigue

Resistant. More comprehensive attempts relating several mechanical

26

 

.0.,A
. «40 P

'.'.‘ L \ 3‘
~c~-».--oa\r~

O ‘ '
1'3: :0 3

l

. ,-, .
t? 1l ‘2 "'
—‘—.£u.nl‘

1
4'“... r
‘ln- \\ l
m ‘..~ .. up.
I

...‘. |
s it“ "
-— lbw)». 53

WE 3:11}, B

I}~"‘Hu y

.._

, 7‘
-~Ab¢u‘ 1‘

I
" I

16:3: of i'

“H. _ .
3.3.3 inc i

i
. v ‘9
x. . ‘n-
‘.“‘ :L‘klb

‘. “1.", .
- ‘ F! .
I kl 55"\T01

::~,,_‘1 ‘
‘8‘?“631

3
fit

a
I

~ 'Y‘V‘y
\cV‘..Vl‘l ‘

 

'0
I:-. ‘
“w. "H o,
-"‘-u 1
-.~ \
'0
‘1‘" ..
\‘.fl ‘_
t“ ‘4.
. A.

characteristics to numerous metabolic capacity measurements were performed by
Barnard (15) and then Peter (239), with the general consensus that three fiber type
populations could be identified: Fast-Oxidative—Glycolytic, Fast—Glycolytic, or
Slow—Oxidative. It is important to emphasize that the tripartite scheme discussed
above is simply a less verbally cumbersome approximation of the in vivo reality.
In his 1968 Ph.D. thesis at Michigan State University, V.R. Edgerton, who later
worked with Barnard, Burke, and Peter, astutely pointed out that an individual
muscle fiber’s metabolic characteristics are more appropriately considered as part
of a continuum (80). The point along the continuum an individual fiber occupies is
a function of its developmental stage (81, 253), motorneuron innervation (35), the
intensity and duration of chronic training (10, 11, 16, 17, 55, 77, 82) or chronic
electrical stimulation (242), volumetric location within the muscle tissue (47, 70,
90) and thyroid hormone level (150, 181, 197). Furthermore, the metabolic capacity
of a muscle’s fiber population resembles a slightly skewed quasi-Gaussian
distribution (242). The degree of skew and standard deviation depends upon the
Specific enzyme used to classify the fiber type (e.g. succinate dehydrogenase,
phosphofructokinase, malate dehydrogenase, hexokinase) and species. In most
cases the quasi—Gaussian distribution nature arises from fiber-specific genetic
expression of varying amounts of enzyme content rather than expression of
different isoforms with different kinetic properties (240). This is based upon a
literature search turning up tissue specific differences but not skeletal muscle
fiber specific differences in most key flux regulating enzymes. (The major
exception was lactate dehydrogenase which showed isoform switching that

promoted the conversion of lactate to pyruvate after aerobic training (156, 163)).

27

fable 1: E

 

—
~ r‘
\ _ . ‘ ‘
.. * fit“.
I F
3‘... ‘1‘...
u_‘
4-. Dub
' v
N ‘
..":l‘\
Q p

rm- ,
‘0 “ ,.

4
"“«un.

,.
"N ch
(",9 .4
“sci
.
‘

v'~...,'.
.£‘\ A
\“'Ju.

 

Table 1: Evolution of fiber type classification schemes

 

 

 

 

Classification Basis of Classification Year, Author
Scheme (Reference)

Slow—Red v. macroscopic inspection of 1873—Ranvier
Fast-White muscle color and measurement (225)

of time to peak tension
Type I—v enzymatic assay of ATPase l960—Dubowitz
Type II—fast activity correlated with (76)

NADHase activity
White—A v. Red—B histochemical metabolic l962—Stein
v. Red—C profiling based upon (280)

differential SDH staining
density

 

Fast Fatigable v.
Fast Fatigue
Resistant v.
Slow Fatigue
Resistant

time to fatigue of different
motor units (that contain
homogenous fiber types) under
continuous electrical
stimulation

1967, l971-Burke,
(36, 38)

 

 

Fast—Twitch—Red v.

Fast-Twitch—White
v. Slow—Twitch—

histochemical metabolic
profiling based upon
differential NADHase staining

197 l-Barnard
(15)

 

 

 

Intermediate density and enzymatic assay of

myosin ATPase activities

correlated with time to peak

tension and half—relaxation

time
Type I v. 'Iype IIA improved on Dubowitz, ‘60, l970—Brooke
v. Type IIB v. differential myosin ATPase (32)
Type IIC stains with preincubation in

. additional LH mediums

Fast—Oxidative— extends Barnard, ‘71, adds 1972—Peter
Glycolytic v. assay for glycogen, myoglobin, (239)
Fast Glycolytic v. and cytochrome contents and

Slow-Oxidative

 

assaying for enzyme activities
of LDH, GPDH, HK, SDH

 

 

 

28

rmaen
{a
.1

P I
I‘ 'fiv“
v F” ‘
.-. 3......

c:7:0urr a."

.I .
“a...“ SAM

' I

0..» Opt}. p ,g
1‘: «Ankh

1".'-~' Ty. -
maul n:

 

"'{v ,q.

I DA

~ a... ansu“
-

Nu. _ ’ I
L.‘ igi’.l-l

..-.. “‘"J‘

5. .

.F..
\ ."~
M...,,Jn(

A‘LAo
.
‘.
\‘P’
4,~‘m'ra
cu

.

K:

 

 

I“ ,
.
QM», :‘

~ - "r.

“ KlU
PM;

‘ P‘s
\“3LE
.‘.'

x" '
\' age“

.g.

\‘m m

a. "r,

"‘0‘:

The mechanical performance characteristics (e.g. time to peak tension,
relaxation time) of a muscle’s fiber population are functions of the binding kinetics
of the thick and thin microfilaments as well as the SR’s calcium release and uptake
capacity. These characteristics have a slightly different distribution than metabolic
capacity. To date, in adult mammalian skeletal muscle, various regimens of gel
electrOphoresis and immunocytochemistry have uncovered four isoenzymes of
myosin heavy chain (268), three isoenzymes with fast and slow subtypes of myosin
light chain (13, 285), fast and slow isoenzymes of each of the three troponin
subunits (121) and fast and slow isoenzymes of the SR calcium ATPase (186, 322).
Curiously, despite the theoretically large number of combinations of the above
protein isoforms that could be assembled in a sarcomere, it appears the frequency
distribution of a muscle’s fibers’ mechanical characteristics, per se, resembles a
bimodal rather than Gaussian lineshape (81, 98, 110, 256).

To summarize, the three lines of investigation converging in the early 19703
documented that PCr kinetics, oxygen consumption kinetics, and the adenine
nucleotide pool are interdependent. This interdependency suggested that
monitoring the temporal changes in one of the three parameters may allow
indirect monitoring of temporal changes in one or both of the others as the muscle
fibers’ metabolic networks relax to a new steady—state after perturbation by
Changes in ATP demand. Moreover, there was accumulating documentation
regarding fiber type heterogeneity with respect to oxidative capacity and ATP
demand rate. This suggested the transient and steady—state responses of PCr, 02,
and the adenine nucleotides to changes in ATP demand may be different for
different fiber types.

PCr and the chemical energy balance method

Exploiting this convergence of ideas, Kushmerick’s lab in the mid-19703 sought

t30 functionally assess the extent and rate of ATP demand during a contraction—

29

 

 

 

/

t

 

..,. o
'1 ‘a‘ or]
«OI-5'. ‘

‘I"’F .‘ ‘-
- \-, _ ,
‘..-.. ”A...

p‘ "" ”'3‘

.- .‘n L":

7'"; 1?

"~.. .5

 

{
v- . . _
a "- ‘ ,ir
1““ u ‘sn
q,
H (‘
4‘ W
h. "- can
t... l
. h'1;y~l
"su’.h‘k“‘
u.._ '
~ 4-
x ‘ 'f . r

.‘I‘r .‘ v];

 

 

 

 

.‘ .
I‘s‘
\ , p.‘
\«I
.
v Jhl
\.
T?- ,
'.' w._
“a" if
s U‘

 

h.

‘ b q

‘-~. '3 , ,
., l

.i‘o

relaxation—recovery cycle and relate this to the extent of recovery metabolism
required to meet these demands (69, 177, 178). Previously, Carlson had shown a
positive linear relationship between the extent of PCr hydrolysis and the isometric
tension-time—integral, TTI (43). (TH, when normalized on a per muscle mass basis
and assuming a constant tissue density, is the time integral of the isometric force
per unit cross—sectional area. Since the development and maintenance of force 3
requires ATP, TTI is an indirect measure of energy cost of contraction that does not 3“".
require a chemical assay as well as a direct measure of force per unit fiber area

(159).) As mentioned above, D.K Hill, Hultman, Margaria, and Piiper had ’

 

independently shown a temporal correlation between PCr resynthesis and oxygen
consumption in a variety of species. Completing the triad of interconnecting PCr
kinetics, 02 consumption, and force development, Stainsby showed oxygen
consumption scales with isometric tension in the in situ dog gastrocnemius (278,
279). There were, however, no studies simultaneously measuring all three.
Addressing this need, Kushmerick and Paul (177, 178) used the experimental
framework called the “chemical energy balance method” which seeks to equate the
extent of high-energy phospate utilization during contraction to the extent of
recovery oxygen consumption above basal levels (AQ02) during recovery.
Phosphate utilization is quantified by the extent of PCr breakdown, and recovery
Oxygen consumption is measured with an oxygen electrode. Their major findings
using frog muscle include: 1) both PCr breakdown and AQ02 have independent
positive linear regressions with TTI that coincidentally have essentially the same
Slope and y-intercept, i.e. APCr/gram versus TTI and AQ02/gram versus TTI are
Sliperimposable plots. 2) both PCr breakdown and AQ02 follow a time course best
described by a monoexponential function 3) AQ02 is proportional to the initial
extent of PCr hydrolysis for tetanic durations between 5—20 seconds. In a later

Paper, Crow and Kushmerick (61) applied these results to mouse muscle and

30

 

was

 

 

 

.'.- r-rw-
. I

.- nin‘

‘1" ”1‘3”-
A
»~~-A~»\o

'. H. .
‘1 '
'13.... oh

idaliw

r’vn

' ‘0 up;

r;"r~

*.~_.‘;~
5

 

 

\

I l. ‘

t.~. ‘

‘ki. \'

‘vl

.

9" V

s

.‘N‘:

.0

". LY.»
“At.

"s.

\Ik‘}.:
.k

 

found fast twitch muscle had a threefold greater cost of briefly maintaining the
same isometric tension than slow twitch muscle (i.e. the fast muscle was less
economical). Interestingly, during longer duration tetani. the fast twitch muscle
reduced its energy usage (i.e. became more economical) to a level only 50% greater
than the slow twitch muscle, which did not change its energy cost. This reduction in

energy cost was explained by a reduction in the myosin ATPase rate.
Oxidative capacity exhibits plasticity in skeletal muscle fibers

During this same time frame of the 19703 there were numerous studies, largely
extending the work of John Holloszy, showing increased oxidative enzyme activities
in response to chronic endurance training. In retrospect, it could be argued
Holloszy ascended to the patriarch of modern exercise biochemistry when he
published his seminal findings that treadmill exercise will increase the
concentration of flux—regulating enzymes in Krebs cycle and electron transport

chain constituents (138). Prior to this paper it was widely believed the

cardiovascular system was the exclusive limiting factor of endurance capacity (292).

Holloszy’s results revealed peripheral muscle adaptations must also be considered.

He also showed both prolonged (120 minutes/day, 5 days/week for 12 weeks) and
Vigorous (>80% VOZmax—based upon treadmill speed of 31m/min for rats from Ref.
( 270)) exercise is required to garner impressive increases in oxidative capacity. This
strenuous experimental protocol was used repeatedly in the 19703 to confirm the
intuitive notions that chronic endurance training has the following consequences:
increased pyruvate oxidation and alanine export for use in the alanine—glucose
Cycle (218), increased citrate synthase activity (97) and NADH—linked
dehydrogenases’ activity in the mitochondrial matrix (140), minimal effect on most
glycolytic enzymes’ activity (12), increased surface area and cytochrome C content
of the inner mitochondrial membrane (97, 139), and prolonged time to exhaustion

as well as sparing liver and muscle glycogen (97 ). Furthermore, it was shown,

31

 

 

~..

....,
. a.
Law”: u

”V'WL‘ a.
'II- 4.
"“Oi‘su

‘
P -
:.""‘Iln
qu,u\' I
u.

‘3.

.J

 

1"! -
‘.
"-u. v.
I
;P.,
,‘w
.
P0
15””;
“uni-l“
v ._
.,
In“,
3
\K‘ A
Ni
\._ I
\ 5‘: .
“q“:
r- .-
“ A "a
H
“|- !.
"4

 

\ 2' .h‘

 

although all fiber types can increase oxidative capacity in response to endurance
training (10), the extent of the increase is fiber type specific, and for oxidative

fibers improvement is generally proportional to the extent of chronic motor unit
recruitment required during the training period (294). Terjung showed in rats who
underwent only the most intense aerobic training did the Fast-Glycolytic (FG)
fibers significantly increase (+60%) their oxidative capacity. On the other hand the
Fast—Oxidative—Glycolytic (FOG) and Slow—Oxidative (SO) fibers increased
oxidative capacity in all training regimens, indicating these fibers were called upon
to contract on a routine basis. These results are a nice corroboration from a
metabolic perspective of Henneman’s Size Principle of motor unit recruitment

( 125).

Phosphorus NMR spectroscopy becomes an important tool in
bioenergetics

A majority of the enlightening experiments mentioned in the previous
subsection all had a major drawback. In most cases these experiments were
conducted under in vitro conditions in which the muscle was excised and then
placed in a bathing medium (AQ02 and force measurements) or frozen and fixated
for serial sectioning (quantitative histochemistry) or ground into a cellular slurry
with a homogenizer (enzymatic assays). It would be preferable if bioenergeticists
had an experimental technique which allowed in vivo or in situ measurements, to
complement in vitro findings.

Fortunately, steady technological improvements since the 19403 allowed the
development and application of biological phosphorous nuclear magnetic
resonance spectroscopy (NMRS) to fill this role by the late 19703. The intricate
details of the quantum physics, electronic instrumentation, and digital signal
processing principles required to execute an NMR experiment are more

competently reviewed by others (2, 5, 31, 107, 212, 299, 300). The following

32

 

 

n a." .0-

.0 ‘3‘“- 5K

y0~y. -,..

{x
D
.
,_

‘ '-
a-bvubs

-§.'

"P mr.,
.. ‘ w .'
'~~m AOA\

s ‘.

-

i _ chi-I
. "4 “so.
‘0

 

\-

..

‘33, 5';
.AJJ F

L

n‘

.l .

a} ‘1‘.
war 1

paragraphs serve as a historical summary and will survey the basic phenomena for
readers who desire to understand NMRS from an end—user’s perspective.

Although the quantum theory of NMRS had been documented by the mid—19403
(27, 247), until the 19703, its application remained largely confined, to physicists
and chemists measuring magnetic moments of nuclei found in crystalline salt
structures and aqueous media. There were a few biological applications in the
19503 in which N MRS was used to assess plant water content (269), mouse blood
flow (273) and the proton resonance amplitude in human red blood cells (231), but
widespread use among biologists was retarded. This was primarily due to low field
strengths of the electromagnets (z 0.1 Tesla) and cumbersome signal acquisition
and processing methods, which conspired to give unacceptably low signal
resolution and low signal—to-noise ratios. Improvements in signal resolution were
aided by material science research in the early 19603, which led to the
development of niobium—titanium wire that superconducts at liquid helium
temperatures (179, 271). New generation, higher field strength (>1.0—10 Tesla)
superconducting magnets were constructed using the niobium—titanium wire
embedded in concentric lamellae of copper. Stronger magnets allowed
experimenters to more clearly discern among individual resonances.

Improvements in the signal—to—noise ratio were substantially aided by computer

numerical methods research during the golden era of mainframe computing in
mid—19603. Cooley and Tukey (59, 224) left an indelible mark in the field of signal
processing by resurrecting the algorithm for calculating a complex Fourier
transform, the so—called Fast Fourier Transform (FFT). Although they were not to
the first to derive the FFT (Carl Friedrich Gauss derived it around 100 years earlier
(224)) their unique approach increased the computational efficiency by reducing
the number of operations on an N—term complex Fourier series from N2 to

N030“). For example, if N = 100, the number of operations is reduced from 100"2

33

 

 

,.._.,.._.
a . r

A-‘~- §

>--.., ,.

1‘..th
a. 0..
. ~ W',
“ at...

 

 

 

,-
I
II
. .
7 'D -‘
‘. \
~~~_~ :‘
.
’s
he”,
a. .
fi..‘-.
4» .
H... l'
‘t‘

 

 

. "H
s

 

 

= 10,000 to 10010g100 = 200 —a 50—fold decrease. Application of the FFT to
biological N MRS allowed experimenters to add time—dependent signals in
computer memory and then simultaneously determine all of the frequency
resonances in the spectrum using the FFT algorithm. As an approximation, if the
number of signals added in memory doubles, the signal-to—noise improves about
41%. Prior to this time, a single frequency spectrum was obtained by continuously
varying the magnet’s field and noting where individual resonances occurred on an
analog oscilloscope (92, 107). This approach did not allow for the signals to be
digitally accumulated in memory and takes to acquire a single spectrum than using
the FFT approach. These technological improvements allowed biological N MRS to
flourish starting in the mid 19703.

The fundamental piece of information any NMRS experiment provides is a
continuous frequency distribution of quasi—Lorentzian shaped “peaks” of the
specific nuclei under investigation (e.g. 31F, 1H, 13C, 15N, or 19F). Each peak in a
spectrum can be characterized by its area, linewidth, and resonant frequency. All
of these characteristics can change as function of time during a biological
experiment by perturbing the metabolic networks through step changes in energy
demand, substrate supply, or hormonal activation. The numerically integrated
area under any single peak is directly proportional to the total number of freely
tumbling nuclei found in similar local electronic environments. Consequently,
peak area measures the relative concentration of a species containing the nuclei of
interest. The observed resonance linewidth of the nuclei within a given molecule
decreases with increasing molecular mobility, increases with increasing magnetic
field inhomogeneities, and can be influenced by the rate of chemical exchange with
Other molecules. The specific frequency at which nuclei resonate depends upon the
interaction between the nuclei nucleons and their local electronic environment.

The local electronic environment depends upon the presence of electron donating

34

 

. .
v- ~3v?~‘\fiv'
\ “ I . "
a..»n~-A4 I

I

,2... m1.

. u , l
—o- IHI “AVA
.
$-p ‘
‘1' I' In a
“I c I“
u

I
.-. .o -
V. i affix.
b‘tfi -c 5.».‘\
‘ l

a."

’A—
F ‘Hl
Mat ~V‘

u. . _ -
r. ....~' ’n.
“ ch-bIIV‘AJ

.
"o. l
;. V") r ‘F
a: will. i

na‘.‘.~ Us
Iéfizple .

336mm

groups (e.g. unshared electron pairs of the oxygen atom in water or the nitrogen
atom in amino groups) and electron withdrawing groups (e.g. free protons, metal
ions, carbonyl carbon atoms). The small differences in resonant frequencies allow
different molecules containing these nuclei to be resolved in a spectrum. For
example, in a 31P—NMR spectrum the phosphorus nucleus bonded to the amino
group of creatine in PCr will resonate at a different frequency than the
phosphorus nucleus of a free phosphate molecule. The three phosphorus nuclei in
ATP will resonate at different frequencies because each is in a slightly different
local electronic environment due to different bonding configurations within the
same molecule. (See Figure 22: Sample high signal—to-noise 31P‘NMR spectra for
an example).

Frequency resonance is achieved in the following manner. When nuclei are
placed in a strong external magnetic field (e.g. the superconducting magnet used in
this dissertation has a field strength (Bo) approximately 100,000—fold greater than
the earth’s magnetic field), they behave like tiny bar magnets designated by the
quantum mechanical property called “spin”. Furthermore, quantum mechanics
restricts the nuclear spin states to only two levels: a low energy state in which the
nuclear spins are aligned parallel to the external magnetic field and a high energy
state in which they are aligned antiparallel (180). Based upon the Boltzmann
distribution at physiological temperatures, there are only about 0.01% more nuclei
found in the low energy state than the high energy state. Nuclei in either energy
state will precess around the longitudinal axis of B0, and, in aggregate, form a net
magnetization vector (M) that is parallel to the external magnetic field. The
magnitude of M is proportional to the number of 31P nuclei in the sample.

Next, a wire coil transmits a very short—duration, broadband pulse (on the order
of microseconds) of magnetic energy to the biological sample (2, 143). This process

is roughly analogous to striking a polytonal tuning fork with a sharp blow. With

35

. 'IL‘n
3M

 

n

the appropriate selection of pulse duration and power, this causes M to rotate 90°
into a plane perpendicular to the longitudinal axis of the magnet. As M spins
around in the plane it induces a time—varying voltage in the wire coil resembling
an exponentially damped sinusoid decaying to zero as time increases. This analog
voltage waveform is called the Free Induction Decay (FID). The FID is then
digitally sampled into discrete time points and stored in computer memory. After a
predetermined number of signals have been accumulated (16 or 32 in this
dissertation) to improve the signal—to—noise ratio, the time domain data is
converted into frequency domain data by the FFT algorithm. A continuous
frequency distribution of peaks is the end result.

The major advantage of 31P—N MRS is it can non—invasively and simultaneously
monitor relative changes in concentrations of the key bioenergetic metabolites
(PCr, ATP, Pi, ICF pH- (144, 222)) over extended periods of time in the same
animal. In vitro based chemical assays require homogenizing one muscle per time
point or per workload intensity level or both. This necessarily increases the
Variability and cost of the measurement since significantly more animals must be
used.

Compared to in vitro methods, the major disadvantage of 31P—NMRS is its
relative insensitivity since an exceedingly small fraction (z 0.01%) of the population
of available nuclei undergo transitions between energy levels. This necessitates
using large tissue sample sizes and signal averaging techniques to increase the
Signal-to—noise ratio. Consequently, 31P—NMRS is unable to reliably measure
cOncentrations below the 100uM level (72, 107, 208, 212). As currently practiced,
however, this is not a serious limitation since most of the energetically important,
fi‘eely—tumbling metabolites (e.g. ATP, Pi, PCr) have intracellular concentrations

greater than lmM.

In 1973, the first published account of a biological application of phosphorous

36

NMRS illustrated that intracellular pH can be non—invasively monitored in red
blood cells by tracking the resonance frequency separation (i.e. chemical shift)
between the two phosphoryl moieties of 2,3-bisphosphoglycerate (222). Building
on this observation a few years later, it was shown over a physiological pH range
in frog muscle, that the chemical shift of Pi, but not ATP or PCr, is a sigmoidal
function of pH (39). Therefore, muscle intracellular pH can easily be determined
by measuring the chemical shift of Pi with respect to PCr and then converting it to
a pH value using a titration curve.

Several of the papers published in the formative years (1973—1982) of biological
31P-NMRS might be characterized as prosperous “fishing expeditions” evidenced by
the relatively generic article titles, copious amounts of data, and nonspecific
hypotheses by recent standards (3, 39, 66, 108, 144, 248). The following paragraph
highlights a few observations from this period relevant to this dissertation, and
illustrates a small fraction of the bioenergetic parameters readily obtainable from
31P—NMRS experiments.

The non-invasive nature of 31P—NMRS allowed investigators to confirm and
refine many key in vitro studies done in the previous two decades. Hoult et al.
(144) were the first to use N MR to verify PCr buffers ATP in accordance with
L0hmann’s and Lipmann’s assertion 40 years earlier. In an unperfused excised rat
leg preparation, ATP levels were maintained for nearly two hours while PCr

deCilined, Pi increased, and hexose phosphates accumulated and then leveled off as
anaerobic glycolysis ground to a halt. These authors were also the first to suggest
the linewidth broadening of the Pi resonance may be due to spatial distribution of
pH in the muscle as a result of an unspecified compartmentation process. Dawson
et, al. (66) found oxygenating a 4°C frog muscle preparation resulted in a
Significantly higher [PCr/Pi] ratio at rest than previous anoxic studies—confirming

Q’{idative metabolism supports PCr synthesis. They also obtained a PCr recovery

37

 

 

 

 

‘wlJ '

 

w.‘ ‘

_, .....

... -.-
» .-

.._'-J-4

u..' q ’3
v

-H“

u
I.A.—c<-'1
O ‘-

-o\. \- -

. -- n,
I 5".
.... .,

v--..-
.

“was

 

H.
'n.

‘ a

'. in.

l

a

o..
.‘M-u

V’I '

 

 

~‘1-
L'“:"‘
., ».,j a
v.

. F
.l‘
'3. .
I ‘- ’
.. .
.“M~L I

 

'1;
’.>
IT,
(I;
1-.

 

time constant of around 14.5 minutes—only a minute longer than Kushmerick
obtained using an in vitro approach (178), and found the Pi linewidth broadened
during recovery. In a later series of papers Dawson showed isometric force is
positively correlated with the free energy of ATP hydrolysis, AGATP, (67) and the
relaxation rate is inversely correlated with AGATP (68). AGATP is the maximum
available work per mole of ATP hydrolyzed (AG=AG°’+RT1 n[ADP] [Pi]/[ATP]),
whose variables are easily estimated from 31P—N MR spectra. Meyer et al. showed
the NMR-measured concentration of free Pi was over 6—fold lower in resting
mammalian fast-twitch muscle than measured by chemical assay methods (212).
This was meaningful since it placed the true [Pi] near the K1 of AMP deaminase
(312) and phosphofructokinase (282), and moved it significantly lower than the Km
of glycogen phosphorylase (54). This suggested that the allosteric effector role of Pi
should be reexamined in contracting skeletal muscle (207). For example, Connett
suggested the Pi released from net PCr hydrolysis during a rest—to—work transition
may play an important role in regulating the second, transient, 100—fold burst in
glycolytic rate that occurs around t= 1 minute during a 4H2 isometric contraction
(57) . Meyer et al. also showed the creatine kinase reaction was indeed at
eqnilibrium in vivo, supporting the earlier works of Kuby and Noda in the 19503
(171, 230). Confirming that the creatine kinase reaction was at equilibrium in vivo
is an essential prerequisite to calculating in vivo values of free [ADP] (185),
phosphorylation potential (305) and the above resting oxygen consumption level

during or after contractions (192, 203, 234).
Transient and steady—state analysis of PCr kinetics

Numerous in vitro studies in the late 19603 and 19703 documented the kinetic
behavior of PCr during exercise and recovery in frog, rat, dog, and human skeletal
Inliscle (73, 84, 120, 146, 177, 178, 244, 245, 259). The major observations were

1actic acid was the primary cause of decreased ICF pH during contractions, PCr

38

‘i‘ ._ -_.1 .13 ‘, I

 

i
fistula-nu-

 

 

4.. u-

' -

 

 

--..

N...“

 

”Wu.

”*2

.
~.‘

‘,

 

 

"J

 

 

 

recovery required oxidative metabolism and PCr recovery timHourses tended to
represent monoexponentials after mild exercise. Additionally, it was shown intense
exercise decreased the ICF pH resulting in slower PCr resynthesis and a
biexponential PCr recovery time course. It was commonly argued that the lowered
pH shifts the creatine kinase equilibrium away from PCr thereby slowing the
recovery kinetics (120, 146, 257, 258).

Toward the mid 19803, exercise and recovery PCr kinetics underwent additional
scrutiny facilitated by continual improvements in the time resolution of 31P—NMRS
and signal gating techniques. From the current vantage point, two approaches
evolved which guided investigators examining the nuances of PCr time courses:
transient analysis and steady-state analysis. The focus of transient analysis is on
quantifying the time constant, T, of PCr during a rest—to—work or work—to—rest step
transition. Variations of the kinetic parameter, 1, abound in the exercise literature.
Two common examples include: the rate constant, k ( = 1/ T) and the half—time, tU2
( = I ln(2)).

The primary focus of steady—state analysis is on quantifying the steady-state
Value of PCr after the transient phase has died out. Despite the fact in the late
19603 DiPrampero demonstrated kinetic data can be used to calculate steady—state
Values (73, 74), these two approaches simultaneously flourished in the 19803
Without widespread realization in the bioenergetic community that they were

retally complementary perspectives of the same approximate first—order
phenomenon.

Britton Chance and his colleagues advocated the steady—state (“work—cost”)
approach in which they found a positive linear relationship between the work rate
0f wrist flexion and the Pi/PCr ratio in the wrist flexor muscles of humans (48, 49,
223). In the steady—state, the macroscopic work rate (i.e. external power output in

Wtiltts) is proportional to the cytoplasmic ATP demand rate which, necessarily, is

39

 

 

 

v

e

"7" I

 

’.: on Q

' I
'- .‘4 ‘-

t
. ~.-
7“

-“n.
a

.- a ‘9‘
O \

oml‘

..-.
“z“,
._._..

-N u- 0.

Hon-
.---’1
....,_

"0.» _
‘.. _ P
.""~0o-L

9"

*‘uck,

 

. ‘n'
..

 

 

 

 

equal to the mitochondrial ATP synthesis rate and hence is proportional to oxygen
consumption rate assuming a constant ATP/02 ratio (61, 142). Chance’s group also
demonstrated this steady—state behavior in isolated rat heart mitochondria and in
in vivo rat leg using 31P—N MRS. In the isolated heart mitochondria they mimicked
increased ATP demand rate by adding a potato enzyme, apyrase A, that hydrolyzes
ATP in the suspension and found a linear dependence between ATPase rate and
the Pi/PCr ratio (115). In the rat leg they observed a linear dependence between
isometric stimulation rate from 0.25Hz to 2.0Hz and Pi/PCr (7). In their human
studies, they went on to show one month of aerobic training increased the slope or
“system gain” of this work-cost relationship (49, 51). The increased slope signified
that a given absolute change in the Pi/PCr ratio from rest—to—work will result in a
larger change in the steady-state power output and hence oxygen consumption-
perfectly consistent with the 19703 research showing increased oxidative capacity
in trained skeletal muscle and the 19603 research on comparative fiber types.

The transient analysis approach toward PCr kinetics was formalized by Meyer
(203) in a first—order analog electric circuit model (parallel RC) based upon earlier
experiments by Kushmerick (175, 178) and Mahler (192). (Digression: The
author’s curiosity has revealed, perhaps, the earliest application of first—order
electrical analog circuit models to cellular phenomena occurred in 1905 when
Hermann modeled the passive spread of a transmembrane potential down an axon

as a transmission cable (126). Sixty years later, Odum used a first—order model

describing the diurnal variation in carbon flow between the competing processes of

Dhotorespiration and photosynthesis in plankton (232).)

The impetus for the Meyer model was to provide a conceptual framework for
Understanding the control of cellular respiration by changes in cytoplasmic
Dhosphorylation potential as an alternative to Britton Chance’s model based upon

the kinetic limitation of mitochondrial oxygen consumption by ADP (49, 50, 53).

40

 

aur-

.-.

-,

.\-

 

. q

.(

budn.‘ \

 

a. .

 

The essential feature of Meyer’s model (cf. 165, 210, 211, 266) is that step changes
in work rate within the aerobic ATP production capacity of the muscle fiber will
change the oxygen consumption rate proportional to the change in cytoplasmic
AGATP- The linearity of this relationship between oxygen consumption and AGATP,
the “error signal”, gives rise to the first-order monoexponential behavior of PCr
kinetics seen in rest—to—work or work—to—rest step transitions (106, 165, 192) .
Furthermore, it is well appreciated PCr acts as a metabolic capacitor, spatially and
temporally buffering ATP through the creatine kinase reaction (188, 213, 318), and
muscle fiber oxidative capacity (hence capacity to support aerobic ATP production)
is largely dependent upon mitochondrial volume density (65, 7 7, 78, 261). As a
consequence of these characteristics, Meyer et a1. asserted, and then
experimentally verified, that the time constant describing the first—order behavior
of PCr kinetics (1130,.) should be independent of workload, depend linearly on total
creatine content (204), and depend inversely on fiber oxidative capacity (234) .
These assertions illustrate an inherent experimental convenience of PCr transient
analysis in that oxidative capacity can be estimated from the time constant of a
single work—rest transition rather than the slope of the steady—state work—cost
relationship which requires quantifying several workload intensities for linear
regression. Recently, Kemp has shown the steady-state analysis and transient
analysis are equivalent constructs, but concluded the steady-state approach is
much more sensitive to small pH changes compared to the more robust transient
approach (165, 166, 167).

There have been numerous 31P——NMRS studies over the past 15 years using
transient analysis, and variations on the theme, toward understanding muscle
bioenergetics. Two papers around 1984 out of George Radda’s group from Oxford
indicated the time course of PCr recovery from a work—rest transition becomes

biexponential if the work intensity exceeds the aerobic ATP production capacity of

41

 

t-‘I‘_ .r— ._..—-_

the muscle (9, 290). Their explanation is that during the work phase, anaerobic
glycolysis was required to help meet the ATP demand and subsequently produced
lactate which lowered the ICF pH. The lower ICF pH was correlated with longer
PCr recovery times and biexponential behavior. These papers also showed the
recovery rates of ADP >> PCr >> pHi.

Blei found the variations in 11301., as measured by 31P—NMRS within a given
muscle, are much greater between individuals than between repeated
measurements within one individual (26). He reported only a 9% variation for
repeated measurements on a single subject, yet nearly a 1.8—fold variation across
eight average humans (4 male, 4 female). This intersubject variability nicely
corroborated decades of histochemical fiber typing which showed, on average, a 2—
fold variation in oxidative capacity in average human skeletal muscle (261).

Achten et al. used recovery kinetics of Pi, rather than PCr, as a measure of
oxidative capacity in human mixed calf muscle (1). They based their premise upon
Pi exhibiting a 1:1 stoichiometry with PCr during PCr resynthesis (205), and the
cause of Pi linewidth broadening being due to intercellular pH inhomogeneity as a
result of different fiber type populations having different oxidative capacities (208).
In their experiment, the Pi peak in spectra of five of seven volunteers split—not just
broadened-into two discernible peaks. This permitted them to track two different
rates of Pi recovery presumably corresponding to the Type I (more oxidative, faster
recovery rate) and Type II (less oxidative, slower recovery rate) fiber populations.
Routine estimation of oxidative capacity by multi—peak Pi recovery kinetics in lieu
of PCr kinetics is limited, however. High intensity workloads which incur acidosis
and fatigue are required to increase the likelihood of Pi peak splitting. This may
not be tolerated well by patient populations with cardiorespiratory or
neuromuscular diseases. Furthermore, even among normal human subjects, as

relative workload intensity increases there will be increased variability of motor

42

 

 

ax;

 

unit recruitment and psychological motivation. This will significantly complicate
the necessary correspondence between the actual working skeletal muscle fiber
population and the volume of muscle tissue interrogated by the NMR transceiver
coil (26). (Personal note: In the author’s experience using a magnet with over six
times the field strength in the Achten study (hence better spectral frequency
resolution), and attempting to integrate the recovery Pi peak in the more
heterogeneous rat mixed calf muscle (8, 261, 319), it has been found the Pi peak ’5
often has very low signal-to—noise and does not reliably split even in the same It
animal over repeated cycles of work and rest on the same experiment day.)

Interestingly, McCully and Chance have realized the benefits of using transient i

 

analysis rather than their steady—state analysis (200). In this human cross— 1 J
sectional study they found a statistically significant positive correlation between
1pc, of the calf muscle (hence decreasing oxidative capacity) and age. They also
showed for elderly (2 80 y/o) volunteers there was no significant effect of gender or
use of medication for common chronic illnesses (diabetes, hypertension,
hypothyroidism) on 11301.. Elderly patients with peripheral vascular disease,
however, had over a 66% longer 1pc, as compared to the “normal” elderly. The loss
in muscle oxidative capacity is likely due to the cumulative effects of decreased
active lifestyle with increased age plus decreased peripheral blood flow capacity
(56).

Lastly, the energetic cost of contraction is another useful parameter that can be
extracted from analysis of 31P—NMRS PCr kinetics. The conceptual basis allowing
ATP cost measurements is summarized next. As mentioned before, the first—order
electric analog model dictates that PCr acts as a metabolic capacitor. This implies
that during a rest—to—work transition the intracellular pool of PCr “discharges” and
Supplies a “current” of phosphoryl groups to the myosin ATPase and SR Ca2+

ATPase. As steady-state is approached, the phosphoryl current from the

43

 

CH.

 

.3.-

u
v“

 

 

 

 

 

 

discharging metabolic capacitor monoexponentially decays to a lower value at the
same time the phosphoryl current from the mitochondrial conductance is
monoexponentially increasing. At a steady-state workload intensity within the
oxidative capacity of the muscle fiber, the cytosolic ATP demand is met by the
mitochondrial ATP supply current with no change in phosphoryl current supplied
by PCr. The significance of this is that taking the time zero derivative of the [PCr]
time course (i.e. the capacitive phosphoryl—charge time course) will provide the
initial rate of change of PCr which reflects the initial energetic cost of contraction
(104). That is dPCr(t)/dt | t=0 = -1/12(PCr0—PCrSS) where I: first order rate constant,
PCrO = resting [PCr] and PCrss = steady-state [PCr]. Within the first several
seconds of the transition to work from rest, net PCr hydrolysis must be supplying
all of the phosphoryl current since glycolytic flux does not reach a maximum for at
least 5-15 seconds (58) and oxidative metabolism has a time constant of at least 30—
70 seconds depending on fiber type, species, and temperature (26, 178, 204, 234).
Foley et al. used this time—zero derivative technique and amazingly showed that
acute but not chronic depletion of ATP by 40—50% will decrease the cost of
isometric twitch and tetanic contractions in rat fast—twitch muscle (102, 103). It was
speculated the acute decrease in ATP favors more IMP production by the near-
equilbrium adenylate kinase reaction during stimulation, and the increased IMP
allosterically affects cross—bridge cycling kinetics such that maximal velocity of
Shorting is reduced, leading to the observed decreased in energetic cost of
Contraction. Since a gradual 9—week reduction in ATP did not alter the cost of
Contraction it was concluded low ATP, per se, was not the cause of the reduced
energy cost (102).

In summary, the role of phosphocreatine in skeletal muscle energy metabolism
is to serve as a phosphagen capacitor to temporally and spatially buffer changes in

ATP thereby preserving a favorable cytosolic AG ATP throughout large swings in

44

 

 

 

workload. The utility of analyzing steady—state and transient behavior of
phosphocreatine kinetics in skeletal muscle is that it permits assessment of
energetic costs of contraction and calcium handling, quantifies economy and
efficiency of contraction, estimates oxygen consumption, and can measure relative
oxidative capacity.

Measuring oxidative capacity via in vivo 31P—NMRS has recently been proposed
as a non—invasive alternative for estimating muscle fiber type distributions. The
promise of 31P—N MRS is it offers real—time tracking of energetically important
intracellular metabolites (PCr, ATP, Pi, pH) within the same muscle. Not only does
this greatly increase time resolution, it also increases statistical power over the
one-muscle—one—time—point (and/or one—geographic—location) limitation of the
muscle biopsy method.

Consequently, a necessary prerequisite would be to correlate a 31P—NMRS
derived parameter to an energy supply or energy demand fiber type parameter.
Previous theoretical and empirical studies have shown that the steady-state PCr
level during stimulation scales with oxygen consumption, and the time constant of
PCr recovery after submaximal stimulation should be directly proportional to total
creatine content and inversely proportional to oxidative capacity (192, 203, 208).
Chapter 3 of this dissertation tests the hypothesis that the rate constant (k = 1/ T)
f0r PCr recovery is linearly dependent on muscle oxidative capacity in a relatively
hOmogeneous fiber population.

Chapter 4 of this dissertation examines the following question: If the PCr
r ecovery rate constant scales with oxidative capacity in a relatively homogeneous
population, can the underlying distribution of fiber types in a heterogeneous

II11.1scle be determined based upon decomposing a global, multicomponent PCr

recovery?

 

CHAPTER 3
Linear dependence of phosphocreatine kinetics
on skeletal muscle oxidative capacity

Introduction

The changes in phosphocreatine (PCr) that occur in skeletal muscle during
transitions between rest and moderate stimulation or exercise follow a
monoexponential time course (192, 203, 296). This behavior can be explained by a
linear metabolic model in which respiration rate is proportional to the cytoplasmic
free energy of ATP hydrolysis (AGATP), and the creatine kinase reaction acts as an
ATP buffer or phosphagen capacitance (203, 213). According to this linear model,
the time constant for PCr changes (1') should be independent of stimulation rate
over submaximal workloads, i.e. workloads that can be maintained in the steady—
state by oxidative ATP production. In addition, the model predicted that I should
depend linearly on total creatine content and inversely on muscle mitochondrial
content or oxidative capacity. Previous studies of rat fast-twitch muscle confirmed
that 17 was independent of stimulation rate over a threefold range of submaximal
rates (203) and directly proportional on total creatine content (204). The purpose of
this study was to test the hypothesis that the rate constant for PCr recovery (k=1/T)
is linearly dependent on muscle oxidative capacity. Also, the effect of intracellular
aCidosis on the PCr recovery rate after stimulation is examined. These results
Provide a framework for the practical application of 31F nuclear magnetic

reSonance spectroscopy (“P—NMRS) data for noninvasive estimation of relative

Skeletal muscle oxidative capacity.

M et h o d 3
Animal care and feeding
Adult male Sprague—Dawley rats (initially 250-350g); Harlan Sprague Dawley

1110., Indianapolis, IN) were housed three per cage in a temperature (=22°C) and

 

humidity (=35%rh) controlled room on a 12:12 hour light—dark cycle. Rats were
provided Purina Rat Chow and tap water ad libitum except for one treatment
group noted below. All experiments were performed during the rat’s nocturnal

cycle.

Treatment groups

Muscle mitochondrial content was decreased in one group of rats (“MMI”) by

treatment with methimazole (1-methyl—2—mercaptoimidazole, MMI; Ref. (60))

v.3
I

administered in the drinking water (0.025% w/v) for 8-10 weeks. In brief, the
pharmacological mechanism of MMI involves inhibiting the peroxidase enzyme—

Which oxidizes dietary iodide into iodine-thereby preventing iodine incorporation

 

‘Kfii‘ii..

into tyrosine within the thyroid follicular cell (122), schematic shown in Appendix F;
E- Methimazole Inhibition). This drastically reduces thyroid hormone (T4 and T3)
synthesis by the follicular cells resulting in systemic hypothyroidism. Chronically
low serum [T3] reduces the nuclear transcription of many structural and enzymatic
proteins found within the mitochondria resulting in decreased mitochondrial
volume percent within skeletal muscle tissue (41, 263). A treatment period of 8—10
weeks was chosen since it has been shown that rat skeletal muscle cytochrome C
Content monoexponentially decreased with a time constant of 11 days after surgical
thYroidectomy (293). Cytochrome C is an electron-carrying, heme—protein found
Within inner mitochondrial membrane. Because methimazole treatment reduced
bOdy mass, the food supply to a second group of rats (“Control—MMI”) was
moderately restricted to 35 gOday‘lecage‘l. Rats in both groups were handled at
least twice per week to monitor changes in body mass (Appendix H— MMI Effects).
Muscle mitochondrial content was increased in a third group of rats (“Trained”)
by a ten week interval training program using a computer controlled running-
Wheel apparatus (311). This interval training program was progressively increased

uIltil reaching a final regimen of 37 m/minute for 60 minutes/day by the start of

47

week nine (Appendix F— Intensity—Duration Protocol). Shepherd has shown the
common laboratory rat running at 35—40m/minute has a whole—body oxygen
consumption of 85—90%V02max (270). The running wheel apparatus provided a
mild constant current electric shock (approximately 0.5 mAmp) if the rat did not
run at the expected velocity. As a group, the rats met or greatly exceeded the
required number of wheel revolutions per day except during week seven when the
incremental duration change was greater than previous weeks. (Appendix Gh—
Training Compliance). All rats were trained under dim red light Monday through
Friday within six hours of the start of their nocturnal (“awake”) cycle. Similar
training regimens using a treadmill nearly doubled the mitochondrial content in
rat white muscle (127, 294). A fourth group of rats (“Control—trained”) was handled
daily for 10 weeks but not run on the wheels. A rat was not trained on the day of
its experiment.
Muscle surgical protocol

Rats were anesthetized with sodium pentobarbital (ip: 50 mg/kg body mass), and
an intraperitoneal catheter was inserted for administering additional anesthetic
(5mg/kg) at approximately 30 minute intervals. The rats were prepared for in situ
stimulation of the right leg on a custom built 31P—NMR probe as previously
described (204) (see also photograph in Appendix A— 31P—NMR Probe). In brief, the
right sciatic nerve of the supine rat was cut and placed in a bipolar platinum
electrode, and the right knee was fixed between two brass posts using a 18G
needle bored through the distal head of the femur. A 201b—test braided filament
fiShing line was threaded through the space between the Achilles tendon and the
distal fibulo—tibular symphysis and then tied to a half—bridge microfoil strain
gange force transducer mounted on an adjustable support in the probe.
Adjustment of this mount placed the right posterior leg 1—2 mm directly above a

1-2 cm diameter, flat, circular surface coil similar to that shown in Appendix B— Leg

48

 

 

" .'

, .4 .
I" \

r! .

“Peck”:-

,fic.

 

 

'0

 

"3;?”

\.~.‘

p
h

._'
a

 

 

 

I

‘_ \

.‘sr’ 1‘ ‘

‘h'w ,1

I

- \

[‘1'-

"‘~ 4:.

. ‘K\

b‘h.’

*1. e
ld\(

 

 

 

 

Cross—Section. Muscle length was optimized to give a maximal, isometric twitch in
response to a supramaximal square-wave pulse (10—20V amplitude, 2ms duration,
Grass stimulator model S48). Passive tension averaged 200—300 grams. Steady—
state isometric twitch force from the entire leg was recorded (Gould recorder model
2200S) at 1 mm/sec chart speed and 500 grams/cm sensitivity. The probe was
ventilated with 100% oxygen and body temperature was continuously monitored
via a rectal thermistor (YSI probe model 402A). Body temperature was maintained
between 37°-39°C by modulating the magnet bore air temperature with a warm—
air blower using a feedback—controller (YSI controller model 73A). This method of
maintaining body—temperature homeostasis was shown to satisfactorily maintain
the posterior leg intra—muscle temperature around 37°C in a separate
supplemental experiment summarized in Appendix C- Temperature Homeostasis.
At the end of each experiment (detailed in next paragraph) the superficial 2—3 mm
sections of the stimulated and contralateral unstimulated gastrocnemius muscles
were excised and immediately clamp—frozen in liquid nitrogen and stored at —80°C.
After all NMR experiments were completed, spectrophotometric assays of citrate
synthase activity (277), cytochrome C content (75), ATP content (22) and total
creatine content (PCr + creatine: Ref. (22)) were performed. The
Spectrophotometer was calibrated each day prior to running any assay. Relative
myosin heavy chain isoform distribution was determined by polyacrylamide—gel
Slab—electrophoresis of the purified, denatured protein (289, 116). The superficial
2~3 mm of the gastrocnemius represents the volume of muscle that is most
effectively sampled by the 1.2cm diameter flat surface coil (175). The remaining
Portion of the stimulated posterior leg muscle group was removed and weighed so

fOrce measurements could be expressed as gram force per gram wet muscle.

49

Spectral acquisition parameters and muscle stimulation protocol
31Phosphorus-NMRS of the right posterior leg was acquired at 162MHz in a
89mm diameter, vertical—bore, Bruker AM400, 9.4 tesla spectrometer. The
transceiver coil was tuned to resonate at 162MHz and impedance matched to 50 Q
for maximum power transfer immediately prior to placing the probe in the magnet
bore. Rat’s head was down. The B0 field was shimmed on the proton signal from
muscle water to a line width of 50—75 Hz. The B1 radio frequency pulse width was
set to 15—20 usecond to optimize the signal—to—noise ratio for the 1.76 second
interpulse interval (TR) used during stimulation. (Note: The BO field is the constant
magnetic field of the superconducting 9.4T magnet; the B1 field is the magnetic
component of the electromagnetic wave emitted by the radiofrequency transmitter
coil.) Before muscle stimulation, a fully relaxed control spectrum (TR = 15 seconds,
16 scans) was acquired. Partially saturated spectra ( TR = 1.76 seconds, 16 spectra+1
dummy scan) were then continuously acquired in 30—second blocks before (2
Spectra), during (16 spectra), and after (16 spectra) eight minutes of submaximal
isometric twitch stimulation at 0.33Hz (MMI study) or 0.75Hz (Trained study). These
Stimulation rates where chosen on the basis of previous studies (142, 203, 204) and
pilot experiments, which demonstrated they could be maintained for eight minutes
Without substantial muscle fatigue or acidification. After a 30 minute rest period
Which was sufficient time for PC r and pH to recover to resting steady-state values,
another series of spectra were acquired before, during, and after eight minutes of
2-0Hz isometric twitch stimulation for all four treatment groups. This stimulation
1‘ ate exceeds the maximum rate that can be maintained in the steady-state by
Oxidative metabolism in superficial gastrocnemius muscle of control rats . 142.:

spectral analysis

The summed free induction decays 'FIDs) were zero—filled to 2K complex data

and multiplied by "i.e. convolved with) a monotonically decreasing exponentia

 

‘ "I‘ll

 

IL,

, ,

no.“ ‘P
|

$.00.“

 

..—. .

a...»
A

4.,- .9‘

any - u

..‘
,-,'

-..

9.,

o.. k.;

I. '
I/t _.

J1
~I.‘__|

 

\
I
'.

 

\~

, l

function in the time domain before Fourier transformation. This results in a 20Hz
linewidth broadening in the frequency domain. Relative changes in PCr were
determined by the Method of Natural Lineshapes (MNL) (124). This method
computes the integral of the peaks in each spectrum relative to the area of the
same peaks in a high signal—to—noise spectrum obtained by adding all 34 spectra in
each stimulation-recovery cycle. (See also Chapter 3-Methods—Spectral Analysis
for additional discussion on the MNL.) Results were expressed as percent of initial
PCr/ZATP. The initial PCr/ZATP and Pi/ZATP were computed by integration of the
fully relaxed control spectrum acquired prior to the start of each experiment.
Intracellular pH was determined by the chemical shift (5) of Pi relative to PCr at -
2.52ppm using the formula: pH=6.7 7+log[(0.89-5)/(5—3.19)] (175). Changes in PCr
during stimulation and recovery were fit to a monoexponential function by an
iterative least-squares algorithm that minimized x2 using two—parameters (I and
Steady-state PCr level). The initial rates of PCr hydrolysis at the onset of
Stimulation were computed from these fits as previously described (104)

S tatistical analysis

All data are reported as means:tSEM. Comparisons of means (treatment versus

Corresponding control) were made by Student’s t-test or Tukey’s Honestly
Significant Difference test, at the P<0.05 level of significance. SigmaPlot 4.0 for
Windows was used for simple linear regression to examine the relationship
between phosphocreatine recovery rate constant and oxidative capacity or end—
Stimulation pH. SigmaPlot uses the Cholesky decomposition to invert the X’Y
matrix.

Results

Treatment effects

Citrate synthase activity of the superficial gastrocnemius muscle was decreased

by 71% in the MMI group and increased 1.8 fold in the Trained group, compared to

51

 

their corresponding control groups (Table 2). Citrate synthase is a key flux—

regulating Krebs cycle enzyme found in the mitochondrial matrix and has been

shown to be a reliable estimator of mitochondrial content in isolated mammalian

skeletal muscle fibers (151). Taken together, the treatments resulted in a 6.7 fold

range of nominal mitochondrial content, as indicated by this particular

mitochondrial marker enzyme.

Table 2: Treatment groups and efi‘ects

 

 

 

 

 

 

 

 

 

 

 

 

L Control- MMI Control- Trained
MMI trained
[_ N 10 9 7 11
__ Body mass (g) 29912 30114 391111 33818*
Jtimulated muscle mass (g) 2310.1 2310.1 3.1101 2.7101
Initial peak isometric 240113 235124 14517 263120*
_‘ twitch force g/g)
Citrate synthase activity 14.7116 4.210.6* 15711.7 28.111.5*
\ (pmole/minute/g)
Cytochrome C content 3.8105 ? (< =2) 3.3104 10.6101"
¥ (nmole/g)

 

 

 

Values are mean1SEM; N, number of rats. MMI, methimazole. The Control—
I\fIMI rats had a moderately restricted food supply. The Control—trained were fed ad
llbitum. Force values are expressed per gram wet weight of stimulated muscle.
*Significant effect of treatment versus corresponding control group (P<0.05).
SiInilarly, cytochrome C, another mitochondrial marker, was over threefold higher
in the trained compared with its corresponding control group. Cytochrome C in the
MMI group was below the level (approximately 2 nmole/g) that could be reliable
measured using our spectrophotometric assay procedure. Neither treatment
r esulted in a significant change in the relative myosin heavy chain homodimer
iS'Oform distribution of the superficial gastrocnemius sections (overall mean 5012%
Type 113, balance Type IIb, with Type I not detected).

There was a significant difference between initial peak isometric twitch force in

the Trained group compared to its corresponding Control-trained group (free fed)

during 0.75Hz stimulation (Table 2). Force in the Trained group, however, was

52

similar to the MMI group and the corresponding Control—MMI group (restricted
food intake). This suggested the force was anomalously low in the Control—trained
group potentially due to a reason related to their larger body mass compared to
the other three groups (Table 2). To resolve this issue, we ordered another group
of rats and interval trained some of them as previously described above in
Methods—Treatment Groups. As before, both of the new (‘) groups (Trained’,
Control-trained’) were handled on a daily basis and fed ad libitium. Mechanical
measurements we made using a standard horizontal bench apparatus for in situ
stimulation (214). In this case, there was no significant difference in any
mechanical characteristic between the Trained’ versus Control—trained’ (free fed)
rats (Table 3). Furthermore, peak isometric twitch force was similar to the MMI,
Control-MMI, and Trained groups obtained in the NMR study (Table 2).
Consequently, it is probable the lower peak isometric twitch force in the Control-
trained rats during the NMR study reflects difficulty in maintaining optimal muscle
length for larger rats within the confined space of the NMR probe (see also

photograph in Appendix A— 31P—NMR Probe).

53

Table 3: Mechanical characteristics-Bench experiment

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

of continuous 2Hz twitch stimulation (g/g)

 

 

Control- Trained’
trained’
N 10 7
Body mass (g) 40615 351110*
L Stimulated muscle mass (g) 3.4102 3.1102
Initial peak isometric twitch force (g/g) 261124 237118
Time to peak twitch force (ms) 2314 2112
Twitch half—relaxation time (ms) 1813 1812
Peak isometric 20 Hz tetanic force (g/g) 387136 356147
Peak isometric 40 Hz tetanic force (g/g) 6031101 711184
Peak isometric 60 Hz tetanic force (g/g) 1068199 10851139
Peak isometric 80 Hz tetanic force (g/g) 13201108 12491154
Peak isometric 100 Hz tetanic force (g/g) 14651116 13661158
Peak isometric 150 Hz tetanic force (g/g) 15181126 14281167
Peak isometric twitch force after 20 minutes 236124 225113

 

Values are mean1SEM; N, number of rats. Both groups were fed ad libitium.

All force values are expressed per gram wet weight of stimulated muscle.
*Significant effect of treatment versus corresponding control group (P<0.05).

Initial resting values of pH, PCr/XATP, and Pi/ZATP were similar to those

reported previously (175, 203), although the Pi/ZATP was significantly higher in
the Control-MMI group compared with the other three groups (Table 4). There

was also a small but significant increase in total creatine content in the MMI

treated group (Table 4).

 

 

 

l

Table 4: Metabolites in resting superficial gastrocnemius

 

 

 

 

 

 

 

 

 

 

 

 

 

Control- MMI Control- Trained
MMI trained
N 10 9 7 11

Intracellular pH 7.091003 7.061001 7.151003 7.0910.02*
PCr/ZATP 3.341016 3.771016 3.531012 3.501007

Pi/XATP 0.141001 0.0810.02* 00910.01 00910.01

ATP content (nmole/g) 7.371012 6.831026 7.041016 7.161012
Total creatine content 38.4108 41.1108* 33.1106 33411.1

(nmole/g)

 

Values are mean1SEM; N, number of rats. pH, PCr, Pi, and ZATP are measured
in fully relaxed spectrum (TR = 15 seconds) where ZATP is the sum of the peak
areas of the a—, B—, and y—ATP resonances. ATP and total creatine content were
measured by chemical assay of contralateral (unstimulated) superficial
gastrocnemius. *Significant effect of treatment versus corresponding control group

(P<0.05).

Effect of submaximal stimulation

 

Figure 1 shows sample spectra acquired before, during, and after stimulation at
the submaximal rates. Figure 2 shows the time course of PCr changes in the four
groups during and after the submaximal stimulation. As expected, PCr decreased
at the onset of stimulation and recovered exponentially afterward. In all groups,
there was a transient alkalinization at the onset of stimulation and transient
acidification at the onset of recovery (data not shown). These transient pH changes
can be attributed to proton consumption and release by net PCr hydrolysis and
reSynthesis, respectively (4). If these transients are ignored, there were only
Iminor changes in intracellular pH by the end of submaximal stimulation (Table 5).
There was no significant decrease in peak twitch force by the end of the eight

Iniflute submaximal stimulation in any treatment group (data not shown).

MMI (hypothyroid)

A .y @O.33Hz

 

Control—trained

B @ 0.75Hz
)4

 

M.
Pi PCr Y—ATP (It—ATP B-ATP

 

Figure 1: Sample 31P—NMR spectra stackplots

Sample spectra acquired before (bottom two spectra in each series), during (next
1_6 Spectra), and after (top 16 spectra) eight minutes of submaximal stimulation. A
single spectrum represents the time—averaged behavior of phosphorus metabolites
Over a period of 29.9 seconds ( 16 FIDs+1dummy scan, TR = 1.76 seconds). X-axis is
r eSonance frequency in parts—per—million (arbitrary scale). Y—axis is resonance
amI_>1itude which is proportion to the number of nuclei in a given local electronic
enVlronrnent. Z—axis is increasing experiment time.

PCr/EAT? 96 of Initial

PCr/EAT}? 95 of Initial

    
  

   

Control-MMI (restricted diet)

MMI treated (hypothyroid)

 

 

 

   
 

B 0.75Hz

 

 

 

I I I I I I I I I I I l I I I I I l I I I I I l I I
4 5 6 7 8 9 10 11 12 13 14 15 16
Experiment Time (min)

Figure 2: Time course of PCr changes (submaximal stim.)

MBan1SEM, Number of rats as in Table 2. Time course of PCr/ZATP during and
after submaximal stimulation.

57

Table 5: PCr kinetics and pH during submax. stimulation

 

 

 

 

 

 

 

 

 

 

 

 

Control- MMI Control- Trained
MMI trained
N 10 9 7 11
Stimulation rate (Hz) 0.33 0.33 0.75 0.75
PCr monoexponential time 1.321029 1.691015 1.171016 0.521008*
constant, I: Onset of
stimulation (minute)
PCr monoexponential time 1.571019 2.651027’“?L 1.011008 0.691007*
constant, I: Onset of
recovery (minute)
Steady—state PCr/ZATP 7314 4813* 5813 6113
during stimulation
_ (% of initial)
Initial rate of PCr hydrolysis, 0.381005 0.481005 0.271005 0.561006*
umole/g/twitch
Intracellular pH at end of 6.971002 6.941002 6.951002 6.901.002
g stimulation

 

 

 

 

 

 

Values are mean1SEM; N, number of rats. *Significant effect of treatment
Versus corresponding control group (P<0.05). T Significant difference between I at
Onset of stimulation versus 1: at onset of recovery corresponding control group
(P<0.05).

The time constant for PCr recovery after submaximal stimulation was
Significantly shorter in the Trained group, and significantly longer in the MMI
group, compared with their corresponding control groups (Table 5). There was no
Significant difference in the time constant for PCr changes at the onset of
stimulation versus the onset of recovery in the Control—trained or Trained groups.
In the MMI group, however, the t at the onset of stimulation was significantly
Shorter than the t at the onset of recovery. Also shown in Table 5 are the steady—
s”Sate PCr levels approached during stimulation and the initial rate of PCr
hydrolysis, both estimated by extrapolation from monoexponential fits. A previous
8121de showed that the initial rate of PCr hydrolysis is a good estimate of the ATP
coat of isometric twitch contractions (104). Consequently, the lower initial rate of
P Cr hydrolysis in the free—fed Control—trained group is consistent with their

anomalously lower initial peak isometric twitch force in the NMR study (Table 2).

58

-‘r'.

"-

 

When the data were pooled from all four treatment groups during submaximal
stimulation, there was a good linear correlation (r = 0.84, n=37, P< 0.01) between
the rate constant of PCr recovery (i.e. k = III) and citrate synthase activity,
normalized to total creatine content (Figure 3). Normalization to total creatine
content is formally required because 1: for PCr recovery also depends on total
creatine as stipulated by the linear metabolic control model of skeletal muscle
Oxidative phosphorylation (203); however, the correction is minor in this case. The
correlation coefficient between U1 and citrate synthase activity without this
normalization was 0.82 (graph not shown). From a different vantage point, it has
been argued that normalization of metabolite content and enzyme activities to total
creatine (as opposed to expressing on a per dry weight or per wet weight or per
gPam of protein basis) minimizes inter—muscle differences, which aids in identifying
true metabolic differences among different muscles (164).

There were no significant correlations in any treatment group between end—
Stimulation pH after a submaximal workload and citrate synthase activity

nOI'malized to total creatine content (data not shown).

59

 

   
 

MMZ

  
 

Control—MMI

  
 

Trained

oeu-
T
o
.I.

 

Trained—control

(Mn—1)
H
N

IBCOV
H
O

k3 1/tau

 

1.40x + 0.27 r = 0.84

0.0 I,
0.0

 

I I I I I I I I

IIIIIII III III III III IIIIIII II
0.2 0.3 0.40.5 0.6 0.70.8 0.9 1.0
Citrate synthase activity
on area no con on

III
0.1

Figure 3: PCr recovery rate constant v. oxidative capacity

Correlation between the rate constant (k: 1/12) for PCr recovery after submaximal
Stimulation and citrate synthase activity, normalized to muscle total creatine
Content. Plotted points are means1SEM for the four treatment groups. Linear
regression was calculated from individual muscle data points (n=37).

60

Effect of 2. 0H2 Stimulation

Figure 4 shows the time course of changes in isometric twitch force, PCr/ZATP,

and ICF pH during 2.0Hz stimulation in the four treatment groups. This
stimulation rate is known to be beyond the maximum rate that can be maintained
in the steady—state by oxidative metabolism in the least-oxidative fibers in
gastrocnemius muscle of sedentary rats (142). Consequently, PCr decreased more
rapidly at the onset of stimulation, and the muscles became more acidic than
during submaximal stimulation. On the other hand, less acidification occurred in
the Trained rats compared to the other groups, as expected from the higher muscle
oxidative capacity (Figure 4C).

Although the PCr changes at the onset of 2.0Hz stimulation were not markedly
different between the four groups (Figure 4B), the recovery of PCr after
stimulation was clearly faster in the Trained group, and slower in the MMI group,
Compared with controls (Figure 5). There was a significant correlation between 1/‘t
for PCr recovery after 2.0Hz stimulation and normalized citrate synthase activity,
but the correlation was weaker (r: 0.70, n=37; graph not shown) than for the same
relationship after submaximal stimulation. This increased variance is in part due to
an independent effect of pH, since there was a significant correlation (r=073, n=53,
P<0.01) between 1/1: for PCr recovery and ICF pH at the end of the 2.0Hz

Stimulation for the two control groups (Figure 6).

61

 

Isometric Twitch Force
(96 of Initial)

 

 

O I I I I I I I I I I I I I I I I I I I I I I I' rI I I I I I I I I
0 1 2 3 4 5 6 7 8
100 _
90 —o— Control—trained
80 —O— Trained B
70
60 —D— Control—MMI

PCr/EAT? 96 of Initial

 

 

ICF pH
O\ m Ch 01 0'\ ON Ox \! \l
w uh U1 0'1 \1 (D \D O H

 

I I I I I I I I I I I I I I I I I I I I ‘I‘I’ I I I I I I I I I I I
0 1 2 3 4 5 6 7 8
Experiment Time (min)

Fig‘ure 4: 2.0Hz Stimulation time courses

Pe_ak isometric twitch force (A), PCr/ZATP (B), and ICF pH (C) during 2.0Hz
11hulation. Values are means1SEM; number of rats as in Table 2.

62

——o—— Control—trained

 

4 0 _ —O— Trained
3 0 i —D— Control—MMI A
2 O _

PCr/EAT? 96 of Initial

- —I—MMI

 

 

 

 

10—
o IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
8 9 1o 11 12 13 14 15 16
7.
7.
7.
6.
:1:
n6-
1.. 6.
o
H 6.
6.
6.
6.
6‘2 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
s 9 10 11 12 13 14 15 16

 

Experiment Time (min)

Figure 5: 2.0Hz Recovery time courses

Pgr/ZATP (A) and ICF pH (B) during recovery after eight minute 2.0Hz
stlInulation. Values are means1SEM; number of rats is as in Table 2.

 

 

 

O
- 0 0.7532 control groups
1 4— ° ° °
0
H . O 2.082 control groups 0 .
I
i
8
:1” a 5
fl L
B
\
I'I
II 5
J4 :
. y = 0.94x - 5.47 r = 0.73 l,
000 I I I I 1 lir I I I j I I I I I T I

 

6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1
ICF pH at end of stimulation

Figure 6: Rate constant v. pH (supramaximal stim.)

Correlation between rate constant for PCr recovery after eight minutes of
isometric stimulation versus ICF pH at end of stimulation in control muscles
stimulated at 0.75Hz or 2.0Hz.

D is c us 3 io n
The main result of this study is that there is a linear relationship between the
global N MR—derived rate constant for PCr recovery (k: 1/1) and muscle oxidative
capacity, as indicated by citrate synthase activity. Taylor et al. were among the first
to explicitly suggest this correlation over 15 years ago (290) and since then
analogous results have been reported by others in rat (296) and human muscle
(individual fibers: (153, 274); whole muscle: (26, 200, 217, 308). This is the first
study, however, to cover over a 6—fold range of citrate synthase activity. The
Practical implication of this result is that noninvasive 31P—NMRS of PCr recovery

transients can be used as an index of oxidative capacity in skeletal muscle.

It should be emphasized that this correlative method is strongly dependent upon
the choice of mitochondrial marker, which is complicated by several factors. First,
the intrinsic constituents of the electron transport system within the inner
mitochondrial membrane (i.e. any cytochrome) are a somewhat better measure of
true oxidative capacity (65, 198, 254) than Krebs cycle enzymes (e.g. citrate
synthase, a—ketoglutarate dehydrogenase) which may have anaplerotic and
amphibolic functions in addition to oxidizing di— and tri—carboxylic acids (28). On
the other hand, compared to Krebs cycle enzymes solubilized in the gel—like
matrix, most electron transport system constituents are tightly integrated within
the inner mitochondrial membrane making them more challenging to routinely
isolate and purify intact with sufficient yield. Second, the various markers of
mitochondrial content do not change proportionally during muscle adaptation (30,
256) or across fiber types (151). For example, the slope of Figure 3 would have
increased if cytochrome C content were used in lieu of citrate synthase activity
since cytochrome C underwent a 3.2—fold increase whereas citrate synthase
increased by only 1.8—fold in the Trained versus Control—trained groups.

The relationship between the PCr recovery rate constant and muscle oxidative
capacity was previously derived from a first—order analog RC circuit model of
muscle respiration (203). This circuit model assumes 1) the cytoplasmic creatine
kinase reaction is in instantaneous and spatially uniform equilibrium (213, 318); 2)
oxygen and substrate supply are not limiting during recovery (259); 3) anaerobic
ATP production is negligible during recovery (62, 178); 4) the mitochondrial ATP/02
and basal rate of muscle Q02 are constant (119, 25); and 5) PCr resynthesis
accounts for all but a negligible fraction of the extra ATP consumed during
recovery (120, 290).

In the context of the circuit model, the use of PCr recovery rate constants to

estimate oxidative capacity depends on four assumptions in addition to the five

65

 

listed above: 1) total creatine content (Tc) must be known because I also depends
linearly on total creatine (204); 2) the PCr measurements must be made over the
range in which the PCr/Tc ratio is directly proportional to AGATP’ i.e., from
approximately 0.2 to 0.7 (203); 3) the model assumes that the cytoplasmic ATPase
rate (almost entirely due to the myosin ATPase (250)) is a step function of the
motorneuron stimulation rate (set by the experimenter or central nervous system),
both at the onset of stimulation and at the onset of recovery; 4) the circuit model
assumes a constant intracellular pH. I

With these additional assumptions, the circuit model predicted that III for PCr
decline at the onset of stimulation should equal l/t during recovery and, therefore,
might also be used to estimate oxidative capacity. In practice, however, the
recovery rate constant is likely to be more reliable than the onset of stimulation
rate constant for several reasons. First, the onset and recovery rate constants are
expected to be equal only for submaximal stimulation, which can be maintained in
a steady-state without fatigue by oxidative metabolism. Consequently, during the
supramaximal 2.0Hz stimulation in this study, PCr depletion was faster than PCr
recovery in all groups (Figure 48 versus Figure 5A). In the MMI group, the onset 1:
Was significantly shorter than recovery I for 0.33Hz stimulation (Table 5),
suggesting that even this low rate exceeded the oxidative capacity of some fibers in
these muscles. Second, the confounding contribution of anaerobic glycolysis to
tOtal ATP production during stimulation can be substantial, particularly in fast—
tWitch muscles. Conversely, PCr recovery is thought to depend almost entirely on
Oxidative metabolism in mammalian muscle (120, 166, 259). Third, the assumption
that ATPase rate is a crisp step—function of stimulation rate is not satisfied if force
is not constant, because the ATP cost of contraction is directly proportional to force
Within a muscle (26, 104, 118). For example, in this study there was a pronounced

Staircase effect during the 2.0Hz twitch stimulation. This would cause variations in

66

wk”;

 

,- 2.1mm“!
,
"4

 

u .,a I.
5 “
nuunul

 

 

 

 

 

 

 

the ATPase rate and result in deviations from the ideal monoexponential time
course of PCr depletion at the onset of stimulation.

Analysis of PCr recovery transients (i.e. 1:) after submaximal workloads (24, 195,
200) is more preferable than analyzing the slope of steady-state PCr/Pi versus
workload, the “work—cost” relationship (7, 49, 223), to estimate oxidative capacity.
In contrast to the PCr recovery rate constant which depends only on oxidative
capacity (under the reasonable assumption total creatine does not change on an
acute basis), the steady-state PCr approached during stimulation also depends on
ATPase rate. This is nicely illustrated in Figure 2B in which both the Trained and
Control—trained groups had comparable steady—state PCr levels during
submaximal stimulation despite the higher oxidative capacity of the Trained group.
This is the result of the lower isometric twitch force (Table 2) and hence lower
ATPase rate of the Control—trained (free fed) group. Furthermore, measuring 1: is
independent of the given single submaximal workload chosen (23, 106, 203). By
definition, constructing a “work-cost” relationship, however, requires several
Workloads (23) over a large enough dynamic range to give different steady—state
PCr/Pi ratios to calculate a slope from linear regression. Consequently, the 1:
method maybe more preferable for patients with cardiovascular disease (296) or
renal disease (235) in which the submaximal workload range may be small and ill—
defined and supramaximal workloads are contraindicated.

The strong correlation between the PCr recovery rate constant and end-
Stimulation ICF pH (Figure 6) suggests workload intensities resulting in significant
intracellular acidosis can potentially confound attempts to estimate relative
OXidative capacity. Decreased PCr recovery rates due to lower ICF pH has been
Shown in healthy human mixed limb muscle (287, 308), malignant hyperthermia-
susceptible humans (221), rat mixed muscle (168), and hypercapnic cat soleus

(119) and is probably related to a non-specific global pH effect on the

67

 

mitochondrial oxidative machinery and/or on substrate delivery to the
mitochondrial matrix from the cytosol.

In summary, the results of this study confirm the rate constant for PCr recovery
in skeletal muscle after submaximal stimulation is linearly dependent on oxidative
capacity as indicated by mitochondrial marker enzymes. These results are
consistent with predictions based upon the linear thermodynamic model of
respiratory control by AGATp (203). The practical implication is that relative
oxidative capacity (depending on the chosen mitochondrial marker) can be
estimated in muscles of human subjects from 31P—NMRS measurements of PCr
recovery. Extrapolating from our results, we suggest clinical measurements be
made after brief exercise of sufficient intensity and duration to cause a 50—60%

decrease in PCr without incurring significant intracellular acidosis.

68

CHAPTER 4
Estimation of skeletal muscle fiber distributions
by phosphocreatine transient analysis

Introduction

Numerous parameters derived from in vivo phosphorus nuclear magnetic
resonance spectroscopy or proton magnetic resonance imaging have been
proposed to non—invasively estimate mammalian muscle fiber-type distributions.
For example, it has been long recognized inter—fiber pH heterogeneity best
explains the line broadening of the inorganic phosphate (Pi) peak in a 31P—NMR
spectrum (66, 208, 290). This phenomenon has been exploited in several studies
whereby Pi peak splitting is observed to occur after intense exercise and has been
interpreted as a differential pH response due to differential recruitment of at least
two distinct fiber type populations in human mixed muscle ( 1, 236, 302, 303). In
other studies, Pi peak splitting has not been documented in a majority of subjects
studied (21, 149), especially if the subjects are endurance athletes (236, 324). It
would appear reliable and repeatable estimation of fiber type distributions in
diverse subject populations based upon Pi peak splitting is questionable.

It has also been proposed that fiber type distribution be non—invasively
estimated by examining steady—state ratios of 31P--NMRS metabolites at rest or
during exercise. Blei et al. has shown a negative correlation between the global
NMR—derived ATPase rate-presumably mirroring a global histochemically
determined ATPase rate—and resting Pi/ATP in human forearm muscle. They did
not find a correlation between ATPase rate and resting PCr/ATP (26). Using spatial
localization in rat leg muscle it has been shown that superficial regions (high
ATPase activity, low oxidative capacity) have a lower resting Pi/ATP ratio than deep
I‘egions (lower ATPase activity, higher oxidative capacity). There was no

sliperficial—to—deep gradient observed for the resting PCr/ATP ratio (46). In

69

 

 

 

..,

.

 

contrast to these studies, Takahashi et al. found a much stronger positive
correlation between resting PCr/ATP and % Type II fibers than between resting
Pi/ATP and % Type II fibers (288). These findings were indirectly supported by an
earlier study which showed sprinters—with presumably higher percentages Type II
fiber population-had higher PCr/ATP ratios in their gastrocnemius than sedentary
controls or an endurance athlete (29).

Results from lH—MRI studies have also been generally equivocal. While some
investigators have shown a correlation between % Type I fibers and the
longitudinal relaxation time, T1, (145) and no correlation between % Type I and the
transverse relaxation time, T2, (145, 288), others have shown no dependence of T1
on fiber type yet observed slow—twitch fibers have longer T2 values than fast—
twitch fibers (89).

Since the time constant of the exponential PCr recovery after a single work—to—
rest step-change is proportional to the relative oxidative capacity of the working
muscle (153, 203, 234, 290) examining PCr transient behavior has been another
popular approach toward non—invasive estimation of fiber type distribution (26,
200, 217 , 296, 308). Unlike the previously mentioned techniques above, there has
been generally consistent agreement among different investigators, including
those who assay muscle biopsies (274), regarding the accuracy and reproducibility
of this method. An interesting corollary to this approach is it may be possible to
identify the proportion and relative oxidative capacity of two or more fiber
Populations in mixed muscle by extracting the amplitude and duration of the
eXponential components comprising the global NMR—derived PCr recovery time
cOnstant. In theory, these components should roughly correspond to the widely—
cited histochemical fiber type categories of Type I versus Type II or Fast—
Oxidative—Glycolytic versus Fast—Glycolytic versus Slow—Oxidative.

It has been well established that rat leg musculature has considerably more

70

 

 

intra-muscle (i.e. fiber type) and inter—muscle heterogeneity with respect to
metabolic capacity (8) and mechanical performance (319) than human muscle (83,
261, 272). Given these observations, the primary objective of this study is to
decompose the components of high signal-to—noise exponential PCr recovery
transients in an attempt to unambiguously identify the distribution of distinct fiber
populations based upon relative oxidative capacity in the rat leg. A secondary
objective of this study is to document the pattern of temporal changes in PCr, pH,
and isometric force during a prolonged bout series of stimulation—recovery cycles.
This is done to determine whether the cyclic temporal changes are sufficiently
disparate to compromise the interpretation of gated-NMR experiments in which
increased signal—to—noise is achieved by adding spectra acquired at identical time
points during repeated stimulation cycles. Lastly, the results of this study are
unique in that they illustrate the utility of Principal Component Analysis for
routine processing‘of very large N MR datasets.

Met h o d 3

Animal care and feeding
Adult male Sprague—Dawley rats (300-400g); Harlan Sprague Dawley Inc.,

Indianapolis, IN) were housed three per cage in a temperature (=22°C) and
humidity (=35%rh) controlled room on a 12:12 hour light—dark cycle. Rats were
Provided Purina Rat Chow and tap water ad libitum. All experiments were

Performed during the rat’s nocturnal cycle.
Muscle surgical protocol
Rats were anesthetized with sodium pentobarbital (ip: 50 mg/kg body mass), and

an intraperitoneal catheter was inserted for administering additional anesthetic
(5mg/kg) at approximately 30 minute intervals. The rats were prepared for in situ
Stimulation of the right leg on a custom built 3 1P—N MR probe as previously

described (204). A photograph is provided in Appendix A— 31P—NMR Probe. In

71

'7' V
' C
‘M 5.4
..>¢ n-

 

v
. .~.,.,6
. v

~ub

Iva‘fi' -

.-
-u but \-
wva .a
‘ i 4

.Lu— _.4

r» «w

.
5 nine
1

.,,
~49; ..
"l. to.‘
’ “5 b

 

‘Q.

"‘“itul.

 

 

 

I

b
x“ 4‘”
u

 

 

I
x.‘
b‘v
\l‘q L?
I ‘M

brief, the right sciatic nerve of the supine rat was cut and placed in a bipolar
platinum electrode, and the right knee was fixed between two brass posts using an
18G needle bored through the distal head of the femur. A 201b—test braided
filament fishing line was threaded through the space between the Achilles tendon
and the distal fibulo—tibular symphysis and then tied to a half-bridge microfoil
strain gauge force transducer mounted on an adjustable support in the probe.
Adjustment of this mount nestled the right posterior leg within a 2.0 cm diameter
saddle—shaped surface coil as shown in Appendix B— Leg Cross—Section. Muscle
length was optimized to give a maximal isometric twitch in response to a
supramaximal square—wave pulse (10—20V amplitude, 2ms duration, Grass
stimulator model S48). Passive tension averaged 200—300 grams. Steady—state
isometric twitch force of the hindlimb was recorded (Gould recorder model 22008)
at 1 min/sec chart speed and 500 grams/cm sensitivity. The probe was ventilated
with 100% oxygen. Since the experiments lasted between 1—10 hours, depending
on stimulation protocol, the body temperature was continuously monitored via a
rectal thermistor (YSI probe model 402A) and maintained between 37°-39°C by
modulating the magnet bore air temperature with a warm—air blower using a
feedback—controller (YSI controller model 73A). This method of maintaining body-
temperature homeostasis was shown to satisfactorily maintain the posterior leg
intra-muscle temperature around 37°C in a separate supplemental experiment
Summarized in Appendix C—Temperature Homeostasis. At the end of the
eXperiment the stimulated posterior leg muscle group was immediately removed
and weighed so force measurements could be expressed as gram force per gram

Wet muscle.
Stimulation-Recovery protocol

Rats were divided into three “treatment” groups based upon stimulation

1:I‘equency (Table 6). The stimulation frequencies chosen are known to be well

72

m

 

 

 

9"

 

within (0.75Hz) or above (2.0Hz and 5.0Hz) the aerobic capacity of rat leg muscle
based upon measurements of steady—state oxygen consumption relative to
maximum oxygen consumption (142). Data from pilot experiments permitted the
recovery duration to be chosen to ensure intracellular pH reliably recovered to a
resting value of approximately 7 prior to the next stimulation period. Note the
number of rats and number of cycles reported in Table 6 was chosen based on the
convenience obtained from analyzing a balanced statistical design and the fact all h
rats were able to endure at least eight cycles without equipment malfunctions, ’
computer memory limitations, or other anomalies. A complete portrayal of the

number of rats per cycle number can be found in Appendix D—N vrs. Cycle

 

Number.

Table 6: Treatment groups

 

 

 

 

 

 

 

 

 

Stimulation frequency (twitches/second) 0.75Hz 2.0Hz 5.0Hz
Number of rats 7 7 7
Minimum number of contiguous 8 8 8
stimulation-recovery cycles per rat

Stimulation duration in one cycle (minutes) 5.7 5.3 2.1
Recovery duration in one cycle (minutes) 5.7 16.0 29.9

 

 

Spectral acquisition parameters

31Phosphorus-NMR spectra of the right posterior leg were acquired at 162MHz
On a 89mm diameter, vertical—bore, Bruker AM400, 9.4 tesla spectrometer as
previously described (234), except a 2cm saddle—shaped surface coil was used to
Sample a larger population of fibers (Appendix B— Leg Cross—Section). The
transceiver coil was tuned to resonate at 162MHz and impedance matched to 50 Q
for maximum power transfer immediately prior to placing the probe (with the rat
1lead-down) in the magnet bore. Rat’s head was down. The B0 field was shimmed
0!) the proton signal from muscle water to a line width of 50—75Hz. The B1 radio

fI‘equency pulse width was set to 20—25useconds to optimize the signal—to—noise

73

r. “‘4‘

"‘4'"

 

.D a
-.....n

o” L
I
“ 'r,
..

 

ratio for the given interpulse interval (Table 7). (Note: The B0 field is the constant
magnetic field of the superconducting 9.4T magnet; the BI field is the magnetic
component of the electromagnetic wave emitted by the radiofrequency transmitter
coil.) The total seconds per block in Table 7 represents the time over which the
partially saturated phosphorus spectra were continuously acquired and averaged

to generate individual time points during and after the isometric twitch stimulation

periods. '
F

Table 7: NMRS Acquisition parameters

Stimulation frequency (twitches/second) 0.75Hz 2.0Hz 5.0Hz

NS—number of spectra in one block = number 16 32 16
of spectra signal averaged together per single
time point

TR— interpulse interval (sec.) = total seconds 1.33 1.00 1.00 '—'—~"'
required to acquire a single spectrum =
relaxation delay+pulse width+acquisition time

 

 

 

 

 

 

 

Total seconds per block 21.33 32.00 16.00
(NS x TR)

Number of blocks acquired during 16 10 8
one stimulation period in one cycle

Number of blocks acquired during 16 30 116

 

 

 

 

 

one recovery period in one cycle

 

Pulse acquisition (8064Hz sweep width, 1K complex data) was gated to periods
immediately before or at least 100 milliseconds after a twitch contraction in order
to minimize motion artifacts. Before the start of each experiment a single fully
relaxed control spectrum of the resting muscle was acquired (TR = 15 seconds, 16
Scans).
Spectral processing

The summed free induction decays (FIDs) were zero—filled to 2K complex data
and multiplied by (i.e. convolved with) a monotonically decreasing exponential
function in the time domain before Fourier transformation. This results in a 20Hz

linewidth broadening in the frequency domain. Each spectrum (approximately

74

 

I
F‘ . r
...—-II

J'J

v

x-tr

l‘ ‘
.4

17,000 in total) was phase corrected and frequency corrected based upon the

Principal Component Analysis (PCA) method of Brown and Stoyanova (33, 281).

What is Principal Component Analysis? (from discussions with R.
Stoyanova, Fox Chase Cancer Center, May 1998)

PCA is a multivariate data analysis technique for decomposing
large datasets along the axes of its largest variations. These axes
are estimated as the eigenvectors of the data—covariance matrix
and are called principal components (PCs). The PCs are ranked by
their contribution to the total variance in the dataset. If there are
N variables of interest in an experiment, each PC represents a
linear combination of the degrees of freedom of the N—
dimensional data set. The first principal component, PC1, explains
the largest amount of variance. The second principal component,
PC2, is orthogonal to PC1 and explains the second largest amount
of variance. That is, PC2 is related to the maximum amount of
residual variability left over after identifying PC1. For example, in
NMR spectral analysis, suppose we have a set of spectra,
containing a single peak whose amplitude varies as a function of
time. Then PC1 reflects the overall lineshape and amplitude of
this peak whereas the rest of the PCs will be noise—related and
are ignored. If in this dataset, besides the amplitude variations,
there are differences in the peak position and phase, then PC2 and
the higher order PCs can be used to estimate the amount of the
frequency shift and phase shift in each spectrum.

In this study, PCA was utilized to preprocess NMR spectral data
by correcting both frequency shifts and phase shifts of each NMR
spectrum prior to Fourier Transformation. This preprocessing
makes subsequent determination of peak areas and peak shifts
much less sensitive to drifting of the BO—field (rats were typically
in the magnet approximately 1—10 hours), fluctuations in the
electrical power grid at 5 PM, or systematic operator error in
integrating peak areas. The latter has been shown to be a
significant source of variability in the in vivo NMR literature (209).

Baseline correction was also performed on every spectrum. For each rat in each
sitimulation protocol, the spectra were added across cycles beginning with cycle #3
to obtain a single high signal—to—noise stimulation—recovery time course of spectra.
Cycles #1 and #2 were not included since the PCr and pH time courses were visibly

different than the other cycles as shown in the Results. The number of spectra in

75

 

 

the high signal—to—noise stimulation—recovery time courses are 32, 40, and 124 for
the 0.75Hz, 2.0Hz, and 5.0Hz frequencies, respectively.
Spectral analysis

For each high signal—to—noise spectrum the relative peak areas were

determined using the Method of Natural Lineshapes (MNL) (25, 26, 124). The MN L
performs a point by point summation of all the spectra (in this case, all the high
signal—to—noise spectra) in a stimulation—recovery series. This generates a single
reference spectrum with a natural lineshape unique to each experimental subject.
The numerically integrated peak areas of the individual lower signal—to—noise
spectra are expressed relative to the single reference spectrum, which in this case
has a very high signal—to—noise. The advantage of MN L over the more common
Lorentzian or Gaussian curve—fitting methods is that it largely removes operator
bias in determining the best lineshape to be integrated, is faster, and is at least as
accurate. The disadvantage of MNL is that it is not suitable for any resonance peak
undergoing significant chemical shifting e.g. inorganic phosphate (124).
Intracellular pH was determined by the chemical shift (5) of Pi relative to PCr at —
2.52ppm: pH=6.77+log[(0.89—5)/(5—3.19)] (175).

We then applied the Non—Negative Least Squares algorithm (no minimum
energy constraint, 200 points within the 1—>500 second time—window) to the PCr
time course data. The NNLS algorithm generates a “spectrum” of recovery PCr
time constants (i.e. relative amplitude vrs. duration) (112, 184, 313). The height of
each peak in the “spectrum” represents the lg APCr term at its corresponding time

Constant, Ii. APCrt=0 is the PCr value at the onset of recovery (end—stimulation).

PCr(t) = APCrt=0 + 2(APCri(1-e't/Ti))

Equation 1: Multicomponent PCr recovery

(i = 1 to n, where n is determined by the algorithm)

76

 

What is NNLS?

In simplistic terms, Non-negative Least Squares analysis is a
regression technique conceptually similar to the traditional Least
Squares method in that both iterative techniques seek to minimize
the collective deviations of individual observations from a user—
supplied function (e.g. exponential) between a dependent and
independent random variable. In the traditional Least Squares
technique the algorithm determines the function’s parameters
(e.g. time constant duration) for a predetermined number of terms
specified by the user. In contrast, the NNLS algorithm does not
specify a fixed number of function parameters a priori thereby
eliminating user bias.

Statistical analysis

All results are reported or plotted as mean1SEM. Prior to comparing means,
data (in 24 stimulation frequency (8) x cycle number (3) of “cells”) was verified for
normality using the Shapiro—Wilks’ test and verified for homogeneity of variance
with Cochran’s C test. In virtually all of the 24 cells, both normality and
homogeneity of variance were satisfactory. This was based upon the Shapiro—
Wilks’ or Cochran’s C test-statistic not reaching the P < 0.05 level of significance.
The statistical design for comparing means is a balanced two—factor experiment
(stimulation frequency x cycle number) with repeated measures on cycle number.
In other words, rats—the random effect—are nested in stimulation frequency and
repeated in cycle number levels. All pair—wise comparisons of group means for the
main effect of stimulation frequency or cycle number were made by Tukey’s
Honestly Significant Difference test at the P < 0.05 level of significance using SPSS
v6.1 for Windows.

Re s u It s
Cyclic changes in metabolites and force

Figure 7 shows sample 31P—NMR spectra of numerous contiguous stimulation—
I‘ewvery cycles of individual rats at 2.0Hz or 0.75Hz. The stimulation-recovery

Protocol and NMR acquisition parameters are detailed in Table 6 and Table 7.

77

Figure 8, Figure 9, and Figure 10 show the time course of relative PCr changes
during the first eight cycles for the 0.75Hz, 2.0Hz, and 5.0Hz rats, respectively. All
values in Figure 8 through Figure 10 are reported as percent of initial PCr, where
“initial” refers to the resting PCr level at the start of the experiment. Literature
values report the range of [PCr] from chemical assays to be approximately 25—30
pmole/g (175). As predicted by a first—order model for the control of oxidative
metabolism for submaximal (i.e. 0.75Hz) workloads (203), the PCr exponentially
decreased to a new steady—state during stimulation and exponentially increased
back to resting levels during recovery. The 2Hz and 5Hz cycles follow a

qualitatively similar time course but with visibly higher—order PCr kinetics.

78

 

 

I l l l l l l l l l l I l l l l
C O I O -3 -‘ -I -I -I.O -12 -16 -1‘ -ll -30 -22 -3I -3I

Figure 7: Sample 31P—NMR spectra stackplots

A single spectrum represents the time—averaged behavior of phosphorus
metabolites over a period of 32.0 seconds (32 FIDs) for the 2.0Hz rat or 21.3 seconds
(16 FIDs) for the 0.75Hz rat. A 5Hz rat, not shown, looks qualitatively similar, but
has a much longer recovery and each spectrum represents 16.0 seconds (16 FIDs).
X—axis is resonance frequency in parts—per—million (arbitrary scale). Y—axis is
resonance amplitude which is proportion to the number of nuclei in a given local
electronic environment. Z—axis is increasing experiment time.

79

110

100

90

80

7O

60

SO

40

[PCr] 96 of Initial

30

20

10

110

100

90

80

7O

60

50
40

[PCr] 96 of Initial

3O

20

10

TI .
I lllllI
lllllmlll m

Cycle 1 & 2

 

r I I T I I I I I I I I I I r f I I I I I I I I I
o 4 8 12 16 20 24
Experiment time (min)

 

Cycle 3 & 4

 

I I I I I I TiI I I I I I I I I I I I I I I I I I
22 26 30 34 38 42 46
Experiment time (min)

Figure 8: Time course of PCr changes (0.75Hz)

Means1SEM (N=7), one cycle = 5.7 minute stimulation, 5.7 minute recovery

80

110

100

70

60

50

40

[PCr] % of Initial

3O

20

[PCr] % of Initial
O\
O

 

Cycle 5 & 6

 

 

Cycle 7 & 8

I I I I I I I ‘T I I I l

l
52 56 60 64
Experiment time (min)

 

75 so 85
Experiment time (min)

Figure 8 (cont’d): Time course of PCr changes (0.75Hz)

Means:SEM (N=7), one cycle = 5.7 minute stimulation, 5.7 minute recovery

81

110
100
90
80
7O
60
50

4O

[PCr] 9:. of Initial

30
2O

10

110

Pa
CD\.OO
000

\l
O

U1
C

[PC2] % Of Initial
p m
o o

w
O

N
O

O

 

 

 

n
‘.
_ , ‘Im
‘Im
Cycle 1 & 2
OIIIIIIIIIIIIIIIIIIII'IIIIIIIIIIIIIIIIIIIIIIII]
0 5 10 15 20 25 3O 35 4O 45
Exparimnt time (min)
,YTYTTT
I: "" . ""’ TyTrTTTITlTT'THTHHTY
: ' ‘ T‘
'5 r'
a v T
- T
5 {TIM ll
3 Cycle 3 & 4
IIIIIIIII'IIIIIIIIIIIIIIlIIIIlIIIIllIIIIIIIII
40 45 50 55 6O 65 7o 75 8O 85

Experiment time (min)

Figure 9: Time course of PCr changes (2.0Hz)

Means:tSEM (N=7), one cycle = 5.3 minute stimulation, 16.0 minute recovery

82

110

TTTTTTTTITTITTITTTTITTT

,7
T

90 T r.‘

100 ,anrTmanTTnTn
11’
T

80 r ’
7o

60

50 ”H NH]

40

[PCr] 9a of Initial

30

20

10 Cycle 5 & 6

0 I I I IT—[ I I I I II I I I l I I I I [I I I I I I I I II I I I I l I I I I l I I I I l
85 9o 95 100 105 110 115 120 125 130
Experiment time (min)

 

110
100 mmmmTITTT’TTTTW TTTmnmmnnmnh

T
r

90 '
8O
7O
60 h
50

ITTTI
4O

[PCr] 96 of Initial

3O

20

1

0

Cycle 7 & 8

I T I I I l I I I I l I I I I I I I I I I I T I I [TI I T] I I I I I I I I I I I I I I l
125 130 135 140 145 150 155 160 165 170
Experiment time (min)

 

0

Figure 9 (cont’d): Time course of PCr changes (2.0Hz)

Means:tSEM (N=7), one cycle = 5.3 minute stimulation, 16.0 minute recovery

83

120

110 T
100 Y m - ‘
90 ff' 1 I

80 5

6O
50
4O

[PCr] 95 of Initial

30
20

10 Cycle 1 & 2

O 5 10 15 20 25 3O 35 40 45 50 55 60 65
Experiment time (min)

120
110
100
90
80
7O
6O

 

50

 

4O

[PCr] 95 of Initial

30

 

20
10

Cycle 3 & 4

65 7o 75 80 85 90 95 100 105 110 115 120 125 130 135
Experiment time (min)

Figure 10: Time course of PCr changes (5.0Hz)

Means:tSEM (N=7), one cycle = 2.1 minute stimulation, 29.9 minute recovery

 

 

[PCr] 9; of Initial

Cycle 5 & 6

 

130 135 140 145 150 155 160 165 170 175 180 185 190 195 200
Experiment time (min)

[PCr] 9; of Initial

 

 

      

Cycle7&8

O IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIllllIllIIl'IIIIlIllllIIIIl
195 200 205 210 215 220 225 230 235 240 245 250 255 260 265
Experiment time (min)

 

Figure 10 (cont’d): Time course of PCr changes (5.0Hz)

Means:tSEM (N=7), one cycle = 2.1 minute stimulation, 29.9 minute recovery

Figure 11A shows the steady—state PCr level (PCrss) attained during stimulation
is proportional to the isometric twitch rate (see also Figure 7). This is consistent
with previous studies showing that steady—state mitochondrial oxygen consumption
scales with workload (192, 203). (Steady—state during stimulation was taken as the
mean PCr level of the last two time points just prior to the onset of recovery for
each rat. I considered the last two time points the minimum number of points
necessary to attain a quasi—steady—state during stimulation. It is acknowledged that
a true steady—state may not have been attained during the 5H2 stimulation.) PCrgs
was significantly different (P < 0.05) across stimulation rates but not across cycles
#1—8. Figure 11B suggests the recovery duration chosen for each stimulation rate
was sufficient for complete resynthesis of PCr prior to the start of the next cycle
since recovery PCrss re—attained a value around 100%. (Steady—state during
recovery was taken as the mean PCr level of the last four time points just prior to
the onset of the next stimulation period for each rat. After visual inspection of each
rat’s recovery time course, the last four time points during recovery appeared to
consistently differ the least from one another. This was taken as evidence of a
steady—state.) There were no significant differences across stimulation rate or
across cycles #1—8 for recovery PCrss. For the two or three rats in the 0.7 5Hz or
2.0Hz groups that successfully completed all 25 cycles (taking five or nine hours,
respectively) there was approximately a 10% decline in the recovery PCrss value,

independent of the stimulation rate, over the course of the experiment (Figure

11B).

86

Steady State PCr during stin.
(10096 O t-O of Cycle #1)

Steady State PCr during recovery
(10096 G t=0 of Cycle #1)

110—
100$
90-
80:
70;
6o:
50‘
4o;
3o:
20;
1o:

 

110—
100:
90$
80:
70:
60:
501
401
3o;
20:
1o:

 

0* *‘P l"O

H—4

1-0

r—b

r-D

.4

DO!
10.!

F9

F’D

FD
r—D r-O
t-D +———O
*0

' l ' l ' I
11 13 15
Cycle #

\14
\0-1

r904

I».-1

+34
vb 0

+00
PD O

00
99

tDO

5.082
2.032

0.7532

+——O

I I I I

l I l l
17 19 21 23 25

Fm-l
kma
r-po4

r-Dol
l-wl
b—DO
*9 O
l-D O

B

 

bd—

I I I I I I

I I
9 ll 13

Cycle #

l I
5 7

III
15 17

I I r I

l l l l
19 21 23 25

Figure 11: Steady-state PCr characteristics vrs. cycle #

Mean:tSEM(N=7 through cycle #8—see Appendix D for N vrs. cycle #). Analysis of
the first eight cycles showed a statistically significant effect (P < 0.05) of stimulation
frequency on PCrss during stimulation but not during recovery. There was no
effect of cycle number or interaction between cycle number and stimulation
frequency on PCrss during stimulation or during recovery.

87

Figure 12, Figure 13, and Figure 14 show the time course of intracellular (ICF)
pH changes during the first eight cycles of the 0.75Hz, 2.0Hz, and 5.0Hz rats,
respectively. As best viewed in the 0.7 5H2 and 2.0Hz rats, there was a transient
intracellular alkalinization at the start of the stimulation and a transient
acidification at the start of recovery. This transient pH behavior is attributed to net
proton consumption by PCr hydrolysis during stimulation or net proton production
by PCr resynthesis during recovery (4). Relative to the stimulation time course, the
generally larger variance of ICF pH in all recovery time courses is due to operator

difficulty in localizing and hence integrating the Pi peak in the NMR spectrum.

,-
‘11.}

ICF pH
\1

x)

O

\D

oo

.5

U)

N

H

O

\O

(D

\)

 

 

 

 

ll
lTllTTT 71, III I
I ITITT TYY I 11111 I I
T l TIT! r l ltll
TlTTI T
_‘ Cycle 1 & 2
q
I I I I I I I I I l I I I r l I I I I I I I I I
o 5 10 15 20
Experiment time (min)
a
: I ”‘n
. l T l r I
j Cycle3&4
'J
I I I l I I T I I I I I I l I I I I I I I I T j
25 30 35 4O 45

Experiment time (min)

Figure 12: Time course of ICF pH changes (0.75Hz)

MeanStSEM (N=7), one cycle = 5.7 minute stimulation, 5.7 minute recovery.

89

Cycle 5 & 6

 

 

6.7 I Ifi I I I I I I l I I I I I I I I I I I I fiI
45 50 55 6O 65
Experiment time (min)

Cycle 7 & 8

 

6‘7 I’T I I I Tfifi rI I I I I fiI I I I l I

I I I I
70 75 80 85 90
Experiment time (min)

Figure 12 (cont’d): Time course of ICF pH changes (0.75Hz)

Means:tSEM (N=7), one cycle = 5.7 minute stimulation, 5.7 minute recovery

m ox \l \l \1 \1
oo \o o H N w
l

lllIlllIlllIllJlulllll

0"
\l

 

Cycle 5 & 6

 

 

45

 

I I I I I I I I I I I I I I I I I I
I l l

I
50 55 60 65
Ebmpuaridnlunt.'tiJme (nmizi)

 

Cycle 7 & 8

 

l I I I I l I I I I l r I I I I I I I I I I
70 75 80 85 90
Ehcsuuriancunt 'tiJne (mniri)

Figure 12 (cont’d): Time course of ICF pH changes (0.75Hz)

Means1SEM (N=7), one cycle = 5.7 minute stimulation, 5.7 minute recovery

7.2 I

ICFl pH
0" \l
(D 0
-—¢
—c
—1
—c

m
0"
IIIIIIIIIIIIJJI
—<
—
-———c
—«
—c
——<

 

 

 

 

 

6.4 cyCIel&2
6-2 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
0 5 10 15 20 25 3o 35 40 45
Experiment time (min)
7.4-.
7.2—
7.0-
:: I
n I
N .-i
U6 8‘
H d
6.6-
6_4_' Cycle3&4
6.2 IIrIlIIIIlIIIIlIIIIlIIIIIIIII1IIII'IIIIIIIIII
40 45 50 55 6o 65 70 75 80 85

Experiment time (min)
Figure 13: Time course of ICF pH changes (2.0Hz)

MeanSiSEM (N=7), one cycle = 5.3 minute stimulation, 16.0 minute recovery

91

 

 

 

7.
:1:
9:
#16
U
H
6.
6.4
Cycle5&6
6'2 IIIIIIIIII'IIII'IIIIIIIIIIIIIIIIIII'IIIIIII—I—Ij
85 90 95 100 105 110 115 120 125 130
Experiment time (min)
7.4-
7.2—
r
7.0-
m .
9' I
p. ..
06 8.
H I
6.6—
6.4-
. Cycle7&8
6.2 IIII'IIIIIIIIIIIIII'IIIIIIIIIIIIII'IIIIIIIII'

 

 

125 130 135 140 145 150 155 160 165 170
Experiment time (min)

Figure 13 (cont’d): Time course of ICF pH changes (2.0Hz)

Means:SEM (N=7), one cycle = 5.3 minute stimulation, 16.0 minute recovery

92

ICF pH

' ' Cycle 1& 2

O
Ul

10 15 20 25 30 35 40 45 50 55 60 65 7o
Experiment time (min)

\I
b

\l
N

\l
O

m
00

 

Ch
ON

llIllJIlllIlllIlllIJ-Lllllll

0'\
up

Cycle 3 & 4

0‘
N

5-0:1TrrrnTrrrrrrrnn1TernfiTrm117me1Trrrqfiwrrrn-FTW
65 7o 75 80 85 9o 95 100 105 110 115 120 125 130 135
Experiment time (min)

 

Figure 14: Time course of ICF pH changes (5.0Hz)

MeanSiSEM (N=7), one cycle = 2.1 minute stimulation, 29.9 minute recovery

93

\J
A

 

 

7.2—"
7.0—-
:5. 6.8;
a. .
U ..
H 6.6-
6.4—-
6.2—~
~ Cycle 5&6
6.0:
WIWWWTWWWW
130 135 140 145 150 155 160 165 170 175 180 185 190 195 200
Experiment time (min)
7.4—
7.21
- A
'1 ll l lllTIlT ll 'l] “H
7.0-‘1 l ‘lll‘lllll, h I, ll H‘ ’, lW‘ W717 I J
-1 l-q' l Irv ‘ , l." llfii‘lllf'lill.||l_"r- " l
a: -' I "
9.6.8- , i‘
a. .
U .
H 6.6-
6.4-:
6.2-:
- Cycle7&8
6.0—-

 

195 200 205 210 215 220 225 230 235 240 245 250 255 260 265
Experiment time (min)

Figure 14 (cont’d): Time course of ICF pH changes (5.0Hz)

Means1SEM (N=7), one cycle = 2.1 minute stimulation, 29.9 minute recovery

94

Figure 15 shows end-stimulation pH is dependent not only upon stimulation
frequency, as expected, but also cycle number. End—stimulation pH was
determined from chemical shift between Pi and PCr in the last spectrum acquired
before the onset of recovery. The higher stimulation frequencies resulted in lower
ICF pH by the end of the stimulation period which is primarily due to increased

glycolytic flux producing lactic acid (257, 260).

 

 

 

 

 

 

7.3—
7.2:
‘ ,_ T- TT 'I'Tvo
- v o - 000 0
11-000000051 00056 ooT o
.. O T o
'8 7.04 0
In 4 *1. A AA v-
0': 6.9- AAA AA AAAA AA A AAAAT
.1
3. 6.8- . 2 o A
'3 67‘ -. ' 2 71'
O ' T "
fi-fi l
p 6.6- A!
In“ ‘
a.” 551 g 5.032
.5
U” 1 A 2.082
Hm 6.3d
6.2-1 0 0.7532
42
6'1I‘Ijl'l‘l'l'l'l'l'l'l'l‘l
135791113151719212325

Cycle #

Figure 15: End-stimulation ICF pH vrs. cycle #

Means:tSEM (N =7 through cycle #8—see Appendix D for N vrs. cycle #). Analysis of
the first eight cycles showed a statistically significant effect (P < 0.05) of stimulation
frequency for a given cycle #. There was also a significant interaction between

cycle number and stimulation frequency.

95

Isometric twitch force normalized per gram wet—weight of stimulated muscle
(gastrocnemius—plantaris-soleus group) is shown in Figure 16, Figure 17, and
Figure 18. All cycles exhibit a positive staircase phenomenon (the early
potentiation of twitch force) which is currently thought to be due to modulation of
the myosin regulatory light chain phosphorylation state by rising levels of calcium
and/or changes in the microfilament lattice spacing (252). The ripples seen in the
5H2 twitch profiles are a result of brief delays (<5 seconds) in the spectrometer
computer’s pulse—acquisition sequence due to writing data to the hard disk prior to

triggering the Grass stimulator.

350

300

250

200

(9/9)

150

100

Force

50

 

Isometric Twitch

 

IIIIIIIIIIIIIIIIIIIIIIIIIIIIII'IIII]

0 l 2 3 4 5 6 7
350

300

 

250

 

200

(9/9)

150 “i

100 Cycle 2

Force

50

Isometric Twitch

 

olIIII'IIIIIIIIIIWIIIIIIII'IIII'IIIII

350 11 12 13 14 15 16 17 18

250 IIT {TIT TTIT rvrT TTTT TTTT Till! I

T
A T Y
C} T
\ 200 ,5!
or

Cycle 3

Twitch

Isometric

 

IIIIIIIIII'IIII'IIII'IIII1TIIIITIIII

22 23 24 25 26 27 28 29
Experiment time (min)

Figure 16: Time course of isometric twitch force (0.7 5Hz)
MeanStSEM (N=7), one stimulation cycle = 5.7 minutes

 

97

350

300

N
W
O

N
O
O

 

H
U1
0

H
O
0

Cycle 4

Force (g/g)

Isometric Twitch
U1
0

 

OlIIII'IIrIrIIIIIIIITTTIII'IIIIrIIIIl

33 34 35 36 37 38 39 4O

 

m
U
’5 Cycle 5
k:

Isometric Twitch
H
L11
0

 

45 46 47 48 49 50 51 52

 

(g/g)

Force
H
O
0

U1
0

Cycle 6

Isometric Twitch
H
U1
0

 

O'IIIITHTFIIIIIIFIHIIIIIII'IIIIIIIII]

56 57 58 59 60 61 62 63
Experiment timen(ndrn

Figure 16 (cont’d): Time course of isometric twitch force
(0.75Hz)

Mean32tSEM (N=7), one stimulation cycle = 5.7 minutes

98

[l

 

 

350

300

250

200

(g/g)

150

 

100

Force

50

Isometric Twitch

 

350

 

300

 

 

r1 Tllll-All'
250 T - ll.

200 E; J
L...—

150

 

 

 

(911/91)

100

Force

Cycle 8

50

Isometric Twitch

 

OrrIIIlIfiITTTITIrIrjI'IIII'IIIIIIIIII

79 80 81 82 83 84 85 86
350

300

250

 

5° Cycle 9

0IIIIIIIIIIIIIIIIIIFIIIIII'IIII'IWII]

9o 91 92 93 94 95 96 97
Experiment time (ner

Isometric Twitch
H
UT
0

 

 

Figure 16 (cont’d): Time course of isometric twitch force
(0.75Hz)

Mean81SEM (N=7), one stimulation cycle = 5.7 minutes

(g/g)

Isometric Twitch
Force

 

  

(g/g)

Isometric Twitch
Force
H
L”
O

 

 

(g/g)

Cycle 3

Isometric Twitch
Force
H
U1
0

 

O
.1

I I I I I I III I I [I 1 I rj I I {IT I [TI II

42 43 44 45 46 47 48 49
Experiment time 0min)

Figure 17: Time course of isometric twitch force (2.0Hz)

Means:tSEM (N=7), one stimulation cycle = 5.3 minutes

100

 

Isometric Twitch
H
Ul
O

 

Isometric Twitch
5...
U1
0

 

 

(g/g)

Force

Cycle 6

Isometric Twitch
H
Ul
O

O

 

I I j I I I I I ‘I I j I I I I I I I l l I I I

l
106 107 108 109 110 111 112
Experiment time 0min)

F igure l7 (cont’d): Time course of isometric twitch force
(2.0Hz)

Means:SEM (N=7), one stimulation cycle = 5.3 minutes

101

:!

 

:Lii

m

350

 

 

 

 

 

 

300 ITIIITIITTT
.c:
B 250
.H"
53200
U)
UVISO
.gm
4633100
50 Cycle7
8‘“ 50
H
0' I I I I I I I ' rt I I I I I I I I I I I I II
127 128 129 130 131 132 133
350
T
300 lTTTTTTTITTT
.c:
B 250
".1"
33200
t»
UVISO
.Qm
4638100 CycleB
EC
0‘“ 50
U)
H
I I]
155
.C:
o
4.)
W4
3
E
o
"-4
n
J.)
(v
E.
0
U)
H
I I—l
170 171 172 173 174 175 176

Experiment time (min)

Figure 17 (cont’d): Time course of isometric twitch force

(2.0Hz)

MeanSiSEM (N=7), one stimulation cycle = 5.3 minutes

102

400
ITT’

 

 

 

 

 

I I n
T'TTII
. I
g 300 'ITTTTIIT
IJ ' ' I I
.r-I" ‘ I 'u, TTTT II T
3\ ‘ ' ll . . III TT‘ IIII III In
3200—: .. '1' H H
o I
.,..| '
98 :
88100d
E -I
0‘“ 4 Cycle 1
m J
H d
O I I I I r I T I III I I I I I
0.0 0 S 1.0 1.5 2 0
400
.TTTTYTT TI
,' TTT
'5 300 H I IIIIIII
. A I II
H m ' 'III m'I TY I” II
E\ III W" I II
I. IT
9200 I. I I”
U I I.
-.-I
58
o *5 100
gm
U) Cycle2
H
O I _I I I I I I I I T l I I I I I I I I I l I I
32.0 32 5 33.0 33 5 34 O
400
£1
0 T" m T in
.fiA 300 II I 'hITTIIIflTT
U) ll I| TNT r n
\ 1 TI rITT
m I ' ”nu "Tl-I'll _IIT
UVZOO ' " "I. 'II
"-1 -
H8 -
3‘33 :
(8);“ 100'“
g : Cycle3
O I I I I I I I I r I I I III I I I r I I I
64.0 64.5 65.0 65.5 66.0

Experiment time (min)

Figure 18: Time course of isometric twitch force (5.0Hz)

Means:tSEM (N=7), one stimulation cycle = 2.1 minutes

103

400

 

 

 

' 77, m?
g 300 II "HI: TWITIT
I WI
"gm '"l TTHITUT T l
9200 l l" l I" ' I' T
0 II
-r-I
H8
33’ 8100
Eu.
0, Cycle 4
H
O I I I I II I I I I I III I I I I I I r I I I
96.0 96.5 97.0 97.5 98.0
400‘
3 .
g 300—I ' TTTT"III .
“A j ' I T I TT In I
'H C) q "I 'mnl I ll n l IImITl
g\ ' " In 'T‘TTII. TTI
U) 200 l II m
g I
H8
‘5 $8 100
8‘“ Cycle 5
U)
H
O I I I I I I I I I I I I I I I I I I I I I I I
128.0 128.5 129.0 129.5 130.0
400
mm.
'T TY" YT?
F8 300 . "n YT T
. IIITT
:3" .I "| IT IT T T
33 l, M. Im ”UT TrrTTTI
99200 "I u 3". 0 J”
3
H 0
U a 100
go
o “4 Cycle 6
U)
H

 

O l I r I f‘r I I I I I I I I I j I I I I I I
160.0 160.5 161.0 161.5 162.0

Experiment time (min)

Figure 18 (cont’d): Time course of isometric twitch force
(5.0Hz)

MeanStSEM (N=7), one stimulation cycle = 2.1 minutes

104

400-

ILL

 

 

 

 

 

13 1TH mm I
U
u 300‘ "M ”In T I
"-18 " I U! l I“ I .
g\ . T” U1 “H, n I
9 ' I III H W In
0 200 I I" H II
“ o
5 H
a [2 100
8
H Cycle 7
O I I I I fl I I I I I T I I I ‘l I I 1 I I I I
192.0 192.5 193.0 193.5 194.0
thpcnminumnt.1:inua (nmhno
4001
3 300: W mm“
B ; 'T. I. n [In I T
"-1 A - 'II II. T T T
C) d In TI .‘ T
g \ l I "I ‘ ”T n ”TIR'} T I
U3 200 ll ‘ n
U .
"-1 ‘1
H 8 :
ff, H 100-
a 8 '
8 2 Cycle 8
H d
O I I I I I I T I I l I I I I ' I I I j I I I
224.0 224.5 225.0 225.5 226.0
EhmpmmriJucurt 1:1nup (knixi)

Figure 18 (cont’d): Time course of isometric twitch force

(5.0Hz)

MeanSiSEM (N=7), one stimulation cycle = 2.1 minutes

105

Analysis of the first eight cycles showed no statistically significant effect (P <
0.05) of within—cycle stimulation frequency on initial peak twitch force (Figure
19A). There was an effect of stimulation frequency on end—stimulation peak twitch
force within a cycle. (Figure 19B, initial peak twitch force was measured at the first
time point for each cycle; end—stimulation peak twitch force was averaged over the
last three time points.) There was a slightly significant effect of increasing cycle
number within the 0.75Hz and 5.0Hz stimulation frequencies (Figure 19A);
however, there was no effect of increasing cycle number on end—stimulation peak
twitch force (Figure 193). Lastly, there was no significant interaction between cycle
number and stimulation frequency for either initial or end—stimulation peak twitch
force.

During the recovery periods of each cycle for most rats, two or three
supramaximal twitches were elicited for the purpose of determining the time-to—
peak tension (TPT) and half—relaxation time (HRT) in milliseconds The TPT and
HRT parameters give a rough global estimate of calcium release and uptake
kinetics (hence activation and de-activation of the cross—bridge machinery),
respectively. It is acknowledged the time required for energy storage and release in
the series elastic components (SEC) of the muscle tissue can potentially confound
the interpretations of TPT and HRT measurements. No attempt was made to
quantify this SEC contribution. Analysis of the first eight cycles showed a
statistically significant effect (P < 0.05) within cycles #2-#5 of stimulation frequency
on twitch HRT when comparing the 2Hz and 5Hz groups (Figure 20A). There were
no other significant effects of stimulation frequency on HRT or TPT within a given

cycle (Figure 20A and B). Across cycles, TPT in cycle #1 was significantly different

106

than the remaining cycles for the 2.0Hz and 5.0Hz groups. Also, the 2.0Hz group
had a significantly longer HRT during the first cycle compared to remaining cycles.

There was no significant difference across cycles for HRT or TPT in the 0.75Hz

group.

107

 

 

.c:
3175
'A o 5.082
“I"
0"150 A 2.032 A
:13

u . 0
35.125 0 07582 I 001
' oo
9'" 0
«:0 T TTT°T Tl l
.2100 up; TTA 022 23300 A 01,
a» galagl Ailfiei 1 AA
00 75 ‘AiToéi 1 9 1*
II- 10. A A AAA
Flee i 1 4 ill
no 1
~40 50
g...
“V
5.3.
“g.25'I'I'I‘I‘I‘I*l'l'I'T‘l'ljl
0° 1 3 5 7 9 11 13 15 17 19 21 23 25
x Cycle #
8 225
3 o 5.082
b 200
u: A 2.032 B [[
“A 175
H: 150 Hl lo l
No l ollllo o
°5‘ 125 l loco o°°° A A $T
a? TT l A AA A A

'3 ooloelgeawwh .‘Hiii‘ 2
II 1 l 1 1
0.; 100 994 AA
fl. 9
u TTITTTTT
3. 75 00 00.00

a?

O
3’33 SO'I'ITI'I'IrrfiI'I'I'I'I'I'I
9V 1 3 5 7 9 11 13 15 17 19 21 23 25
3 Cyc1.#

Figure 19: Initial and end-stimulation peak twitch force

Mean:SEM(N =7 through cycle #8-see Appendix D for N vrs. cycle #). All values
are expressed relative to the force measured at t=0 of cycle #1 within given
stimulation frequency.

108

Twitch Half-Relaxation
Tina (nsoc)

Twitch Tima-to-Peak
Tension Cnsac)

 

 

 

A I :3:
lillilliliill °° l
P's-ECOLLZAA 18:? A AAAfATAAAlAO
9.0-: ll‘ilill l filly“) l ill

22-

20

18

ILLALLIJJJIIIIIIIIII

 

12

l l '
l 3 5 7 9

0—1

'l'lfil'l'l'ljr

' l

11 13 15 17 19 21 23 25

Cyclenif
I 5.032
B A 2.082
0 0.7532 T
‘A A A A

 

1 3 S 7 9

l'l'l‘r‘l

'I'l'I
11 13 15 17 19 21 23

Cycle #

'l
25

Figure 20 Force kinetics of single twitches vrs. cycle #

MeaniSEM(N=7 through cycle #8—see Appendix D for N vrs. cycle #).

109

 

Utility of Principal Component Analysis
Figure 21 shows a typical “before and after” output of the PCA method applied

to a region of 129 points around the PCr peak for a single rat. The first four
principal components roughly correspond to the overall lineshape (PCl),
frequency shift (PC2), linewidth (PC3), and phase shift (PC4) of the PCr peak. The
PC magnitude represents the relative amount of variance that can be explained by
the given PC component. For example, Figure 21A illustrates P01 in the original
uncorrected NMR dataset, can explain approximately 89.6% of the variance in the
PCr peak. After five iterations of phase and frequency correction, PC1 was able to
explain 99.5% of the variance whereas the remaining PCs explained proportionally
less variance in the PCr peak (Figure 21B). The robustness of the PCA spectral
correction method is nicely shown in cycle #5. In this cycle there is an abrupt
discontinuity in the resting steady—state PCr level as indicated in the PCI (i.e.
overall lineshape) time course in the original dataset (Figure 21A). This abrupt
change in the resting PCr level appears to be largely due to a sudden frequency
shift as shown in PC2. The exact cause of this discontinuity is mysterious since the
muscle is at rest and presumably in a metabolic steady—state. A single isolated
malfunction of the spectrometer computer, superconducting magnet, or Grass
stimulator over the five hour experiment seems unlikely since events abruptly
returned to their normal pattern. Interestingly, this discontinuity occurred around
5 PM, so one potential rational is that an abnormal fluctuation in the building or
campus electrical power grid occurred as most people left their offices. Regardless
of the cause, the PCA method was able to easily correct this abnormality (P01 in
Figure 21B).

110

A Original dataset

 

 

 

 

PC shape PC magnitude PC time course
11 = 89.5670°/o .
a l '1 e
.. 1A WWW WW"
'1 2 3 4 5 6 7 8
cycle #
pc2 1% k2 = 8.13010/0 I h
\ 1 W13 L
\/

.. O

p C3 1A“ A3 - 15037 ADM/WWW-

‘\] \/

PC4/\ 14 = 0.4041°/o
/\ l

 

BCorrected dataset

x1 = 99.54237. .
w, W .(T'W

45678

cycle #
12 = 0.3217%

pc2 A l

 

 

 

13 = 0.0325%
H PC3 fl ’1 ,me.
PC4 14 = 0.0210°/o W
/\ [\A '1

Figure 21: Spectral correction by PCA

The first four principal components (PCs) of the PCr peak from a single 5Hz rat
before (A) and after (B) five corrections by the Principal Component Analysis
method. PC 1, PC2, PC3, and P04 roughly correspond, respectively, to the overall
lineshape, frequency shift, linewidth, and phase shift of the PCr peak. Lambdas
are the eigenvalues of the data covariance matrix and represent the relative
contribution of each principal component to the overall variance in the dataset.

111

Decomposition of global PCr time constants

After phase and frequency correction by the PCA method and then baseline
correction, a high signal—to—noise 31P—NMR time course was obtained by summing
across cycles #3—#8 for each rat (Figure 22). Next, the NNLS algorithm was applied
to each rat’s PCr recovery time course data points (Figure 23A) thereby generating
a “spectrum” of time constants (Figure 238). The relative amplitude (APCri) and
time constant duration (Ti) of the PCr transients are summarized in Table 8 for all
rats. (APCri and Ti are the iterated parameters from Equation 1.) For convenience,
all of the time constants were rank ordered and assigned to arbitrary bins based
upon visually apparent groupings. Comparisons among means was performed on
the four cells in Table 8 that had N25. There are two general trends which reached
statistical significance (P3005): within a given stimulation frequency, there was
always one component that had a larger relative amplitude than the rest; and
secondly, the duration of the 5Hz—Long, 5Hz-Short—Medium, 2.0Hz-Medium, and

0.75Hz-Medium were significantly different from one another.

112

 

 

PCr 'y-ATP a-M-p

Figure 22: Sample high signal-to—noise 31P-NMR spectra

Individual spectra (such as shown in Figure 7) were summed across cycles for a
given rat to generate a single high signal—to—noise stimulation—recovery time
course. The above 32 spectra are from a single 0.75Hz rat summed across #3—-#8
cycles.

113

9a of Initial PCr

Amplitude (95 of initial PCr)

100 *

95

90

85

80

75

70

A

PCr Recovery Time Course
(rat mixed muscle, 0.75Hz)

 

 

 

 

 

 

 

 

Monoexponential fit Tau = 33.1 s
I I _I I I I I I I I T I I I I I I I I j l I I I I
0 50 100 150 200 250
Time (sec)
B NN LS Analysis of PCr Recovery
25 1 (rat mixed muscle, 0.75Hz)
20: Computed mean Tau = 24.1 s
j Tau = 15.1 sec
: Amp = 0.198
.15-
‘ Tau = 62.4 sec
1 Amp = 0.188
10:
+ H
.05:
'00 IIII'IIII'IIII'IIIT'IIIIlIIII'IIII'IIjII
0 10 20 30 40 50 60 70 80

PCr Recovery Time Constant (sec)

Figure 23: NNLS applied to a PCr recovery transient

A: The 5.7 minute PCr recovery time course of a single rat mixed muscle stimulated
at 0.75Hz for 5.7 minutes. A monoexponential fit using the Marquardt-Levenberg
algorithm (194) is superimposed.

13: The resulting “spectrum” of time constant values obtained by applying the

N NLS algorithm to Figure 23A.

114

C PCr Recovery Time Course
(rat mixed muscle, 0.7 5Hz)

 

 

 

 

100 ,
95
§ 90
H 85
d
-d
'3 80 . . .
a Biexponential fit from NNLS output
75 Taul = 15.1 sec
% Tau2 = 62.4 sec
* 7O
65
60 r I I I I I I I I I I I I I I I I I I I I I I I I
O 50 100 150 200 250
Time (sec)

Figure 23 (cont’d) NNLS applied to PCr recovery transient

C: The 5.7 minute PCr recovery time course of a single rat mixed muscle stimulated
at 0.75Hz for 5.7 minutes. A biexponential fit is superimposed using the tau and
amplitude parameters from Figure 23B .

115

Table 8: Time constants from NNLS output

 

 

 

 

Twitch Very Short Short— Medium Long Very
Stim. Short Medium Long
Rate

0.75Hz 1.0 15.1 30.0 51.113.43 107.9 186.5

(7) 0.01 0.20 0.16 02710.06 0.06 0.11
(2) (1) (2) (6) (1) (1)
2.0Hz 1.0100 14.4121 none 43.8109 none 226.4
(7) 00110.00 0 0310 02 observed in 0.341005 observed in 0.02
(3) (3) this range (7) this range (1)
5.0Hz 2311.3 16.1119 33.1128 none 83514.9 322.9
(7 ) 0. 0410. 00 0 1410 02 0.281008 observed in 0.531010 0.89
(3) (3) (5) ““5 range (6) (1)

 

All values are mean1SEM. For each table cell: top number is time constant
duration (seconds), middle italic number is relative magnitude of time constant (%
of initial PCr), bottom number is N. Bold indicates dominant time constants.

Modelling of fiber type distributions

The global time course of PCr recovery for a hypothetical mixed muscle was

modeled using two diverse distributions of fiber oxidative capacities: discrete

bimodal versus quasi—Gaussian. In the discrete bimodal model, the mixed muscle

consisted of 1000 cells composed of 50% high-oxidative capacity fibers and 50%

low-oxidative capacity fibers (Figure 24A). In the quasi—Gaussian model the mixed

muscle consisted of 1000 cells with a broad normal distribution of oxidative

capacities (Figure 24B). In both models the PCr recovery time course from
individual muscle fibers was assumed to be monoexponential, rising from a steady—

state end-stimulation level that is linearly dependent on tau, where tau is

independent of submaximal workload (203). A four—minute global time course of

PCr recovery after a submaximal workload for the whole mixed muscle was

computed from the sum of the individual muscle fiber PCr recoveries for each

hypothetical distribution (Figure 25). The resulting global PCr recovery transient

was then decomposed using the NNLS algorithm thereby generating a “time

constant spectrum” for each distribution (Figure 26).

116

A Two Cell Populations (Tau = 30 and 84 s)

Mean = 57 5

Number of Cells
8

 

 

 

 

O t v . r 1 r 4’.

0 20 40 60 BO 100 120
PCr Recovery Time Constant (s)

B Gaussian Distribution of Cell PCr Recovery

301-

Mean = 57 3

Number of Cells

 

 

0 20 40 60 80 100 120
PCr Recovery Time Constant (s)

Fig‘ure 24: Hypothetical fiber type distributions

Discrete bimodal (A) and quasi—Gaussian (B) distributions of fibers were
constructed with equal values of mean PCr recovery tau (57 seconds). The discrete
burlOdal model has 500 high—oxidative capacity muscle fibers with PCr tau = 30
Seconds and 500 low—oxidative capacity muscle fibers with PCr tau = 84 seconds

e qufisi—Gaussian model has 1000 muscle fibers with 100 distinct PCr recovery
taus (57:18 seconds, mean1SD).

117

A Two Equal Cell Populations (tau: 30, 84 s)

 

    
 

 
 
 

 

 

‘°° - - - 2 a a s 5
90 l
:2; 00 ‘ Single lit tau = 64 s
E vs. True mean = 57s
o\°
‘- 7
0 ° ‘
ll
6° 1
so . . . . .
o 50 100 150 200 250
Time (s)

B Gaussian Cell Population (mean tau = 57 s)

 

 

 

 

 

71‘
1‘: Single fit tau = 59 s
o\°
5 70 a
0.

3° 1

so . . . .

0 50 100 150 200 250
Time (s)

Figure 25: Synthesized global PCr recovery time—courses

Four-rninute global PCr recovery time courses after four different submaximal
“’9" 0ads were calculated for the bimodal and Gaussian distribution. Artifical
“0156 Was added to all recovery time courses. In each distribution, a

monoexponential curve fit 13 superimposed over the synthesized recovery time
POints along with the monoexponential tau value.

118

A NNLS Analysis of PCr Recovery
(2 cell populations)

70 w

Computed Mean Tau = 565

6° 1 (vs. True Mean 57s)
50 1

Tau = 78 s
40 4 Amp = 32.7

Cell Fraction = 58%

Amplitude (% initial PCr)
8

 

 

 

2° 1 Tau .-. 26s E 1
m Amp=775 1
‘ Cell Fraction = 42%
0 . AL
10 1:0 '
PCr Recovery Time Constant (s) 1
_ 1

 

B NNLS Analysis of PCr Recovery
(Gaussian Cell P0pulation)

70 a.

so . Computed Mean Tau = 57 s
50 «

Tau = 63 3
‘° ‘ Amp = 37.4

Cell Fraction = 83%

2“ Tau=25s

Amp = 3.1
Cell Fraction = 17 °/o

04 AP

10~

Amplitude (°/. initial PCr)
8

 

 

10 100

PCr Recovery Time Constant (s)

Figure 26: NNLS output of hypothetical distributions

The “spectrum” of time constant values was obtained by applying the NNLS
algorithm to the synthesized global PCr recovery time course after the highest
sPbmaximal workload. Note both underlying hypothetical distributions have
8111lilar spectrums. The relative cell fraction for the fast and slow component was
calculated for a given amplitude weighted by its tau value. This resulted in a
computed mean tau virtually identical to the assumed true mean of 57 seconds.

119

 

Dis c us 3 io n
Decomposition of PCr recovery transients

This study has shown that application of the unbiased N on—N egative Least
Squares algorithm to high signal-to—noise PCr recovery transients of rat mixed
muscle yields multiple time constants for both submaximal (0.75Hz) and
supramaximal (2.0 and 5.0Hz) workloads (Table 8). The general trend seen in
Table 8 is that faster recovery time constants are recorded more often as
stimulation frequency increases. Though not the central observation in other
studies, this pattern does appear when results of these studies are considered in
aggregate. For example, in studies using a single combination of stimulation
intensity and duration which placed the workload in the submaximal domain, the
PCr recovery time course was best fit by a monoexponential function in frog (192,
65), rat (203), cat (176), and human (149, 195, 323) muscle. In other studies using
only supramaximal stimulation intensity and duration, the PCr recovery time
cOllrse was best fit by a biexponential function in rat (214, 243) and human (120,
259, 274) muscle. Curiously, in a few reports, if the muscle contains a slow—
OXidative fiber distribution it exhibits PCr recovery kinetics resembling a slightly
nuClerdamped second—order response after either submaximal stimulation (cat
$01eus (176); creatine—depleted rat gastrocnemius (204)) or supramaximal
StiInulation (isolated human Type I fibers (274)). Lastly, a few human studies using
submaximal and then supramaximal workloads have noted monoexponential and
then biexponential PCr recovery time courses, respectively (9, 287, 308).

Over twenty years ago an explanation for this stimulation—dependent pattern of
time constants was put forward by Sahlin’s group based upon human biopsy data
(120, 258). They suggested that as workload intensity increases, the increase in

anaerobic glycolytic flux produces an increased [H+] which shifts the creatine

120

kinase equilibrium reaction to the right (as written below) by the beginning of
recovery.
PCr + ADP + H” <—-> ATP + Cr

Throughout the subsequent recovery, this mass action effect of elevated but
decreasing [H+] results in a lower A[PCr]/Atime which would explain any slow
eXponential components that may be observed. As mechanistic support for their
eXplanation, they cited in vitro experiments in the mid—19508 which showed a
strong dependence of the creatine kinase equilibrium constant and reaction
velocity on pH. This is due to the adenine nucleotides’ changing binding affinities
since pH modulates the ionization states of their Mg2+ and/or K+ complexes. More
recently, this proton mass—action explanation for multicomponent PCr recovery
kinetics has also been directly or implicitly stated in numerous human in vivo 31P—
NMRS studies (6, 9, 21, 217, 291,308).

The above mass—action argument is somewhat weakened on two accounts. First,
it is implicitly assumed the muscle is composed of a cellular homogenous
POPUIation of fibers with regard to oxidative capacity and total creatine content
well that pH effects are the sole modulator of the creatine kinase equilibrium and
reaction velocity. But it is well documented humans and animals have significant
intra‘1"nuscle and inter-muscle fiber type heterogeneity (261). Second, the
argument assumes the creatine kinase reaction is acting as an isolated equilibrium
System so that increases in [H*] lead to predictable stoichiometric decreases in
[Perl ~ Although it is established that the creatine kinase reaction is at equilibrium
during all but the most severe workloads (165, 201, 213, 318), it is also well

recognized this reaction is not isolated; it is functionally coupled by ATP and ADP
to Several non—equilibrium reactions (the Na+/K+—, Ca2+-, and myosin—ATPases;
mitochondrial ATP synthetase) (169, 17 2, 211). This implies, for example, that the

net . . . .
PCr resynthes1s reactlon occumng during recovery

121

 

creatine kinase reaction: ATP + Cr <-——> PCr + ADP + H”

mitochondrial ATP synthetase: ADP + Pi —> ATP
Net reaction during recovery: Cr + Pi —> PCr + H+

is also not at equilibrium. This complicates a simple and explicit mass action
relationship between changes in [H+] and changes in [PCr]. Experimentally, data
from in situ resting cat slow-twitch muscle, has shown hypercapnic acidosis to pH
6.5 increases, not decreases, the steady—state [PCr] by approximately 8—10% (206,
118, 119). Other studies have shown decreased pH, primarily due to increased lactic
acidosis, prolongs the PCr recovery in rat (234) and human mixed muscle (287, 308)
but not in dogfish white muscle (62). Taken together, these results suggest
quantitatively or qualitatively forecasting the precise effect of changes in [H+] on
lPCr] is extremely challenging and cannot be extrapolated from examining the
creatine kinase reaction in isolation.

An alternative explanation for the global N MR—observed multicomponent
behaVior of PCr recovery kinetics is the well documented existence of intra—muscle
and iIlter—muscle heterogeneity of fiber type distributions based upon muscle
bi0psy data (109, 261, 241). As commonly practiced, the non—invasive nature of
311LNMRS dictates that the PCr signal arising from a given volume of muscle
r epreSents the global average of individual muscle fiber PCr transients.
Theoretically, it is possible for every muscle fiber to exhibit unique PCr kinetics
Since each cell may have slightly different total creatine content and mitochondrial
Volume density. In reality, the intra—muscle fiber type variability is somewhat
reduced since muscle fibers within a given motor unit have essentially the same

biochemical and mechanical characteristics (110, 123, 37).

Comparison of the 5.0Hz versus the 0.75Hz data in Table 8 reveals that the
higher workload resulted in more frequent appearance of exponential components
with Significantly longer and shorter durations than the lower workload. Since the

d .
uratlon of the recovery PCr time constant is inversely proportional to

122

 

.‘1

un

ti

 

 

mitochondrial content (203, 234), these results suggest that the longer time
constant (i.e. the “long” 83.5 seconds component) is due to the presence of fibers
with low relative oxidative capacity. It is likely this fiber population has an
attenuated PCr recovery rate due a lower ICF pH (234, 287, 308) since it has been
shown in cat soleus that hypercapnic acidosis can increase the PCr recovery time
constant by 2.7—fold (119). Harkema et al. speculated this attenuation was due to
perturbations in cytoplasmic substrate delivery to the mitochondria and/or
deviations from the mitochondrial enzymes’ pH optima which thereby decreased
mitochondrial ATP synthesis rate and hence decreased the PCr recovery rate (119).
The shorter time constants (i.e. the “short” 16.1 seconds, and “short—medium”
33.1 seconds components in Table 8) are more frequently observed at higher
workloads and maybe explained by a preferential increase in blood flow (in both
relative and absolute terms) to fibers with higher oxidative capacity. (191, 295,
182). The increased blood flow would enhance the rate of oxidative recovery
metabolism and increase the PCr recovery rate resulting in shorter time constants.
The explanation of the occasionally observed “very short” PCr recovery time
conStant from is problematic but raises an intriguing possibility concerning
glycolYtic regulation. This very fast recovery component (approximately two
seconds) would correspond to an unattainable mitochondrial ATP synthesis rate 5—
15 tiInes greater than the calculated ATP synthesis rate at maximal oxygen
conSurnption in the rat hindlimb (Q02max =66 mM/minute from Table 5 of Ref
(234)). If oxidative metabolism cannot explain this very fast component, the only
Other quantitatively significant and high flux rate source of ATP during the initial
Phage of recovery is anaerobic glycolysis. In effect, the glycolytic flux would exhibit
a “momentum” quality which would carry it into the early phases of recovery. This
couje(rture, of course, runs counter to the conventional wisdom, that states when

St“ .
111111lation stops, glycolytic flux will abruptly stop due to removal of allosteric

123

‘

facilitation of phosphofructokinase and glycogen phosphorylase. Unfortunately,
the spectral time resolution used in this study (see Table 7) was insufficient for us
to conclude that the pH change during the first 40 seconds of recovery was due to
continued lactic acidosis superimposed upon the expected transient acidification
from net PCr resynthesis (4, 167, 276). Consequently, at this time, a cogent
explanation for this very fast component remains elusive.

Lastly, as an independent test of the NN LS algorithm’s ability to decompose PCr
recovery transients, we digitally scanned in the raw data points (SigmaScan 2.0 for
Windows) from a previously published human study by Walter et al. (Figure 3A of
Ref (308)). They reported a monoexponential recovery time constant of
approximately 34 seconds in the medial gastrocnemius after a submaximal
workload (9 seconds of rapid plantar flexions at 30% maximal voluntary
contraction which caused only a modest decrease in ICF pH to 6.8). Applying the
NNLS algorithm to the scanned data points, a fast (22 seconds) and slow (90
seconds) component were resolved (i.e. biexponential recovery) suggesting two
fiber populations with different oxidative capacities were contributing the global
NMR—derived PCr time course. At a longer duration workload which resulted in
Significant intracellular acidosis to pH 6.2 they superimposed a biexponential curve
on their recovery data points but did not report time constants. Applying the NNLS
aIgorithm to their higher workload data confirmed their biexponential curve fit and

shOWed that the two components were prolonged (39 seconds, 130 seconds).

Temporal metabolite patterns during prolonged intermittent exercise

This study documents the temporal pattern of PCr, pH, and isometric force
dul‘ing numerous, prolonged, intermittent stimulation—recovery cycles for
sllbmaximal and supramaximal workloads in the common laboratory rat. This type

0f stimulation protocol more closely mimics the cyclic work-rest locomotion

124

patterns of humans and many animals (123, 255, 283). To the knowledge of the
author, there are only three other studies in the past 30 years of the muscle
energetics literature that have used a remotely similar stimulation—recovery
protocol (virtually all other studies employ a single bout or, more rarely, a second
bout). Curtin et a1. (62) measured heat and 31P—NMR metabolites during nine
cycles of an 18 second isometric tetanus plus two hour recovery in ex vivo dogfish
white muscle. Edwards et al. (84) measured ATP, PCr, and lactate via muscle
biopsy throughout five cycles of 15 second dynamic contractions at 66% maximum
voluntary contraction followed by 20 second recovery in the human quadriceps.
Finally, Marsh et al. (195) measured 31P—NMR metabolites during six cycles of five
minute submaximal dynamic wrist flexions with a five minute recovery in humans.
This group did not record the temporal patterns of pH changes and chose to plot
only the cyclic PCr and Pi data from one of six subjects.

A key finding in the current study is the end—stimulation pH during the first
cycle was significantly lower (approximately 6.6 or 6.15) than the remaining cycles
for the 2.0Hz and 5.0Hz but not 0.75Hz workloads (Figure 15). After the first cycle
01' two, the end—stimulation pH attained essentially same value (approximately
6.95 or 6.8) as cycle number increased. This pattern is similar to the one observed
by Edwards et al. in human quadriceps (84), except they measured lactate, but not
by Curtin et al. (62) in dogfish white muscle. In Curtin’s paper the end—stimulation
PH of the first cycle was higher than the remaining eight cycles. A possible
explanation for this large initial deviation in our pH data is there may be a delay
distributing the blood to the more oxidative fibers (182, 295) thereby requiring
these fibers to use anaerobic glycolysis to a larger extent during the first two
cycles. As the experiment continues, local metabolic factors (e.g. H”, K+, adenosine,
aCetylcholine spill over from synapses, (183) continue to accumulate and open

dormant capillary anastomoses such that subsequent stimulation cycles begin with

125

”I”

n

better perfusion.

Regardless of the precise underlying mechanism of the pH deviation, these
results have two practical implications. First, they indicate the importance of
“warming-up” before engaging in any form of physical activity (especially exercises
requiring only one or two short—duration, high—intensity power bursts) since the
lactic acidosis would reduce the oxidative capacity of the working muscle during the
first few bouts (119, 234). Second, in gated N MR experiments, where signal—to—
noise and time resolution is enhanced by summing spectra acquired at identical
time points during repeated bouts, there is an assumption that the metabolic
events are identical from cycle to cycle. Although this appears to be a reasonable
assumption for submaximal workloads (Figure 8 and Figure 15), it does not appear
to hold for supramaximal workloads (Figure 14 and Figure 15). It would seem

prudent not to include data from at least the first cycle in gated NMR experiments.

Viability of in vivo fiber typing by 3’ P—NMRS
Can phosphocreatine transient analysis estimate skeletal muscle fiber

distributions? From a global functional standpoint, yes, but not at the cellular
level as has been proposed by others. Since recovery PCr time constants can be
used to assess relative oxidative capacity of skeletal muscle (234), the results of
and Figure 23 would suggest applying the unbiased NNLS method to PCA—
cOl'l‘ected 31P—NMR data can potentially resolve at least two fiber type
Populations. A major result of this study, however, is that we could not
unambiguously identify the distribution of distinct fiber populations based solely
uDon a single global NMR derived parameter since essentially equivalent results
are obtained from PCr transients from two diverse, hypothetical, fiber type
distributions (Figure 26). This is at odds with the prevailing tendency in the in vivo

31P—NMRS muscle energetics literature to correlate various NMR parameters with

126

histochemical fiber types usually based upon ATPase staining methods which give
bi— or tri—partitite distributions (217, 308, 304, 236). Although these correlations
are useful first approximations, they tend to grossly oversimplify the diverse
metabolic profiles of mixed skeletal muscle tissue and thereby perpetuate the idea
that only two or three discrete fiber type populations exist (e.g. Type I vrs. II or
Type I vrs. IIA vrs. IIB). This is in spite of the fact that over the past three decades,
invasive biopsy techniques of human and animal muscle have repeatedly provided
profuse evidence that fiber—type distributions represent a continuum of energy-
supply and energy—demand properties (e.g. ATPase activity, oxidative capacity,
glycogen depletion, capillary density, etc.) rather than discrete stereotypes (83, 85,
187, 272, 284, 109, 261, 267). For example, Pette concluded that fiber typing based
upon ATPase activity (i.e. Type I vrs. IIA vrs. IIB) and oxidative capacity (e.g. based
upon succinate dehydrogenase activity) are not necessarily correlated since he
observed considerable overlap of SDH activity between I, IIA, and IIB fibers—and
inter—fiber variability (241). Additionally, Guth has shown a collection of putatively
homogeneous fibers can exhibit different ATPase staining intensities (i.e. an intra—-
fiber variability) since the stain does not distinguish between myofibrillar ATPase,
the SR Ca—ATPase, or the mitochondrial ATPase (113, 114). Furthermore, since
these diverse fibers are arranged in a random spatial distribution within healthy
Ilinscle (152, 160, 284), the biopsy literature suggests completely non—invasive
attempts to identify Type I and 11 fiber populations (an energy demand
Classification scheme) based solely upon an energy supply classification scheme
(e.g. PCr recovery tau, Pi peak splitting) are inherently imprecise and can be
misleading. This is further seriously complicated during in vivo experiments by
Variable inter-subject motor unit recruitment strategies (25, 26, 71, 37) and the
inability to precisely define the muscle volume interrogated by the NMR
transceiver coil (154, 155, 226).

127

On the other hand, comprehensive whole-muscle fiber type classification based
upon invasive biopsies would require reporting several dozen metabolic, cellular,
and functional parameters (e.g. protein isozymes profiles, mitochondrial content,
enzymatic activities, time—to—fatigue, time—to—peak—tension, etc.) from several
different geographic locations within the muscle. Since this would require
numerous tissue samples and hence subject discomfort, it would be valuable to
develop a taxonomy exploiting the non—invasive nature of 31P—NMRS while
attempting to acknowledge the continuum nature of fiber properties.

Although current technological limitations precludes 31P—NMRS from resolving
individual fiber type properties (107), a suggested first step would be to describe
the global functional characteristics of a given volume of muscle by both an
energy—supply parameter and an energy—demand parameter in response to a
standardized workload. As discussed in this dissertation, the inherent high signal—
to—noise of the PCr resonance coupled with the unbiased determination of time
constants by the NNLS algorithm, make analyzing PCr recovery transients a
suitable method for assessing global relative oxidative capacity as the energy-—
supply parameter. ATP cost of contraction determined by the time—zero derivative
of [PCr] at the onset of stimulation (104) or by the slope of [PCr] versus time
during ischemic stimulation (25, 26) could serve as the energy—demand parameter.
(In order to minimize the confounding effects of inter-subject differential motor
unit recruitment and fatigue responses on ATP cost, it is necessary to use brief
bouts of suprathreshold motor unit stimulation by either direct nerve contact in
animal studies or via percutaneous electrodes in human studies.) After measuring
these two non-invasive parameters in different animal models over a wide cross—
section of subjects using standardized workloads, the goal would be to plot
individual relative oxidative capacity versus ATP cost and look for clustering. If

data point clusters did emerge, this would provide a basis for a completely non—

128

invasive, N MR—centric method of “fiber typing” which is relieved of the constraints
imposed by confining global NMR parameters to specific enzymatic profiles
obtained by biopsy. It is certainly acknowledged this proposed method does not
actually “fiber type” in the cellular sense and would require agreement as to what
constitutes a “standard workload”. Its advantage, however, is it attempts to
correlate two key functional parameters during in vivo whole—muscle exercise.
This could provide a framework for systematically quantifying the effects of
various athletic training regimens or neuromuscular pathologies. It should also be
of use to investigators who wish to link the whole—body V02 slow—component
(seen at workloads above the plasma lactic acid threshold) and fiber type
distribution in exercises that activate a large fraction of the total muscle mass
(246).

In summary, although previous 31P—NMRS studies by others have shown the
multicomponent nature of the PCr recovery time course after stimulation is most
easily explained by fiber type heterogeneity, we have not found it possible to
unambiguously identify distinct fiber type population distributions based
exclusively on global NMR—dervived time constants since bimodal and Gaussian
distributions give similar results. This certainly does not preclude the use of NMR

to quantify more global parameters of muscle function.

129

CHAPTER 5
SUMMARY AND RECOMMENDATIONS

S umma ry

Previous comparative and human cross-sectional studies have suggested a link
between PCr kinetics and skeletal muscle oxidative capacity over limited ranges of
mitochondrial content. Data from the first investigation in this dissertation
(Chapter 3) confirms that the PCr recovery rate constant is directly proportional to
oxidative capacity over a 6—fold range of mitochondrial content in mammalian
muscle. The practical application of this result is that relative skeletal muscle
oxidative capacity can be assessed, non—invasely, by analysis of PCr recovery
transients obtained via 31P—N MRS. This data also suggests that submaximal
workloads (i.e. workloads that can be maintained within the oxidative capacity of
the muscle fibers without fatigue or acidosis) should be used since intracellular
acidosis depresses the PCr recovery rate constant thereby confounding its
correlation to relative oxidative capacity. Lastly, these results are consistent with
the linear control model of oxidative phosphorylation by the cytoplasmic free
energy of ATP hydrolysis.

Given the outcome of the first investigation, an attempt was made (Chapter 4) to
Unambiguously identify the distribution of distinct fiber populations by
decomposing the global NMR-derived PCr recovery time constant into its
Constituent components. High signal—to—noise 31P—NMRS data was obtained by
Spectral processing with the Principal Component Analysis algorithm and then
Summing across repeated stimulation—recovery cycles. The PCr recovery time
Constant from the high signal—to—noise dataset was decomposed using the Non-
negative Least Squares (NN LS) algorithm, which eliminates investigator bias by
not specifying the number of components a priori. This extensive data processing

resolved at least two components for both submaximal or supramaximal

130

 

to:

stimulation rates. Furthermore, it was argued that the presence of multiple
components is best explained by fiber type heterogeneity rather than a proton—
induced mass—action shift in the creatine kinase equilibrium reaction, as is
commonly reported. Unfortunately, a modelling analysis comparing a discrete
bimodal fiber type distribution versus a quasi—Gaussian fiber type distribution
showed that similar results are obtained by the N NLS algorithm. This indicates
that unambiguous fiber type determination at the cellular level is not currently
possible with even high signal—to—noise 31P-N MRS data. This conclusion runs
contrary to the tendency in the in vivo N MR literature which suggests that various

steady state and transient parameters can distinguish Type I verus ’lype II skeletal

muscle fibers.

Recommendations for future studies

“As the island of knowledge expands, so does the shore of the unknown.”
Anonymous

Perhaps the most intriguing discovery in this dissertation was identification of a
very short PCr recovery time constant (approximately two seconds) using the
NNLS algorithm (Table 8). Such rapid recovery components have not been
reported in the mammalian muscle literature. A logical, but to date empirically
unsupported, origin of this very fast component would be continued glycolytic flux
into the early stages of recovery. If tenable, then resolving this rapid component is
Of interest to bioenergeticists on two counts. First, it contradicts the prevailing
dOg‘ma which stipulates that significant glycolytic flux occurs only during
Stimulation and then abruptly ceases when stimulation stops. Second, the increase
in intracellular protons from lactic acidosis in early recovery would necessarily
Change the cytosolic redox potential and possibly the redox potential in the
mitochondrial matrix. This in turn could influence the mitochondrial ATP synthesis

rate which is dependent upon the difference between the mitochondrial redox

131

All.

 

potential and the cytosolic AG ATP- Since the spectral time resolution used in this
study was insufficient for us to conclude that the pH change during early recovery
was due to continued glycolytic flux, it is incumbent to increase the time resolution
to approximately one second/spectrum without sacrificing signal—to—noise by using
gated 31P—N MR experiments. If better time resolution suggests that the pH
changes during early recovery are consistent with continued glycolytic flux, then
corroboration from analyzing intracellular lactate kinetics would be required. This
could be accomplished with 13C—NMRS measurements of 13C—labeled glucose—6—
phosphate.

Another interesting finding in this dissertation was the identification of an
increasing trend in end—stimulaiton pH as a function of cycle number after
supramaximal workloads (Figure 15). It was postulated that increased blood flow
during the later cycles was responsible for the attenuation of end—stimulation pH.
This attenuation could be experimentally manipulated through the use of locally
injected nitric oxide agonists or antagonists, which would vasodilate or vasoconstrict
the vascular smooth muscle, respectively. If this pharmacological treatment alters
the pattern of end—stimulation pH as a function of cycle number, this would
support the hypothesis that blood flow (hence convective 02 delivery) plays an
important role in determining the skeletal muscle’s redox state and dependence on
anaerobic metabolism. Also, using the repeated stimulation—recovery protocol
described in Chapter 4, the effects of aerobic training, which would presumably
increase 02 extraction capacity by increasing mitochondrial content, could be
investigated. Aerobic training would be expected to attenuate the drop in end—
stimulaiton pH during the first three cycles of supramaximal stimulation.

It would also be of interest to rigorously define the three—dimensional B1 field
pattern from the saddle—shaped surface coil used in Chapter 4. This would require

using antenna theory along with computer numerical methods due to the complex

132

geometry of the transceiver coil. Once defined, the B1 field pattern would be used
in a computer simulation to interrogate a hypothetical, heterogenous muscle. The
purpose would be to catalog the effects of different spatial distributions of fiber

types on the transient and steady—state behavior of the global, NMR—derived, PCr

time constant.

133

APPENDICES

134

Appendix A

Figure 27: 31P-NMR Probe Photographs

 

 

 
 

/I/n/.. I 7" N: «1 7
Plexiglas cradle with brass sheath (top), brass base with instrument connectors
(left) and brass adjustable force transducer mount (right).
BOTTOM: Close up of box with LC—tank circuit with saddle shaped transceiver coil,
adjustable force transducer mount and rigid brass posts to secure femur head.

 

TOP:

135

Appendix B
Figure 28 & Table 9: Leg cross-section

l.— :1'2 cm __.I anterior ,

T

     
     
  

proximal

posterior

Tibia

Fibula

Tibialis Anterior—White
Tibialis Anterior—Red
Ext. Digitorum Longus
Peroneals

Flexor Hallicus Longus
Tibialis Posterior
Flex. Digitorum Longus
Soleus

Plantaris

10 Gastrocnemius—Red

11 Gastrocnemius—Mixed

12 Gastrocnemius—White

 

 

Wtb~lmlnfinwl0F‘Nii

1 2.5 cm

approximate
interrogation
volume

 

6
Z
fi-—flf1

 

 

 

 

4h
’fl Frequency Tuning
& Impedance

Radiorre enc Transceiver Coil . . .
qu y Matching Circuit

 

 

Posterior Leg Fiber FOG FOG FG % FG SO % SO
Muscle mass % mass mass mass
(mg) (n_1_g) (mg) (mg)
Plantaris 368 50 155 41 206 9 25
Gastroc—Red 135 62 76 8 12 30 47
Gastroc-White 116 16 10 84 106 0 0
Gastroc—Mixed 1764 28 310 65 1372 7 82

 

Above table is an excerpt of Table 2 from Ref. (8) illustrating the inter—muscle
and intra—muscle fiber type heterogeneity of the posterior rat leg. Total mass of rat
1:18 = 5500mg. Only mean values are shown. SEM is typically 10—15% of mean

=6).

136

flit

It II I. IEI‘E‘III

 

iv

)-
.

 

Appendix C
Figure 29: Temperature Homeostasis

+ Rectal

—0— Surface of gastrocnemius
”811818314 .
-----o- Air-Magnet Bore

Intra—gastrocnemius

'C

Mean Temperature

 

 

 

hours

In this supplemental experiment, four rats of similar body mass to the main
experiments (300—400g) underwent the identical surgical protocol as previously
described in Chapter 4—Methods except that the bipolar electrode was not placed
across the nerve. Since the nerve was not directly stimulated, force was not
recorded. 31P—NMR spectroscopy was continuously acquired (TR = 1.00 second, NS
= 16) but not saved. In addition to the usual rectal temperature probe (YSI model
402A), a “banjo” temperature probe (YSI model 408) was secured underneath the
Skin directly on the epimysium of the gastrocnemius belly and a fine—tip
temperature probe (YSI model 520) was bored =3—5mm into the gastrocnemius
belly. Also, the ambient magnet bore air temperature was measured on the medial
side of the hindlimb with a YSI probe model 405. The temperature controller used
feedback from the rectal probe. Each probe underwent a separate 5—point
Salibration (310°, 345°, 355°, 370°, 400“) against an NIST certified partial
mlmersion thermometer (OMEGA model ASTM—lC —20°— +150°C). in a
temperature regulated water bath. Probe temperature (101°C) was digitally
reCorded every 30 minutes for 10 hours. (Cole-Palmer temperature recorder model
8402—10). Note, for reference purposes, the duration of a single stimulation—
n9(30very cycle is 11.4, 21.3, and 32.0 minutes for 0.75Hz, 2.0Hz and 5.0Hz
respectively.

137

Appendix D
Figure 30: N versus Cycle Number

7
35
£5

3

0.7 5Hz

.5

Number of
H N

O
l 3 5 7 9 11 13 15 17 19 21 23 25
9
8
7
n
+36
35
'H
04
3
2
g, 2.0Hz
0 . |

1 3 5 7 9 11 13 15 17 19 21 23 25

 

Number of Rats

 

138

Appendix E
Figure 31: Methimazole Inhibition

 

 

l -
[meow/T3 "TBS + T4/T3 p asma I
ISF
, I'-ATPase
. 9" um
T4 + T3 thyr01d "‘1 p p
follicular
cell 1'

H202
T peroxidase
T4‘-. ’I'TB

:0 H206 WI

 

 

T9
iodination of
tyrosines on
IflMI
colloid
space
. coupling of
Thyroglobulln (Tg) iodotyrosine
I ! monomers on
DI;| DIT D11l M T T9
+ +
T4 T3

Dietary iodide is actively taken up by the thyroid follicular cell from the plasma
via an ATPase pump and oxidized into iodine by the peroxidase enzyme found in
the cytosol. One or two iodine atoms are then incorporated into tyrosine amino
acids on the thyroglobulin molecule forming monoiodotyrosine or diiodotyrosine,
respectively. Both the oxidation step and iodine incorporation step are blocked by
methimazole. In the normally functioning thyroid follicular cell, iodine is added to
the phenolic group of tyrosine molecules bound to thyroglobulin (Tg) forming
monoiodotyrosine (MIT) or diiodotyrosine (DIT) monomers. The coupling of MITs
and DITs forms thyroid hormone (MIT+DIT=T3, triiodotyrosine; DIT+DIT=T4,
tetraiodotyrosine) and takes place within the colloid space on the thyroglobulin
molecule. The thyroid cells export mostly T4 and a little T3 both of which bind to
Thyroid Binding Globulin (TBG) in the plasma. T4 is converted into the more

biologically active form of T3 by deiodinases located within the interstitial fluid of
muscle and most other tissues.

139

Appendix F

Figure 32: Intensity-Duration Protocol

 

 

 

 

 

 

 

 

42
3’
ff
2’1
3 37-
g E
\ p.
H
5 a:
>1 5
u
.1 1- i A z:-
a
'5 mm ' , - g
g 1mm 5 5 -20
u 27-_ ’ i A
o I-
g I Expected Velocity 10 E
1.
D Total Work Time _ E
22 I I I T I o

 

l r I l
0 5 10 15 20 25 30 35 40 45 50
Training Day

The above is a graphical representation of the controlled running wheel
intensity—duration parameters used over the 10—week interval training period.
These parameters nearly doubled the mitochondrial content in the superficial
white gastrocnemius of the rat based upon citrate synthase activity. Modified from
Hickson et al. (127). Rats running at 37m/min roughly corresponds to an intensity
of 85-90% V021,”x (270).

140

Appendix G

Figure 33: Training Compliance
19o -

180-1
170-:Tl'rT —e—- 96 Shock Free Time/day

—o— 95 Expected Revolutions/day

Percent

 

 

O 5 10 15 20 25 30 35 4O 45 50
Training Day

All values are mean1SEM (N=12). Graph indicates, as a group, the rats met or
greatly exceeded the expected number of wheel revolutions per day for virtually
the entire 10 week protocol. The training intensity and/or duration was
progressively increased throughout the 10 week regimen as shown in Appendix F.
The apparatus also recorded the amount of time per animal a mild constant
current (z 0.5mAmp) was applied to the aluminum rungs of the running wheel to
motivate the rat to run. This allowed calculation of % shock free time per day. The
dip in compliance seen between days 30—36 correlates with the larger jump in
training duration seen in Appendix F. Rats were not trained within 24 hours of
start of their NMR experiment.

141

(grams)

Body Mass

(ml/rat)

Mean Water consumed

Appendix H

Figure 34: Methimazole Effects

450 -

425 -

400 d

375 -

350

325 -

300

Methimazole Effect
on Body Mass

   
  

 

 

O
325 -

300-
275-
250-
225-
2004
175-
150-

 

125

I T I I I I I

I
123456789

Methimazole Effect
on 1120 Consumption

 

 

 

—Cl— Control-MMI-Free Fed

 

(nu-18)

+ MMI (n-18)

—-O— Control-MMI (nu-15)

Points are means1SEM: TOP GRAPH, All rats from each group where weighed
every Monday and Thursday within six hours of the start of their nocturnal cycle.
The weekly body mass for one rat was taken as the average of the two values
recorded and was averaged with others in the group. Rats in the Control-MMI
group were limited to 35 g/day/cage of Purina Rat Chow to mirror the decline in
bOdy mass of the MMI rats. BOTTOM GRAPH, Total weekly water consumption
for each cage was measured on Thursdays based upon the difference method using
a graduated cylinder. This number was divided by the number of rats per cage and
averaged with others. Square symbols in bottom graph represent all rats from
both Control groups (n=33).

142

LIST OF REFERENCES

143

LIST OF REFERENCES

1. Achten, E., M. VanCauteren, R. Willem, R. Luypaert, W. J. Malaisse, G.
VanBosch, G. Delanghe, K. DeMeirleir, and M. Osteaux. 31P—NMR
spectroscopy and the metabolic properties of different muscle fibers. Journal of
Applied Physiology 68(2): 644—649, 1990.

2. Ackerman, J. J. H. Surface (local) coils as NMR receivers. Concepts in Magnetic
Resonance 2: 33—42, 1990.

3. Ackerman, J. J. H., P. J. Bore, D. G. Gadian, T. H. Grove, and G. K. Radda.
N.m.r. studies of metabolism in perfused organs. Phil. Transactions of the Royal
Society of London B 289: 425-436, 1980.

4. Adams, G. R., J. M. Foley, and R. A. Meyer. Muscle buffer capacity estimated
from pH changes during rest-to—work transitions. Journal of Applied Physiology
69(3): 968-972, 1990.

5. Andrew, E. R., Introduction to nuclear magnetic resonance. In: NMR in
Physiology and Biomedicine, edited by R. J. Gillies, Orlando, 1994, pp. 1—23.

6. Argov, Z., N. DeStefano, and D. L. Arnold. ADP recovery after a brief
ischemic exercise in normal and diseased human muscle—a 31P MRS study. NMR in
Biomedicine 9(4): 165-172, 1996.

7. Argov, Z., J. Maris, L. Damico, M. Koruda, Z. Roth, J. S. Leigh, and B.
Chance. Continuous, graded steady—state muscle work in rats studied by in vivo
SIP-NMR. Journal of Applied Physiology 63(4): 1428—1433, 1987.

8. Armstrong, R. B., and R. O. Phelps. Muscle fiber type composition of the rat
hindlimb. American Journal of Anatomy 171: 259—272, 1984.

9. Arnold, D. L., P. M. Matthews, and G. K. Radda. Metabolic recovery after
exercise and the assessment of mitochondrial function in vivo in human Skeletal
muscle by means of 31P N MR. Magnetic Resonance in Medicine 1: 307—315, 1984.

10. Baldwin, K. M., G. H. Klinkerfuss, R. L. Terjung, P. A. Mole, and J. O.
Holloszy. Respiratory capacity of white, red, and intermediate muscle: adaptive
response to exercise. American Journal of Physiology 222(2): 373—378, 1972.

11. Baldwin, K. M., and C. M. Tipton. Work and metabolic patterns of fast and
slow twitch skeletal muscle contracting in situ. European Journal of Physiology
334: 345—356, 1972.

12. Baldwin, K. M., W. W. Winder, R. L. Terjung, and J. O. Holloszy.

Glycolytic enzymes in different types of skeletal muscle: adaptation to exercise.
American Journal of Physiology 225(4): 962-966, 1973.

144

 
 
 

13. Bar, A., J.-A. Simoneau, and D. Pette. Altered expression of myosin light—
chain isoforms in chronically stimulated fast—twitch muscle of the rat. European
Journal of Biochemistry 178(3): 591-594, 1989.

14. Barker, R. D. J., T. G. Cartledge, F. Dewhurst, and R. O. Jenkins,
Principles of Cell Energetics. edited by G. D. Weston, Biotechnology by Open
Learning, Butterworth—Heinemann, Oxford, 1992.

15. Barnard, R. J., V. R. Edgerton, T. Furukawa, and J. B. Peter.
Histochemical, biochemical, and contractile properties of red, white, and
intermediate fibers. American Journal of Physiology 220(2): 410—414, 1971.

16. Barnard, R. J., V. R. Edgerton, and J. B. Peter. Effect of exercise on
Skeletal muscle 11. Contractile properties. Journal of Applied Physiology 28(6):
767—770, 1970.

17. Barnard, R. J., V. R. Edgerton, and J. B. Peter. Effect of exercise on
skeletal muscle I. Biochemical and histochemical properties. Journal of Applied
Physiology 28(6): 762—766, 1970.

18. Barstow, T. J., A. M. Jones, P. H. Nguyen, and R. Casaburi. Influence of
muscle fiber type and pedal frequency on oxygen uptake kinetics of heavy
exercise. Journal of Applied Physiology 81(4): 1642—1650, 1996.

19. Bassett, D. R., and E. T. Howley. Maximal oxygen uptake: "classical"
versus "contemporary" vieWpoints. Medicine & Science in Sports & Exercise 29(5):
591—603, 1997.

20. Beard, H. H., Creatine and Creatinine Metabolism. Chemical Publishing Co.,
Brooklyn, 1943.

21. Bendahan, D., S. Confort-Gouny, G. Kozak—Reiss, and P. J. Cozzone.
Heterogeneity of metabolic response to muscular exercise in humans: New criteria
of invariance defined by in vivo phosphorus—31 NMR spectroscopy. FEBS Letters
272(1—2): 155—158, 1990.

22. Bergmeyer, H. U., Ed, Metabolites 2: Tri- and Dicarboxylic Acids, Purines,
Pyrimidines and Derivatives, Coenzymes, Inorganics, In: Methods of Enzymatic
Analysis, Vol. VII, VCH, Weinheim, 1985.

23. Binzoni, T., and P. Cerretelli. Bioenergetic approach to transfer function
of human skeletal muscle. Journal of Applied Physiology 77(4): 1784—1789, 1994.

24. Binzoni, T., G. Ferretti, K. Schenker, and P. Ceretelli. Phosphocreatine

hydrolysis by 31P—NMR at the onset of constant—load exercise in humans. Journal
of Applied Physiology 73(4): 1644—1649, 1992.

145

i“

0.!

al-.

w...

it‘-

25. Blei, M. L., K. E. Conley, and M. J. Kushmerick. Separate measures of
ATP utilization and recovery in human skeletal muscle. Journal of Physiology 465:
203—222, 1993.

26. Blei, M. L., K. E. Conley, I. R. Odderson, P. C. Esselman, and M. J.
Kushmerick. Individual variation in contractile cost and recovery in a human

skeletal muscle. Proceedings of the National Academy of Science USA 90(August):
7396-7400, 1993.

27. Bloch, F. Nuclear induction. Physical Review 70(7, 8): 460—474, 1946.

28. Bodner, G. M. Metabolism Part II: The Tricarboxylic Acid (TCA), Citric Acid,
or Krebs Cycle. Journal of Chemical Education 63(8): 673-677, 1986.

29. Boicelli, C. A., A. M. Baldassarri, C. Borsetto, and F. Conconi. An
approach to noninvasive fiber type determination by NMR. International Journal
of Sports Medicine 10: 53—54, 1989.

30. Booth, F. W., and D. B. Thomason. Molecular and cellular adaptations of
muscle in response to exercise: Perspectives of various models. Physiological
Reviews 71(2): 541—585, 1991.

31. Bracewell, R. N., The Fourier Transform and Its Application. 2nd, Revised
Ed., edited by S. W. Director, Circuits and Systems, McGraw—Hill, New York, 1986.

32. Brooke, M. H., and K. K. Kaiser. Muscle fiber types: How many and what
kind? Archives of Neurology 23(October): 369—379, 1970.

33. Brown, T., and R. Stoyanova. NMR spectral quantitation by principal—
component analysis. II Determination of frequency and phase shifts. Journal of
Magnetic Resonance Series B 112: 32—43, 1996.

34. Buchner, E. Alkoholische gahrung ohne hefezellen. Berlin d. Deutsch
Chem. Gesellsch 30: 117-124, 1897.

35. Buller, A. J., J. C. Eccles, and R. M. Eccles. Interactions between
motoneurones and muscles in respect of the characteristic speeds of their
responses. Journal of Physiology 150: 417—439, 1960.

36. Burke, R. E. Motor unit types of cat triceps surae muscle. Journal of
Physiology 193: 141—160, 1967.

37. Burke, R. E., Motor units: anatomy, physiology, and functional organization.
In: Handbook of Physiology, Section 1, The Nervous System, edited by V. B. Brooks,
Bethesda, American Physiological Society, 1981, Vol. 2 (part 1), pp. 345—422.

146

 
 
 
 
 
 

38. Burke, R. E., D. N. Levine, F. E. Zajac, P. Tsairis, and W. K. Engel.
Mammalian motor units: Physiological-histochemical correlation in three types in
cat gastrocnemius. Science 174(November 12): 709—712, 1971.

39. Burt, C. T., T. Glonek, and M. Barany. Analysis of phosphate metabolites,
the intracellular pH, and the state of adenosine triphosphate in intact muscle by

phosphorous nuclear magnetic resonance. Journal of Biological Chemistry 251(9):
2584-2591, 1976.

40. Cain, D. F., and R. E. Davies. Breakdown of adenosine triphosphate during
a single contraction of working muscle. Biochemical and Biophysical Research
Communications 8(5): 361-366, 1962.

41. Caiozzo, V. J., and F. Haddad. Thyroid hormone: Modulation of muscle,

structure, function, and adaptive responses to mechanical loading. Exercise Science
and Sports Review 24: 321-361, 1996.

42. Canfield, P., and G. Marechal. Equilibrium of nucleotides in frog sartorius
muscle during an isometric tetanus at 20°C. Journal of Physiology (London) 232:
453-466, 1973.

43. Carlson, F. D., D. Hardy, and D. R. Wilkie. The relation between heat
produced and phosphorylcreatine split during isometric contraction of frog's
muscle. Journal of Physiology 189: 209—235, 1967.

44. Carlson, F. D., D. J. Hardy, and D. R. Wilkie. Total energy production and

phosphocreatine hydrolysis in the isotonic twitch. Journal of General Physiology
46: 851—882, 1963.

45. Carlson, F. D., and A. Siger. The mechanochemistry of muscular
contraction I. The isometric twitch. Journal of General Physiology 44: 33-60, 1960.

46. Challiss, R. A. J., M. J. Blackledge, and G. R. Radda. Spatial
heterogeneity of metabolism in Skeletal muscle in vivo studies by 31P—NMR

spectroscopy. American Journal of Physiology (Cell Physiology) 254(23): C417-
C422, 1988.

47. Chanaud, C. M., C. A. Pratt, and G. E. Loeb. Functionally complex
muscles of the cat hindlimb: V. The roles of histochemical fiber—type
regionalization and mechanical heterogeneity in differential muscle activation.
Experimental Brain Research 85: 300—313, 1991.

48. Chance, B., S. Eleff, J. S. Leigh, D. Sokolow, and A. Sapega.
Mitochondrial regulation of phosphocreatine/inorganic phospate ratios in
exercising human muscle: A gated 31P NMR study. Proceedings of the National
Academy ofScience USA 78(11): 6714-6718, 1981.

147

Vt."

i

 

 

I/f

49. Chance, B., J. S. Leigh, B. J. Clark, J. Maris, J. Kemt, S. N ioka, and D.
Smith. Control of oxidative metabolism and oxygen delivery in human skeletal
muscle: A steady—state analysis of the work/energy cost transfer function.
Proceedings of the National Academy of Sciences USA 82: 8384-8388, 1985.

50. Chance, B., J. S. Leigh, J. Kent, and K. McCully. Metabolic control
principles and 31P—N MR. Federation Proceedings 45: 2915—2920, 1986.

51. Chance, B., J. S. Leigh, J. Kent, K. McCully, S. Nioka, B. J. Clark, J. M.
Maris, and T. Graham. Multiple controls of oxidative metabolism in living
tissues as studied by phosphorous magnetic resonance. Proceedings of the National
Academy of Science USA 83: 9458—9462, 1986.

52. Chance, B., and G. R. Williams. Respiratory enzymes in oxidative
phosphorylation. Journal of Biological Chemistry 217: 395-451, 1955.

53. Chance, B., and G. R. Williams. The respiratory chain and oxidative
phosphorylation. Advances in Enzymology 17: 65-134, 1956.

54. Chasiotis, D., K. Sahlin, and E. Hultman. Regulation of glycogenolysis in
human muscle at rest and during exercise. Journal of Applied Physiology
(Respiratory, Environmental, Exercise Physiology) 53(3): 708-715, 1982.

55. Close, R. 1. Dynamic properties of mammalian Skeletal muscle.
Physiological Reviews 52(1): 129-197, 1972.

56. Coggan, A. R., R. J. Spina, and D. S. King. Histochemical and enzymatic
comparison of the gastrocnemius muscle of young and elderly men and women.
Journal of Gerontology: Biological Sciences 47(3): 371—376, 1992.

57. Connett, R. J. Glycolytic regulation during an aerobic rest—to—work

transition in dog gracilis muscle. Journal of Applied Physiology 63(6): 2366—2374,
1987.

58. Connett, R. J., and K. Sahlin, Control of glycolysis and glycogen
metabolism. In: Handbook of Physiology Section 12: Exercise: Regulation and
Integration of Multiple Systems, edited by L. B. Rowell, and J. T. Shepherd, New
York, Oxford University Press, 1996, pp. 870—911.

59. Cooley, J. W., and J. W. Tukey. An algorithm for the machine calculation of
complex Fourier series. Mathematics of Computation 19(April): 297-301, 1965.

60. Cooper, D. S., D. Kieffer, V. Saxe, H. Mover, F. Maloof', and E. C.

Ridgway. Methimazole pharmacology in the rat: studies using a newly developed
radioimmunoassay for methimazole. Endocrinology 114(3): 786—793, 1984.

148

61. Crow, M. T., and M. J. Kushmerick. Chemical energetics of slow— and
fast—twitch muscles of the mouse. Journal of General Physiology 79: 147—166,
1982.

62. Curtin, N. A., M. J. Kushmerick, R. W. Wiseman, and R. C. Woledge.
Recovery after contraction of white muscle fibres from the dogfish Scyliorhinus
canicula. Journal of Experimental Biology 200: 1061—1071, 1997.

63. Curtin, N. A., and R. C. Woledge. Energy changes and muscular
contraction. Physiological Reviews 58(3): 690—761, 1978.

64. Dainty, M., A. Kleinzeller, A. S. C. Lawrence, M. Miall, J. Needham, D.
M. Needham, and S.-C. Shen. Studies on the anomalous viscosity and flow—
birefringence of protein solutions. Journal of General Physiology 27 : 355—399,
1944.

65. Davies, K. J. A., L. Packer, and G. A. Brooks. Biochemical adaptation of
mitochondria, muscle, and whole-animal respiration to endurance training.
Archives of Biochemistry and Biophysics 209(2): 539+554, 1981.

66. Dawson, M. J ., D. G. Gadian, and D. R. Wilkie. Contraction and recovery
of living muscles studied by 31P nuclear magnetic resonance. Journal of
Physiology 267: 703—735, 1977.

67. Dawson, M. J., D. G. Gadian, and D. R. Wilkie. Muscle fatigue
investigated by phosphorus nuclear magnetic resonance. Nature 274(August 31):
861—866, 1978.

68. Dawson, M. J ., D. G. Gadian, and D. R. Wilkie. Studies of the
biochemistry of contracting and relaxing muscle by the use of 31P n.m.r. in
conjunction with other techniques. Philosophical Transactions of the Royal Society
of London B 289: 445—455, 1980.

69. Defuria, R. R., and M. J. Kushmerick. ATP utilization associated with
recovery metabolism in anaerobic frog muscle. American Journal of Physiology
(Cell Physiology) 232(1): C30—C36, 1977.

70. DeRuiter, C. J., A. deHaan, and A. J. Sargeant. Repeated force production
and metabolites in two medial gastrocnemius muscle compartments of the rat.
Journal of Applied Physiology 79(6): 1855—1861, 1995.

71. DeRuiter, C. J., P. E. M. H. Habets, A. DeHaan, and A. J. Sargeant. In
vivo ID( and IIB fiber recruitment in gastrocnemius muscle of the rat is
compartment related. Journal of Applied Physiology 81(2): 933—942, 1996.

72. Diehl, P., E. Fluck, H. Gunther, R. Kosfeld, and J. Seelig, Eds., In: In-

Vivo Magnetic Resonance Spectroscopy III: In-Vivo MR Spectroscopy-Potential and
Limitations, Vol. 28, Springer—Verlag, Berlin, 1992.

149

73. DiPrampero, P. E., and R. Margaria. Relationship between 02
consumption, high energy phosphates and the kinetics of the 02 debt in exercise.
European Journal of Physiology 304: 11—19, 1968.

74. DiPrampero, P. E., and R. Margaria. Mechanical efficiency of phosphagen
(ATP + CP) splitting and its speed of resynthesis. European Journal of Physiology
308: 197—202, 1969.

75. Druyan, R., B. DeBernard, and M. Rabinowitz. Turnover of cytochromes

labeled with y—aminolevulinic acid—3H in rat liver. Journal of Biological Chemistry
244(21): 5874-5878, 1969.

76. Dubowitz, V., and A. B. Everson—Pearse. A comparative histochemical
study of oxidative enzyme and phosphorylase activity in skeletal muscle.
Histochemie 2: 105-117, 1960.

77. Dudley, G. A., W. M. Abraham, and R. L. Terjung. Influence of exercise
intensity and duration on biochemical adaptations in skeletal muscle. Journal of

Applied Physiology (Respiratory, Environmental, Exercise Physiology) 53(4): 844—
850, 1982.

78. Dudley, G. A., P. C. Tullson, and R. L. Terjung. Influence of
mitochondrial content on the sensitivity of respiratory control. Journal of
Biological Chemistry 262(19): 9109—9114, 1987.

79. Ebashi, S. Calcium binding activity of vesiculare relaxing factor. The Journal
of Biochemistry 50(3): 236—244, 1961.

80. Edgerton, V. R., Ph.D. Thesis, Histochemical changes in rat skeletal muscle
after exercise, Michigan State University, 1968.

81. Edgerton, V. R. Mammalian muscle fiber types and their adaptability.
American Zoologist 18: 113—125, 1978.

82. Edgerton, V. R., L. Gerchman, and R. Carrow. Histochemical changes in
rat skeletal muscle after exercise. Experimental Neurology 24: 110—123, 1969.

83. Edgerton, V. R., J. L. Smith, and D. R. Simpson. Muscle fibre type
populations of human leg muscles. Histochemical Journal 7(3): 259—266, 1975.

84. Edwards, R. H. T., R. C. Harris, E. Hultman, and L.-O. Nordesjo.
Phosphagen utilization and resynthesis in successive isometric contractions,

sustained to fatigue, of the quadriceps muscle in man. Journal of Physiology
(London) 224(1): 40P—41P, 1972.

85. Elder, G. C. B., K. Bradbury, and R. Roberts. Variability of fiber type

distributions within human muscles. Journal of Applied Physiology (Respiratory,
Environmental, Exercise Physiology) 53(6): 1473—1480, 1982.

150

86. Embden, G., and E. Grafe. Uber den einflub der muskelarbeit auf die
phosphorsaureausscheidung. Zeitschrift f Physiological Chemistry 113: 108-137,
1921.

87. Engelhardt, W. A., and M. N. Idubimowa. Myosine and
adenosinetriphosphatase. Nature 144: 668-669, 1939.

88. Engelman, T. W. On the nature of muscular contraction. Proceedings of the
Royal Society of London Serial B 411: 411-435, 1895.

89. English, A. E., M. L. G. Joy, and R. M. Henkelman. Pulsed NMR
relaxometry of striated muscle fibers. Magnetic Resonance in Medicine 21: 264—281,
1991.

90. English, A. W., S. L. Wolf, and R. L. Segal. Compartmentalization of
muscle and their motor nuclei: The partitioning hypothesis. Physical Therapy 73:
857-867, 1993.

91. Ennor, A. H., and J. F. Morrison. Biochemistry of the phosphagens and
related guanidines. Physiological Reviews 38(October): 631—674, 1958.

92. Farrar, T. C., and E. D. Becker, Pulse and Fourier Transform NMR:
Introduction to Theory and Methods. Academic Press, New York, 1971.

93. Fenn, W. O. The relation between the work performed and the energy
liberated in muscular contraction. Journal of Physiology 58: 373-395, 1923.

94. Fiske, C. H., and Y. Subbarow. The nature of the inorganic phosphate in
voluntary muscle. Science 65(1686): 401-403, 1927.

95. Fiske, C. H., and Y. Subbarow. The isolation and function of
phosphocreatine. Science 67(Feb. 10): 169—170, 1928.

96. Fiske, C. J., and Y. Subbarow. Phosphocreatine. Journal of Biological
Chemistry 81: 629—679, 1929.

97. Fitts, R. H., F. W. Booth, W. W. Winder, and J. O. Holloszy. Skeletal
muscle respiratory capacity, endurance, and glycogen utilization. American

Journal of Physiology 228(4): 1029—1033, 1975.

98. Fitts, R. H., and J. J. Widrick. Muscle mechanics: Adaptations with
exercise—training. Medicine and Science in Sports and Exercise 24: 427—473, 1996.

99. Fletcher, W. M., and F. G. Hopkins. Lactic acid in amphibian muscle.
Journal of Physiology 35: 247—309, 1907.

151

100. Fletcher, W. M., and F. G. Hopkins. The respiratory process in muscle and
the nature of muscular motion. Proceedings of the Royal Society of London Serial
B 89: 444—467, 1917.

101. Flinn, R. A., and P. K. Trojan, Engineering Materials and Their
Applications. 3rd Ed, Houghton Mifflin Co., Boston, 1986.

102. Foley, J. M., G. R. Adams, and R. A. Meyer. Different effects of gradual vs.
acute adenine nucleotide depletion on ATP cost of muscle contraction. American
Journal of Physiology (Cell Physiology) 267(36): C1177—C1184, 1994.

103. Foley, J. M., S. J. Harkema, and R. A. Meyer. Decreased ATP cost of
isometric contractions in ATP—depleted rat fast-twitch muscle. American Journal
of Physiology (Cell Physiology) 261(30): C872—C881, 1991.

104. Foley, J. M., and R. A. Meyer. Energy cost of twitch and tetanic contractions
of rat muscle estimated in situ by gated 31P—NMR. NMR in Biomedicine 6: 32—38,
1993.

105. Fruton, J. S., Molecules and Life: Historical Essays on the Interplay of
Chemistry and Biology. Wiley—Interscience, New York, 1972.

106. Funk, C. I., A. J. Clark, and R. J. Connett. A simple model of aerobic
metabolism: applications to work transitions in muscle. American Journal of
Physiology (Cell Physiology) 258(27): C995—01005, 1990.

107. Gadian, D. G., NMR and its application to living systems. 2nd Ed., Oxford
University Press, Oxford, 1995.

108. Gillies, R. J., K. Ugurbil, J. A. Den_Hollander, and R. B. Shulman. 31P
NMR studies of intracellular pH and phosphate metabolism during cell division
cycle of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences
USA 78(4): 2125—2129, 1981.

109. Gollnick, P. D., R. B. Armstrong, C. W. Saubert, K. Piehl, and B.
Saltin. Enzyme activity and fiber composition in skeletal muscle of untrained and
trained men. Journal of Applied Physiology 33(3): 312—319, 1972.

110. Gordon, T., and M. C. Pattullo. Plasticity of muscle fiber and motor units
types. Exercise Science and Sports Review 21: 331—362, 1993.

111. Gower, D., and K. M. Kretzschmar. Heat production and chemical change

during isometric contraction of rat soleus muscle. Journal of Physiology 258: 659—
671, 1976.

112. Graham, S. J ., P. L. Stanchev, and M. J. Bronskill. Criteria for analysis

Of multicomponent tissue T2 relaxation data. Magnetic Resonance in Medicine 35:
370~378, 1996.

152

113. Guth, L. Fact and artifact in the histochemical procedure for myofibrillar
ATPase. Experimental Neurology 41: 440—450, 1973.

114. Guth, L., and H. Yellin. The dynamic nature of the so—called "fiber types"
of mammalian skeletal muscle. Experimental Neurology 31: 277—300, 1971.

115. Gyulai, L., Z. Roth, J. S. Leigh, and B. Chance. Bioenergetic studies of
mitochondrial oxidative phosphorylation using 31phosphorus N MR. Journal of
Biological Chemistry 260: 3947—3953, 1985.

116. Hames, B. D., Gel Electrophoresis of Proteins. 2nd Ed., edited by D.
Rickwood, and B. D. Hames, The Practical Approach Series, IRL Press, Oxford,
1990.

117. Harden, A., and W. J. Young. The alcoholic ferment of yeast—juice.
Proceedings of the Royal Society of London Serial B 77: 405—420, 1906.

118. Harkema, S. J., G. R. Adams, and R. A. Meyer. Acidosis has no effect on
the ATP cost of contractions in cat fast— and slow—twitch skeletal muscles.
American Journal of Physiology (Cell Physiology) 272(41): C485—C490, 1997.

119. Harkema, S. J., and R. A. Meyer. Effects of acidosis on control of
respiration in skeletal muscle. American Journal of Physiology (Cell Physiology)
272(41): C491-C500, 1997.

120. Harris, R. C., R. H. T. Edwards, E. Hultman, L.-O. Nordesjo, B.
Nylind, and K. Sahlin. The time course of phosphorylcreatine resynthesis
during recovery of the quadriceps muscle in man. European Journal of Physiology
367: 137-142, 1976.

121. Hartner, K.-T., and D. Pette. Fast and slow isoforms of troponin I and
troponin C: Distribution in normal rabbit muscles and effects of chronic
stimulation. European Journal of Biochemistry 188: 261—267, 1990.

122. Haynes, R. C., Thyroid and antithyroid drugs. In: Goodman and Gilman's
The Pharmacological Basis of Therapeutics, (8th Ed.), edited by A. Goodman—

Gilman, T. W. Rall, A. S. Nies, and P. Taylor, New York, Pergamon Press, 1990, pp.
1373—1376.

123. Heckman, C. J ., and T. G. Sandercock. From motor unit to whole muscle
properties during locomotor movements. Exercise Science and Sports Review 24:
109—133, 1996.

124. Heineman, F. W., J. Eng, B. A. Berkowitz, and R. S. Balaban. NMR

Spectral analysis of kinetic data using natural lineshapes. Magnetic Resonance in
Medicine 13: 490-497, 1990.

153

125. Henneman, E., and C. B. Olson. Relations between structure and function
in the design of skeletal muscles. Journal of Neurophysiology 28: 581—598, 1965.

126. Hermann, L. Beitrage zur physiologie und physik des nerven. European
Journal of Physiology 109: 95—144, 1905.

127. Hickson, R. Skeletal muscle cytochrome c and myoglobin, endurance, and
frequency of training. Journal of Applied Physiology (Respiratory, Environmental,
Exercise Physiology) 51(3): 746—749, 1981.

128. Hill, A. V. The energy degraded in the recovery processes of stimulated
muscles. Journal of Physiology 46: 28—80, 1913.

129. Hill, A. V. Thermodynamics in physiology. Nature 113(2850): 859—862, 1924.

130. Hill, A. V. The recovery heat—production in oxygen after a series of muscle
twitches. Proceedings of the Royal Society of London Serial B 103: 183—191, 1928.

131. Hill, A. V. The revolution in muscle physiology. Physiological Reviews 12:
56—67, 1932.

132. Hill, A. V. The mechanical efficiency of frog's muscle. Proceedings of the
Royal Society of London Serial B 127: 434-451, 1939.

133. Hill, A. V. A discussion on muscular contraction and relaxation: their
physical and chemical basis. Proceedings of the Royal Society of London Serial B
137: 40—87, 1950.

134. Hill, A. V. A challenge to biochemists. Biochimica et Biophysica Acta 4: 4—11,
1950.

135. Hill, A. V., Trails and Trials in Physiology. Williams & Wilkins Co.,
Baltimore, 1965.

136. Hill, D. K. The time course of the oxygen consumption of stimulated frog's
muscle. Journal of Physiology 98: 207-227, 1940.

137. Hohorst, J. J., M. Reim, and H. Bartels. Studies on the creatine kinase
equilibrium in muscle and the significance of ATP and ADP levels. Biochemical
and Biophysical Research Communications 7(2): 142-146, 1962.

138. Holloszy, J. 0. Biochemical adaptations in muscle: Effects of exercise on
mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle.
Journal of Biological Chemistry 242(9): 2278—2282, 1967.

139. Holloszy, J. O., and L. B. Oscai. Effect of exercise on a—glycerophosphate

dehydrogenase activity in skeletal muscle. Archives of Biochemistry and
Biophysics 130: 653—656, 1969.

154

140. Holloszy, J. O., L. B. Oscai, I. J. Don, and P. A. Mole. Mitochondrial citric
acid cycle and related enzymes: Adaptive response to exercise. Biochemical and
Biophysical Research Communications 40(6): 1368—1373, 1970.

141. Homsher, E., and C. J. Kean. Skeletal muscle energetics and metabolism.
Annual Review of Physiology 40: 93-131, 1978.

142. Hood, D. A., J. Gorski, and R. L. Terjung. Oxygen cost of twitch and
tetanic isometric contractions of rat skeletal muscle. American Journal of
Physiology (Endocrinol. Metab.) 250(13): E449—E456, 1986.

143. Hoult, D. I. The magnetic resonance myth of radio waves. Concepts in
Magnetic Resonance 1: 1—5, 1989.

144. Hoult, D. I., S. J. W. Busby, D. G. Gadian, G. K. Radda, R. E. Richards,
and P. J. Seeley. Observation of tissue metabolites using 31P nuclear magnetic
resonance. Nature 252(November 22): 285—287, 1974.

145. Houmard, J. A., R. Smith, and G. L. Jendrasiak. Relationship between
MRI relaxation time and muscle fiber composition. Journal of Applied Physiology
78(3): 807—809, 1995.

146. Hultman, E., J. Bergstrom, and N. M. Anderson. Breakdown and
resynthesis of phosphorylcreatine and adenosine triphosphate in connection with

muscular work in man. Scandinavian Journal of Clinical Laboratory
Investigations 19: 56—66, 1967.

147. Hunter, A., Creatine and Creatinine. Longmans, Green and Co. LTD,
London, 1928.

148. Huxley, H., and J. Hanson. Structural changes in muscle during
contraction: Changes in the cross—striations of muscle during contraction and
stretch and their structural interpretation. Nature 173(4412): 973—97 6, 1954.

149. Iotti, S., R. Lodi, C. Frassineti, P. Zaniol, and B. Barbiroli. In vivo
assessment of mitochondrial functionality in human gastrocnemius muscle by 31P
MRS. NMR in Biomedicine 6(4): 248—253, 1993.

150. Izumo, S., B. Nadal-Ginard, and V. Mahdavi. All members of the MHC
multigene family respond to thyroid hormone in a highly tissue—specific manner.
Science 231: 597-600, 1986.

151. Jackman, M. R., and W. T. Willis. Characteristics of mitochondria isolated
from type I and type IIb skeletal muscle. American Journal of Physiology (Cell
Physiology) 270(39): C673—C678, 1996.

152. James, N. T. The distribution of type I and type II fibres in muscles. Journal
ofAnatomy 108: 612—613, 1971.

155

153. Janssen, E., G. A. Dudley, B. Norman, and P. A. Tesch. Relationship of
recovery from intense exercise to the oxidative potential of skeletal muscle. Acta
Physiologica Scandinavica 139(1): 147—152, 1990.

154. Jeneson, J. A. L., S. J. Nelson, D. B. Vigneron, J. S. Taylor, J. Murphy-
Boesch, and T. R. Brown. 'IVvo—dimensional 31P—chemical shift imaging of
intramuscular heterogeneity in exercising human forearm muscle. American
Journal of Physiology (Cell Physiology) 263(32): C357—C364, 1992.

155. Jeneson, J. A. L., J. O. vanDobbenburgh, C. J. A. vanEchteld, C.
Lekkerkerk, W. J. M. J anssen, L. Dorland, R. Berger, and T. R. Brown.
Experimental design of 31P MRS assessment of human forearm muscle function:

Restrictions imposed by functional anatomy. Magnetic Resonance in Medicine 30:
634—640, 1993.

156. J i, L. L., F. W. Stratman, and H. A. Lardy. Chronic exercise training alters
kinetic properties of rat skeletal muscle and myocardial lactate dehydrogenase.
FEBS Letters 208(2): 297—300, 1986.

157. Jobsis, F. F. Energy utilization and oxidative recovery metabolism in
skeletal muscle. Current Topics in Bioenergetics 3: 279—349, 1969.

158. Jobsis, F. F., and J. C. Duffield. Oxidative and glycolytic recovery
metabolism in muscle. Journal of General Physiology 50: 1009—1047, 1967.

159. Jobsis, F. F., and J. C. Duffield. Force, shortening, and work in muscular
contraction: Relative contributions to overall energy utilization. Science 156(June
9): 1388—1392, 1967.

160. Johnson, M. A., J. Polgar, D. Weightman, and D. Appleton. Data on the
distribution of fibre types in thirty—Six human muscles: An autopsy study. Journal
of the Neurological Sciences 18: 111-129, 1973.

161. Jones, J. H., and S. L. Lindstedt. Limits to maximal performance. Annual
Review of Physiology 55: 547—569, 1993.

162. Kalckar, H. M., Biological Phosphorylations: Development of Concepts.
Prentice—Hall, Englewood Cliffs, 1969.

163. Karlsson, J., B. Sjodin, A. Thorstensson, B. Hulten, and K. Frith. LDH
isozymes in skeletal muscles of endurance and strength trained athletes. Acta
Physiologica Scandinavica 93: 150—156, 1975.

164. Kelso, T. B., D. R. Hodgson, A. R. Visscher, and P. D. Gollnick. Some

properties of different skeletal muscle fiber types: comparison of reference bases.
Journal of Applied Physiology 62(4): 1436—1441, 1987.

156

 

165. Kemp, G. J. Interactions of mitochondrial ATP synthesis and the creatine
kinase equilibrium in skeletal muscle. Journal of Theoretical Biology 170: 239—
246, 1994.

166. Kemp, G. J., D. J. Taylor, and G. K. Radda. Control of phosphocreatine
resynthesis during recovery from exercise in human skeletal muscle. NMR in
Biomedicine 6: 66-72, 1993.

167. Kemp, G. J., D. J. Taylor, and G. K. Radda. The production, buffering, and
efflux of protons in human skeletal muscle during exercise and recovery. NMR in
Biomedicine 6: 73-83, 1993.

168. Kemp, G. J., C. H. Thompson, A. L. Sanderson, and G. K. Radda. pH
control in rat skeletal muscle during exercise, recovery from exercise, and acute
respiratory acidosis. Magnetic Resonance in Medicine 31: 103—109, 1994.

169. Krebs, H. A., and R. L. Veech. Equilibrium relations between pyridine
nucleotides and adenine nucleotides and their roles in the regulation of metabolic
processes. Advances in Enzyme Regulation 7: 397—413, 1969.

170. Kretzschmar, K. M., and D. R. Wilkie. A new approach to freezing tissues
rapidly. Journal of Physiology (London) 202: 66P—67P, 1969.

171. Kuby, S. A., L. Noda, and H. A. Lardy. Adenosinetriphosphate—creatine
transphosphorylase III: Kinetic Studies. Journal of Biological Chemistry 210: 65—
83, 1954.

172. Kushmerick, M. J., Energetics of muscle contraction. In: Handbook of
Physiology, Section 10, Skeletal Muscle, edited by L. H. Peachey, R. H. Adrian, and
S. R. Gieger, Bethesda, American Physiological Society, 1983, pp. 189—236.

173. Kushmerick, M. J., and R. E. Davies. The chemical energetics of muscle
contraction: II. The chemistry, efficiency and power of maximally working sartorius
muscles. Proceedings of the Royal Society of London Serial B 174: 315—353, 1969.

174. Kushmerick, M. J., R. E. Larson, and R. E. Davies. The chemical
energetics of muscle contraction: I. Activation heat, heat of shortening and ATP
utilization for activation-relaxation processes. Proceedings of the Royal Society of
London Serial B 174: 293-314, 1969.

175. Kushmerick, M. J ., and R. A. Meyer. Chemical changes in rat leg muscle
by phosphorus nuclear magnetic resonance. American Journal of Physiology (Cell
Physiology) 248: C542—C549, 1985.

176. Kushmerick, M. J., R. A. Meyer, and T. R. Brown. Regulation of oxygen

consumption in fast— and slow-twitch muscle. American Journal of Physiology
(Cell Physiology) 263(32): C598—C606, 1992.

157

 

177. Kushmerick, M. J., and R. J. Paul. Relationship between initial chemical
reactions and oxidative recovery metabolism for single isometric contractions of
frog sartorius at 0°C. Journal of Physiology 254: 711—727, 197 6.

178. Kushmerick, M. J., and R. J. Paul. Aerobic recovery metabolism following
a single isometric tetanus in frog sartorius muscle at 0°C. Journal of Physiology
254: 693—709, 1976.

179. Langone, J., Superconductivity. Contemporary Books, Chicago, 1989.

180. Lapaglia, S. R., Molecular spectra and molecular symmetry—Introduction to

magnetic resonance spectroscopy. In: Introduction to Quantum Chemistry, 1971,
pp.238—255.

181. Larsson, L., X. Li, A. Teresi, and G. Salviati. Effects of thyroid hormone
on fast— and slow-twitch skeletal muscles in young and old rats. Journal of
Physiology 481(1): 149—161, 1994.

182. Laughlin, M. H., and R. B. Armstrong. Muscular blood flow distribution
patterns as a function of running speed in rats. American Journal of Physiology
(Heart and Circulatory Physiology) 243(12): H296—H306, 1982.

183. Laughlin, M. H., R. J. Korthuis, D. J. Duncker, and R. J. Bache,
Control of blood flow to cardiac and skeletal muscle during exercise (Chapter 16).
In: Handbook of Physiology Section 12: Exercise: Regulation and Integration of
Multiple Systems, edited by L. B. Rowell, and J. T. Shepard, New York, Oxford
University Press, 1996, pp. 7 05—769.

184. Lawson, C. L., and R. J. Hanson, Solving least squares problems. Prentice—
Hall, Inc., Englewood Cliffs, 1974.

185. Lawson, J. W. R., and R. L. Veech. Effects of pH and free Mg2+ on the Keq
of the creatine kinase reaction and other phosphate hydrolyses and phosphate
transfer reactions. Journal of Biological Chemistry 254(July 25): 6528—6537, 1979.

186. Leberer, E., and D. Pette. Immunochemical quantification of sarcoplasmic
reticulum Ca—ATPase, of calsequestrin and of parvalbumin in rabbit Skeletal
muscles of defined fiber composition. European Journal of Biochemistry 156: 489—
496, 1986.

187. Lexell, J., D. Downham, and M. Sjostrom. Distribution of different fibre
types in human skeletal muscles: Fiber type arrangement in m. vastus lateralis
from three groups of healthy men between 15 and 83 years. Journal of the
Neurological Sciences 72: 211-222, 1986.

188. Lipmann, F. Metabolic generation and utilization of phosphate bond energy.
Advances in Enzymology and Related Areas of Molecular Biology 1: 99—162, 1941.

158

189. Lohmann, K. The chemistry of muscle contraction (translated from
German). Naturwiss 22: 409—414, 1934.

190. Lundsgaard, E. The significance of the phenomenon "alactacid muscle
contractions" for an interpretation of the chemistry of muscle contraction
(translated from German). Danske Hospitalstidende 75: 84—95, 1932.

191. Mackie, B. G., and R. L. Terjung. Blood flow to different Skeletal muscle
fiber types during contraction. American Journal of Physiology (Heart Circ.
Physiology) 245(14): H265—275, 1983.

192. Malher, M. First-order kinetics of muscle oxygen consumption, and an
equivalent proportionality between Q02 and phosphorylcreatine level:
implications for the control of respiration. Journal of General Physiology 86: 135—
165, 1985.

193. Margaria, R., H. T. Edwards, and D. B. Dill. The possible mechanisms of
contracting and paying the oxygen debt and the role of lactic acid in muscular
contraction. American Journal of Physiology 106(3): 680—715, 1933.

194. Marquardt, D. W. An algorithm for least squares estimation of parameters.
Journal of the Society of Industrial and Applied Mathematics 11: 431—441, 1963.

195. Marsh, G. D., D. H. Paterson, J. J. Potwarka, and R. T. Thompson.
Transient changes in muscle high—energy phosphates during moderate exercise.
Journal of Applied Physiology 75(2): 648—656, 1993.

196. Martinas, K., L. Ropolyi, and P. Szegedi, Thermodynamics: History and
Philosophy, Conference: History of Thermodynamics—Facts, Trends, Debates,
World Scientific Publishing Co., Veszprem, Hungary, 1990, p. 527.

197. McAllister, R. M., R. W. Ogilvie, and R. L. Terjung. Functional and
metabolic consequences of skeletal muscle remodeling in hypothyroidism.
American Journal of Physiology (Endocrinology Metabolism) 260(23): E272—E279,
1991.

198. McAllister, R. M., and R. L. Terjung. Acute inhibition of respiratory
capacity of muscle reduces peak oxygen consumption. American Journal of
Physiology (Cell Physiology) 259(28): C889—C896, 1990.

199. McAllister, R. M., and R. L. Terjung. Training—induced muscle
adaptations: increased performance and oxygen consumption. Journal of Applied
Physiology 70(4): 1569—1574, 1991.

200. McCully, K. K., M. A. Forciea, L. M. Hack, E. Donlon, R. W. Wheatley,
C. A. Oatis, T. Goldberg, and B. Chance. Muscle metabolism in older subjects
using 31P magnetic resonance spectroscopy. Canadian Journal of Physiology and
Pharmacology 69: 576-580, 1991.

159

 

201. McFarland, E. W., M. J. Kushmerick, and T. S. Moerland. Activity of
creatine kinase in a contracting mammalian muscle of uniform fiber type.
Biophysical Journal 67(5): 1912—1924, 1994.

202. Mendel, L. B., and W. C. Rose. Experimental studies on creatine and
creatinine. Journal of Biological Chemistry 10: 213—264, 1911.

203. Meyer, R. A. A linear model of muscle respiration explains monoexponential
phosphocreatine changes. American Journal of Physiology (Cell Physiology)
254(23): C548—C553, 1988.

204. Meyer, R. A. Linear dependence of muscle phosphocreatine kinetics on total
creatine content. American Journal of Physiology (Cell Physiology) 257(26):
C1149—C1157, 1989.

205. Meyer, R. A., and G. R. Adams. Stoichiometry of phosphocreatine and

inorganic phosphate changes in rat skeletal muscle. NMR in Biomedicine 3(5):
206—210, 1990.

206. Meyer, R. A., G. R. Adams, M. J. Fisher, P. F. Dillon, J. M. Krisanda, T.
R. Brown, and M. J. Kushmerick. Effects of decreased pH on force and
phosphocreatine in mammalian Skeletal muscle. Canadian Journal of Physiology
and Pharmacology 69: 305—310, 1991.

207. Meyer, R. A., T. R. Brown, B. L. Krilowicz, and M. J. Kushmerick.
Phosphagen and intracellular pH changes during contraction of creatine-depleted
rat muscle. American Journal of Physiology (Cell Physiology) 250(19): C264—C274,
1986.

208. Meyer, R. A., T. R. Brown, and M. J. Kushmerick. Phosphorus nuclear
magnetic resonance of fast— and slow—twitch muscle. American Journal of
Physiology (Cell Physiology) 248(17): C279—C287, 1985.

209. Meyer, R. A., M. J. Fisher, S. J. Nelson, and T. R. Brown. Evaluation of
manual methods for integration of in vivo phosphorus NMR spectra. NMR in
Biomedicine 1(3): 131—135, 1988.

210. Meyer, R. A., and J. M. Foley. Testing models of respiratory control in
skeletal muscle. Medicine and Science in Sports and Exercise 26(1): 52—57, 1994.

211. Meyer, R. A., and J. M. Foley, Cellular processes integrating the metabolic
response to exercise (Chapter 18). In: Handbook of Physiology Section 12: Exercise:
Regulation and Integration of Multiple Systems, edited by L. B. Rowell, and J. T.
Shepard, New York, Oxford University Press, 1996, pp. 841—869.

212. Meyer, R. A., M. J. Kushmerick, and T. R. Brown. Application of SIP—

NMR spectroscopy to the study of striated muscle metabolism. American Journal
of Physiology (Cell Physiology) 242(11): C1-C11, 1982.

160

213. Meyer, R. A., H. L. Sweeney, and M. J. Kushmerick. A simple analysis of
the "phosphocreatine shuttle". American Journal of Physiology (Cell Physiology)
246(15): C365—C377, 1984.

214. Meyer, R. A., and R. L. Terjung. Differences in ammonia and adenylate
metabolism in contracting fast and slow muscle. American Journal of Physiology
(Cell Physiology) 237(6): C111—C118, 1979.

215. Meyerhoff, 0., The transformation of energy in muscle. Chemical Dynamics
of Life Phenomena, Lippincott Comp., Philadelphia, 1925.

216. Mitchell, P. Coupling of phosphorylation to electron and hydrogen transfer
by a chemio—osmotic type of mechanism. Nature 191(4784): 144—148, 1961.

217. Mizuno, M., N. H. Secher, and B. Quistorff. 31P—NMR Spectroscopy,
rsEMG, and histochemical fiber types of human wrist flexor muscles. Journal of
Applied Physiology 76(2): 531—538, 1994.

218. Mole, P. A., K. M. Baldwin, R. L. Terjung, and J. O. Holloszy. Enzymatic
pathways of pyruvate metabolism in skeletal muscle: adaptations to exercise.
American Journal of Physiology 224(1): 50—54, 1973.

219. Mommaerts, W. F. H. M. Energetics of muscular contraction. Physiological
Reviews 49(3): 427—508, 1969.

220. Mommaerts, W. F. H. M., and J. C. Rupp. Dephosphorylation of adenosine
triphosphate in muscular contraction. Nature 168(4283): 957, 1951.

221. Monsieurs, K., L. Heytens, C. Kloeck, J.-J. Martin, F. Wuyts, and L.
Bossaert. Slower recovery of muscle phosphocreatine in malignant
hyperthermia—susceptible individuals assessed by 31P—MR spectroscopy. Journal
of Neurology 255(10): 651—656, 1997.

222. Moon, R. B., and J. H. Richards. Determination of intracellular pH by 31P
magnetic resonance. Journal of Biological Chemistry 248(20): 7276-7278, 1973.

223. Naqui, A., and B. Chance. Steady—state kinetic approaches to complex
biological systems. Cell Biophysics 11(December): 159—176, 1987.

224. Nash, S. G., A history of scientific computing. ACM Press History Series,
Addison-Wesley Publishing Co., New York, 1990.

225. Needham, D. M., Machina Carnis: The biochemistry of muscular contraction
in its historical development. Cambridge University Press, Cambridge, 1971.

161

226. Nelson, 8., J. S. Taylor, D. B. Vigneron, J. Murphy-Boesh, and T. R.
Brown. Metabolite images of the human arm: Changes in spatial and temporal

distribution of high energy phosphates during exercise. NMR in Biomedicine 4:
268—273, 1991.

227. Newton, K., W. Steeds, and T. K. Garrett, The Motor Vehicle. 12th Ed.,
Butterworth—Heinemann, Oxford, 1996.

228. Noakes, T. D. Challenging beliefs: ex Africa semper aliquid novi. Medicine &
Science in Sports & Exercise 29(5): 571—590, 1997.

229. Noakes, T. D. Maximal oxygen uptake: "classical" versus "contemporary"
viewpoints: a rebuttal. Medicine & Science in Sports & Exercise 30(9): 1381—1398,
1998.

230. Noda, L., S. A. Kuby, and H. A. Lardy. Adenosinetriphosphate—creatine
transphosphorylase IV: Equilibrium studies. Journal of Biological Chemistry 210:
83—95, 1954.

231. Odeblad, E., and G. Lindstrom. Some preliminary observations on the
proton magnetic resonance in biologic samples. Acta Radiologica 43: 469—475,
1955.

232. Odum, H. T., R. J. Beyers, and N. E. Armstrong. Consequences of small
storage capacity in nanoplankton pertinent to measurement of primary production
in tropical waters. Journal of Marine Research 21(3): 191—198, 1963.

233. Padykula, H. A., and G. F. Gauthier. Cytochemical studies of adenosine
triphosphatases in skeletal muscle fibers. The Journal of Cell Biology 18: 87-107,
1963.

234. Paganini, A. T., J. M. Foley, and R. A. Meyer. Linear dependence of
muscle phosphocreatine kinetics on oxidative capacity. American Journal of
Physiology (Cell Physiology) 272(41): C501-C510, 1997.

235. Painter, P. L. Exercise in end—stage renal disease. Exercise Science and
Sports Review 16: 305-339, 1988.

236. Park, J. H., R. L. Brown, C. R. Park, K. McCully, M. Cohn, J.
Haselgrove, and B. Chance. Functional pools of oxidative and glycolytic fibers in
human muscle observed by 31P magnetic resonance spectroscopy during exercise.
Proceedings of the National Academy of Science USA 84: 8976—8980, 1987.

237. Paton, D. N. Creatinle] excretion in the bird and its Significance. Journal of
Physiology 39: 495—504, 1910.

162

238. Pekelharing, C. A., and C. J. C. VanHoogenhuyze. Die bildung des
kreatins im muskel beim tonus und bei der starre. Zeitschrift f Physiological
Chemistry 64: 262—293, 1910.

239. Peter, J. B., R. J. Barnard, V. R. Edgerton, C. A. Gillespie, and K. E.
Stemple. Metabolic profiles of three fiber types of Skeletal muscle in guinea pigs
and rabbits. Biochemistry 11(14): 2627—2633, 1972.

240. Pette, D. Activity—induced fast to Slow transitions in mammalian muscle.
Medicine and Science in Sports and Exercise 16(6): 517—528, 1984.

241. Pette, D. Metabolic heterogeneity of muscle fibers. Journal of Experimental
Biology 115: 179-189, 1985.

242. Pette, D., and R. S. Staron. Cellular and molecular diversities of
mammalian skeletal muscle fibers. Review of Physiology. Biochemistry and
Pharmacology 116: 1-76, 1990.

243. Phillips, S. K., M. Takei, and K. Yamada. The time course of phosphate
metabolites and intracellular pH using 31P NMR compared to recovery heat in rat
soleus muscle. Journal of Physiology 460(January): 693—704, 1993.

244. Piiper, J., P. E. DiPrampero, and C. P. Oxygen debt and high—energy
phosphates in gastrocnemius muscle of the dog. American Journal of Physiology
215(3): 523-531, 1968.

245. Piiper, J., and P. Spiller. Repayment of 02 debt and resynthesis of high—
energy phosphates in gastrocnemius muscle of the dog. Journal of Applied
Physiology 28(5): 657—662, 1970.

246. Poole, D. C., T. J. Barstow, G. A. Gaesser, W. T. Willis, and B. J. Whipp.
V02 slow component: physiological and functional significance. Medicine & Science
in Sports & Exercise 26: 1354-1358, 1994.

247. Purcell, E. M., H. C. Torrey, and R. V. Pound. Resonance absorption by
nuclear magnetic moments in a solid. Physical Review 69: 37—38, 1946.

248. Radda, G. K., and P. J. Seeley. Recent studies on cellular metabolism by
nuclear magnetic resonance. Annual Review of Physiology 41: 749—69, 1979.

249. Radmer, R. J ., and B. Kok, Light conversion efficiency in photosynthesis. In:
Encyclopedia of Plant Physiology, edited by A. Pirson, and M. H. Zimmermann,
Berlin, Springer—Verlag, 1977, Vol. 5, pp. 125-135.

250. Rall, J. A. Energetics of skeletal muscle contraction: Implications of fiber
types. Exercise Sports Science Review 13: 33—74, 1985.

163

251. Ranvier, M. L. Proprietes et structures differentes des muscles rouges et des
muscles blancs, chez les lapins (French: Difference in the properties and structure
of the red and white muscle of rabbits). Comptes Rendus Academie des Sciences 77:
1030—1034, 1873.

252. Rassier, D. E., L. A. Tubman, and B. R. Maclntosh. Length—dependent
potentiation and myosin light chain phosphorylation in rat gastrocnemius muscle.
American Journal of Physiology (Cell Physiology) 273(42): C198—C204, 1997.

253. Reiser, P. J., and B. T. Stokes. Development of contractile properties in
avian embryonic skeletal muscle. American Journal of Physiology (Cell
Physiology) 242(11): C52—C58, 1982.

254. Robinson, D. M., R. W. Ogilvie, P. C. Tullson, and R. L. Terjung.
Increased peak oxygen consumption of trained muscle requires increased electron
flux capacity. Journal of Applied Physiology 77(4): 1941—1952, 1994.

255. Rome, L. C., R. P. Funke, R. McNeill—Alexander, G. Lutz, H. Aldridge,
F. Scott, and M. Freadman. Why animals have different muscle fibre types.
Nature 335(October 27): 824-827, 1988.

256. Roy, R. R., K. M. Baldwin, and V. R. Edgerton. The plasticity of skeletal
muscle: Effects of neuromuscular activity. Exercise Science and Sports Review 19:
269—312, 1991.

257. Sahlin, K. Intracellular pH and energy metabolism in skeletal muscle of
man. Acta Physiologica Scandinavica Suppl. 455: 1—56, 1978.

258. Sahlin, K., R. C. Harris, and E. Hultman. Creatine kinase equilibrium
and lactate content compared with muscle pH in tissue samples obtained after
isometric exercise. Biochemical Journal 152: 173—180, 1975.

259. Sahlin, K., R. C. Harris, and E. Hultman. Resynthesis of creatine
phosphate in human muscle after exercise in relation to intramuscular pH and

availability of oxygen. Scandinavian Journal of Clinical Laboratory Investigations
39: 551-558, 1979.

260. Sahlin, K., R. C. Harris, B. Nylind, and E. Hultman. Lactate content and
pH in muscle samples obtained after dynamic exercise. European Journal of
Physiology 367: 143—149, 1976.

261. Saltin, B., and P. D. Gollnick, Skeletal muscle adaptability: significance
for metabolism and performance. In: Handbook of Physiology, Section 10, Skeletal
Muscle, edited by L. D. Peachey, R. H. Adrian, and S. R. Gieger, Bethesda, American
Physiological Society, 1983, pp. 555-631.

164

 

262. Saltin, B., and S. Strange. Maximal oxygen uptake: "old" and "new"
arguments for a cardiovascular limitation. Medicine & Science in Sports & Exercise
24(1): 30—37, 1992.

263. Samuels, H. H., B. M. Forman, Z. D. Horowitz, and Z. S. Ye. Regulation

of gene expression by thyroid hormone. Annual Review of Physiology 51: 623—639,
1989.

264. Sandberg, J. A., and F. D. Carlson. The length dependence of
phosphorylcreatine hydrolysis during and isometric tetanus. Biochemische
Zeitschrifi‘ 345: 212—231, 1966.

265. Sandow, A. Excitation-contraction coupling in muscular response. Yale
Journal of Biology and Medicine 25: 176-201, 1952.

266. Sapega, A. A., D. P. Sokolow, T. J. Graham, and B. Chance. Phosphorus
nuclear magnetic resonance: a non—invasive technique for study of muscle

bioenergetics during exercise. Medicine and Science in Sports and Exercise 19(4):
410—420, 1987.

267. Schiaffino, S., V. Hanzlikova, and S. Pierobon. Relations between
structure and function in rat skeletal muscle fibers. The Journal of Cell Biology
47: 107-119, 1970.

268. Schiaffino, S., and C. Reggiani. Myosin isoforms in mammalian skeletal
muscle. Journal of Applied Physiology 77(2): 493—501, 1994.

269. Shaw, T. M., and R. H. Elsken. Nuclear magnetic resonance absorption in
hygroscopic materials. Journal of Chemical Physics 18: 1113—1114, 1950.

270. Shepherd, R. E., and P. D. Gollnick. Oxygen uptake of rats at different
work intensities. European Journal of Physiology (362): 219-222, 1976.

271. Simon, R., and A. Smith, Superconductors. Plenum Press, New York, 1988.
272. Simoneau, J.—A., and C. Bouchard. Human variation in skeletal muscle
fiber-type proportion and enzyme activities. American Journal of Physiology

(Endocrinology and Metabolism) 257(20): E567—E572, 1989.

273. Singer, J. R. Blood flow rates by nuclear magnetic resonance
measurements. Science 130: 1652-1653, 1959.

274. Soderlund, K., and E. Hultman. ATP and phosphocreatine changes in
single human muscle fibers after intense electrical stimulation. American Journal
of Physiology (Endocrinology and Metabolism) 261(24): E737—741, 1991.

275. Sommerville, J., and U. Sheer, Electron Microscopy in Molecular Biology:
A Practical Approach. IRL Press, Oxford, 1987.

165

 

276. Spriet, L. L., K. Soderlund, J. A. Thomson, and E. Hultman. pH
measurement in human skeletal muscle samples: effect of phosphagen hydrolysis.
Journal of Applied Physiology 61(5): 1949—1954, 1986.

277. Srere, P. A., Citrate synthase. In: Methods in Enzymology, 1969, Vol. 13, pp.
3—11.

278. Stainsby, W. N. Oxygen uptake for isotonic and isometric twitch contractions
of dog skeletal muscle in situ. American Journal of Physiology 219(2): 435-439,
1970.

279. Stainsby, W. N., and J. T. Fales. Oxygen consumption for isometric tetanic
contractions of dog skeletal muscle in situ. American Journal of Physiology 224(3):
687—691, 1973.

280. Stein, J. M., and H. A. Padykula. Histochemical classification of individual
skeletal muscle fibers of the rat. American Journal of Anatomy 110: 103—115, 1962.

281. Stoyanova, R., A. C. Kuesel, and T. R. Brown. Application of principal-
component analysis for NMR spectral quantitation. Journal of Magnetic Resonance,
SeriesA 115: 265—269, 1995.

282. Sugden, P. H., and E. A. Newsholme. The effects of ammonium, inorganic
phosphate and potassium ions on the activity of phosphofructokinases from muscle

and nervous tissues of vertebrates and invertebrates. Biochemical Journal 150:
113-122, 1975.

283. Sullivan, T. E., and R. B. Armstrong. Rat locomotory muscle fiber activity
during trotting and galloping. Journal of Applied Physiology (Respiratory,
Evironmental, Exercise Physiology) 44(3): 358—363, 1978.

284. Susheela, A. K., and J. N. Walton. Note on the distribution of histochemical
fibre types in some normal human muscles: A study on autopsy material. Journal
of the Neurological Sciences 8: 201—207, 1969.

285. Sweeney, H. L., M. J. Kushmerick, K. Mabuchi, F. A. Sreter, and J.
Gergely. Myosin alkali light chain and heavy chain variations correlate with
altered shortening velocity of isolated skeletal muscle fibers. Journal of Biological
Chemistry 263(18): 9034-9039, 1988.

286. SzentGyorgi, A. Studies on muscle. Acta Physiologica Scandinavica
8(Suppl. 25): 3-115, 1945.

287. Takahashi, H., M. Inaki, K. Fujimoto, S. Katsauta, 1. Anna, M. Niitsu,
and Y. Itai. Control of the rate of phosphocreatine resynthesis after exercise in

trained and untrained human quadriceps muscles. European Journal of Applied
Physiology 71(5): 396-401, 1995.

166

288. Takahashi, H., S. Kuno, S. Katsuta, H. Shimojo, K. Masuda, H.
Yoshioka, 1. Anna, and Y. Itai. Relationships between fiber composition and
NMR measurements in human Skeletal muscle. NMR in Biomedicine 9: 8-12, 1996.

289. Talmadge, R. J., and R. R. Roy. Electrophoretic separation of rat skeletal

muscle myosin heavy-chain isoforms. Journal of Applied Physiology 75(5): 2337—
2340, 1993.

290. Taylor, D. J., P. J. Bore, P. Styles, D. G. Gadian, and G. Radda, K.
Bioenergetics of intact human muscle a 31P nuclear magnetic resonance study.
Molecular Biology in Medicine 1: 77—94, 1983.

291. Taylor, D. J., P. Styles, P. M. Matthews, D. A. Arnold, D. G. Gadian, P.
Bore, and G. K. Radda. Energetics of human muscle: exercise—induced ATP
depletion. Magnetic Resonance in Medicine 3: 44-54, 1986.

292. Taylor, H. L., E. Buskirk, and A. Henschel. Maximal oxygen intake as an
objective measure of cardio—respiratory performance. Journal of Applied
Physiology 8: 73—80, 1955.

293. Terjung, R. L. Cytochrome C turnover in skeletal muscle. Biochemical and
Biophysical Research Communications 66(1): 173-178, 1975.

294. Terjung, R. L. Muscle fiber involvement during training of different
intensities and durations. American Journal of Physiology 230(4): 946-950, 1976.

295. Terjung, R. L., and B. Mackie—Engbretson. Blood flow to different rat
skeletal muscle fiber type sections during isometric contraction
in situ. Medicine and Science in Sports and Exercise 20(5): 8124-8130, 1988.

296. Thompson, C. H., G. J. Kemp, A. L. Sanderson, and G. K. Radda.
Skeletal muscle mitochondrial function studied by kinetic analysis of postexercise
phosphocreatine resynthesis. Journal of Applied Physiology 78(6): 2131-2139,
1995.

297. Thompson, W. H. The metabolism of voluntary muscle. I: The effect of
prolonged excitation of motor nerves on the creatine content of limb muscles.

Quarterly Journal of Experimental Physiology 11(3): 223—230, 1917:

298. Thunberg, T. Zur kenntis des kreatins (To the knowledge the creatine).
Zentralblatt fur Physiologie 20: 915—916, 1911.

299. Traficante, D. D. Impedance: what it is and why it must be matched.
Concepts in Magnetic Resonance 1: 73—92, 1989.

300. Traficante, D. D. Introduction to transmission lines. Concepts in Magnetic
Resonance 5: 57—86, 1993.

167

 

301. Uyeno, K., and T. Mitsuda. Creatine formation during tonic muscle
contractions. Journal of Physiology (London) 57: 313—317, 1923.

302. Vandenborne, K., K. McCully, H. Kakihira, M. Prammer, L. Bolinger,
J. A. Detre, K. DeMeirleir, G. Walter, B. Chance, and J. S. Leigh. Metabolic
heterogeneity in human calf muscle during maximal exercise. Proceedings of the
National Academy of Science USA 88(July): 5714—5718, 1991.

303. Vandenborne, K., G. Walter, J. S. Leigh, and G. Goelman. pH
heterogeneity during exercise in localized spectra from single human muscles.
American Journal of Physiology (Cell Physiology) 265(65): C1332—C1339, 1993.

304. Vandenborne, K., G. Walter, L. Ploutz—Synder, R. Staron, A. Fry, K.
DeMeirleir, G. A. Dudley, and J. S. Leigh. Energy rich phosphates in slow and

fast human skeletal muscle. American Journal of Physiology (Cell Physiology)
268(37): C869—C876, 1995.

305. Veech, R. L., J. W. R. Lawson, N. W. Cornell, and H. A. Krebs. Cytosolic
phosphorylation potential. Journal of Biological Chemistry 254(14): 6538-6547,
197 9.

306. Vincent, A., and J. McD.Blair. The coupling of the adenylate kinase and
creatine kinase equilibrium. Calculation of substrate and feedback signal levels in
muscle. FEBS Letters 7(3): 239-244, 1970.

307. Wagner, P. D. Determinants of maximal oxygen transport and utilization.
Annual Review of Physiology 58: 21-50, 1996.

308. Walter, G., K. Vandenborne, K. K. McCully, and J. S. Leigh.
Noninvasive measurement of phosphocreatine recovery kinetics in single human
muscle. American Journal of Physiology (Cell Physiology) 272(41): C525—C534,
1997.

309. Wasserman, K. Coupling of external to cellular respiration during exercise:
the wisdom of the body revisited. American Journal of Physiology (Endocrinology
and Metabolism) 266(29): E519—E539, 1994.

310. Weber, A., and H. H. Weber. Zur thermodynamik der kontraktion des
fasermodells. Biochimica et Biophysica Acta 7: 339—348, 1951.

311. Wells, R. L., and W. W. Heusner. A controlled running wheel for small
animals. Laboratory Animal Science 21(6): 904—910, 1971.

312. Wheeler, T. J., and J. M. Lowenstein. Adenylate deaminase from rat
muscle. The Journal of Biological Chemistry 254(18): 8994—8999, 1979.

313. Whittall, K. P., and A. L. Mackay. Quantitative interpretation of NMR
relaxation data. Journal of Magnetic Resonance 84: 134—152, 1989.

168

 

314. Wilkie, D. R. Thermodynamics and the interpretation of biological heat
measurements. Progress in Biophysics and Biophysical Chemistry 10: 259—298,
1960.

315. Wilkie, D. R. Heat, work, and phosphorylcreatine break—down in muscle.
Journal ofPhysiology 195(1): 157—183, 1968.

316. Wilkie, D. R., Muscle as a thermodynamic machine. In: Ciba Foundation
Symposium, Amsterdam, Elsevier, 1975, Vol. 31, pp. 327—339.

317. Winegrad, S. Intracellular calcium movements of frog skeletal muscle
during recovery from tetanus. Journal of General Physiology 51: 65—83, 1968.

318. Wiseman, R. W., and M. J. Kushmerick. Creatine kinase equilibration
follows solution thermodynamics in Skeletal muscle. Journal of Biological
Chemistry 270(21): 12428—12438, 1995.

319. Woittiez, R. D., G. C. Baan, P. A. Huijing, and R. H. Rozendal.
Functional characteristics of the calf muscles of the rat. Journal of Morphology
184: 375—387, 1985.

320. Woledge, R. C. Heat production and chemical change in muscle. Progress in
Biophysics and Molecular Biology 22: 37—74, 1971.

321. Woledge, R. C., N. A. Curtin, and E. Homsher, Energetic Aspects of Muscle
Contraction. edited by P. F. Baker, Monographs of the Physiological Society, Vol.
41, Academic Press, San Diego, 1985.

322. Wu, K.-D., and J. Lytton. Molecular cloning and quantification of
sarcoplasmic reticulum Ca2+—ATPase isoforms in rat muscles. American Journal
of Physiology (Cell Physiology) 264(33): C333—C341, 1993.

323. Yoshida, T., and H. Watari. 31P-nuclear magnetic resonance spectroscopy
study of the time course of energy metabolism during exercise and recovery.
European Journal of Applied Physiology 66: 494-499, 1993.

324. Yoshida, T., H. Watari, and K. Tagawa. Effects of active and passive

recoveries on splitting of the inorganic phosphate peak determined by 31P—nuclear
magnetic resonance spectroscopy. NMR in Biomedicine 9: 13—19, 1996.

169

INDEX

—A—

A.V. Hill, 11, 13, 15, 21, 24

acetylcholine, 125

Achilles tendon, 48, 72

Achten, 42, 144

acidification, 3, 50, 55, 61, 88, 124

actin, 12, 17

adenine nucleotides, 22, 25, 121, 157

adenosine, 7, 12, 125, 147, 155, 161,
162

adenosine triphosphate, 12, 147, 155,
161

ADP, 7, 14, 15, 25, 38, 42, 121, 122,
144, 154

aerobic capacity, 1, 73

aerobic training, 2, 32, 40, 132

alactacid oxygen debt, 24

alkalinization, 55, 88

amino groups, 35

AMP, 10, 15, 25, 38

AMP deaminase, 38

amphibolic, 65

analog, 34, 40, 65

anaplerotic, 65

anastomoses, 125

apyrase A, 40

artifactual hydrolysis, 16

ATP, vii, 1, 2, 10, 12, 15, 21, 23, 29, 30,
35, 40, 45, 46, 49, 51, 54, 55, 57, 58,
61, 62, 63, 65, 69, 121, 122, 123, 125,
128, 130, 146, 149, 150, 152, 153,
154, 157, 165, 167

ATP consumption, 17

ATP demand, 1, 14, 23, 29, 40

ATP hydrolysis, 21, 38, 46, 130

ATP supply, 1, 14, 26, 44

ATP synthesis, 40, 123, 157

ATPase, 1, 12, 18, 22, 26, 28, 29, 31, 40,
66, 69, 127, 139, 153, 158, 169

—B—

Bl, 50, 74, 132
Barnard, 27, 28, 145, 163

basal metabolic rate, 24
beer, 8

170

biexponential, 39, 115, 120, 124

bimodal, 3, 29, 116, 117, 118, 129, 131

biopsies, 1, 2, 70, 128

bisphosphoglycerate, 37

Blei, 42, 69, 146

blood flow, 16, 25, 33, 43, 123, 132,
158

B0, 35, 50

Boltzmann, 35

Brooke, 26, 28, 146

Bruker, v, 50, 73

Buchner, 8, 146

buffer, 9, 17, 46, 144

Burke, 26, 28, 146, 147

_c_

Cain, 14, 15, 147

calcium, 18, 20, 29, 45, 96, 106, 169

Calcium Theory, 18

calf, 42, 168, 169

calsequestrin, 20, 158

capacitor, 11, 15, 41, 44

capillary, 4, 125, 127

capillary density, 4, 127

carbonyl, 35

cardiovascular, 1, 31, 67, 165

Carlson, 17, 30, 147, 165

Carnot, 5

cat, 6, 67, 120, 122, 123, 146, 147, 153

Challenge to Biochemists, 13

Chance, 13, 39, 144, 147, 148, 153, 159,
161, 162, 165, 168

charge, 11, 44

chemical energy balance method, vii,
30

chemical potential, 10, 12, 25

chemical shift, 37, 51, 76, 95, 156

chemiosmotic hypothesis, 25

chemomechanical, 15

Cholesky decomposition, 51

circuit model, 40, 65

citrate synthase, 2, 31, 49, 59, 60, 61,
64, 140

Clausius, 5

Clayeperon, 5

C02, 7, 23

computational efficiency, 33

Connett, 38, 148, 152

continuum, 27, 127

Cooley, 33, 148

copper, 33

Cori, 8

creatine, 3, 6, 9, 12, 15, 25, 35, 39, 45,
46, 49, 55, 59, 65, 120, 121, 122, 131,
154, 157, 158, 160, 162, 164, 167, 168

creatine kinase, 9, 14, 15, 25, 38, 39,
46, 65, 121, 122, 131, 154, 157, 158,
160, 168

Curtin, 125, 149, 169

cytochrome C, 47, 49, 52, 65

cytochrome oxidase, 24

_1)_

D.K. Hill, 24, 30

Dainty, 12, 149

Dalton, 5

Davies, 14, 22, 147, 149, 157
Dawson, 37, 149
developmental stage, 27
diaphragm, 18
diiodotyrosine, 139
DiPrampero, 39, 150, 163
dog,30,38,148,163,166
dogfish, 122, 125, 149
Dubowitz, 26, 28, 150

—E—

Ebashi, 18, 150

Edgerton, 27, 145, 150, 163, 164

Edwards, 125, 150, 153, 159

efficiency, vii, 15, 20, 23, 33, 45, 150,
154, 157, 163

eigenvectors, 75

elastic component, 21, 106

elderly, 43, 148

electron microscopy, 18

electron pairs, 35

electron transport chain, 31

electron withdrawing groups, 35

Embden, 6, 151

endergonic, 10

endurance capacity, 31

endurance training, 31, 149

energy balance, vii, 19, 23, 30

energy cost, 15, 30, 44, 148

171

energy currency, 14

energy demand, 34, 45, 127

Engelmann, 5

engine, 5, 6, 20

enthalpy, 15, 20, 23

epoxy, 16

equilibrium, 15, 25, 38, 39, 65, 121,
122, 131, 154, 157, 164, 168

ethanol, 8

exercise biochemistry, 31

exergonic, 10

explained enthalpy, 19

external work, 9, 17 , 21

extraction, 16, 132

—F—

FAD, 15

Fast Fourier Transform, 33

fast twitch muscle, 31

fatigue, 4, 9, 26, 28, 42, 50, 66, 128,
130, 149, 150

FDNB, 14, 16, 22

femur, 48, 72

Fenn, 17, 151

Fenn Effect, 17

fermentation, 8

FFT, 33

FG, 2, 32, 136

fiber recruitment, 1, 149

fiber type distributions, 3, viii, x, 2, 45,
69, 116, 117, 122, 126, 150

fiber type heterogeneity, 29, 121, 129,
131, 136

fiber typing, viii, 1, 2, 42, 126, 127

FID, 36

First Law of Thermodynamics, 19

fish, 26

Fisk, 9

Fletcher, 8, 151, 152

fluorodinitrobenzene, 14

FOG, 2, 32, 136

Foley, v, 44, 144, 152, 160, 162

follicular cells, 47

Fourier transform, 3, 33, 51, 74

free energy, 15, 21, 26, 38, 46, 130

Free Induction Decay, 36

frequency distribution, 29, 34

frequency domain, 36, 51

frog, 13, 16, 21, 30, 37, 38, 120, 147,
149, 154, 158, 169

—G—

G6P, 11

gastrocnemius, 2, ix, 3, 30, 49, 50, 52,
55, 61, 70, 96, 120, 124, 137, 140,
147, 148, 149, 155, 163, 164

gating, 39

Gauss, 33

Gaussian, 3, 27, 29, 76, 116, 117, 118,
129, 131

gel electrophoresis, 2, 29

Gibbs, 5

glycogen, 8, 24, 38, 124, 127, 148, 151

glycogen depletion, 127

glycogen phosphorylase, 38, 124

glycolysis, 9, 14, 16, 21, 26, 37, 42, 66,
123, 125, 148

glycolytic flux, 9, 44, 95, 123, 131

glycolytic pathway, 7

Gower, 20, 152

Guth, 127, 153

—H—

Hanson, 14, 155, 158

Harden, 8, 153

Harkema, 123, 152, 153

heart, 7, 40

heat, v, vii, 5, 6, 16, 21, 23, 125, 147,
154, 157, 163, 169

heat capacity, 16

heat engine, 5, 6, 21

Helmholtz, 5

Henneman, 32, 154

Hermann, 40, 154

hexokinase, 27

hexose, 8, 37

hexose monophosphates, 8

Hill, 6, 11, 13, 15, 21, 24, 30, 146, 154

hindlimb, 6, 72, 123, 137, 144, 147

Holloszy, 31, 144, 151, 154, 155, 161

homogeneity of variance, 77

Hopkins, 8, 151, 152

HRT, 106

Hultman, 24, 30, 148, 150, 153, 155,
164, 165, 166

human, 3, 4, 24, 33, 40, 64, 69, 120,
121, 122, 124, 125, 127, 130, 144,

172

145, 146, 147, 148, 150, 155, 156,
157, 158, 161, 162, 164, 165, 166,
167, 168

Huxley, 14, 155

hypercapnic acidosis, 122, 123

hypothyroidism, 2, 43, 47 , 159

_1_

ICF, x, 36, 39, 61, 62, 63, 64, 67, 88, 89,
90, 91, 92, 93, 94, 95, 123

IIA, 26, 28, 127

IIB, 26, 28, 127, 149

IIC, 26, 28

immunocytochemistry, 29

inner mitochondrial membrane, 25,
47, 65

inorganic phosphate, 6, 10, 69, 76,
151, 160, 166, 169

intermediary metabolism, 10

interpulse interval, 50, 74

interval training, 47 , 140

intracellular pH, 2, 37, 55, 66, 73, 147,
152, 160, 161, 163

iodoacetate, 9, 16

isoenzymes, 1, 29

isometric, 2, x, 17, 21, 30, 38, 40, 49,
50, 52, 54, 58, 61, 62, 64, 67, 71, 72,
74, 86, 97, 98, 99, 100, 101, 102, 103,
104, 105, 124, 147, 150, 152, 155,
158, 164, 165, 166, 167

isometric tension, 17, 30

isothermal, 19, 21

isotonic, 17, 21, 147, 166

isovolumetric, 19

._J_
Joule, 5

—K—

Kelvin, 5

Kemp, 41, 157, 167

Krebs cycle, 26, 31, 52, 65

Kuby, 38, 157, 162

Kushmerick, 22, 30, 38, 40, 146, 149,
157, 158, 160, 161, 166, 169

—L—
labile phosphate, 16

 

lactacid oxygen debt, 24

lactate, 8, 9, 19, 25, 42, 125, 132, 156,
164

lactate dehydrogenase, 27, 156

lactate hypothesis, 8, 9

lactic acid, 7, 10, 20, 24, 95, 122, 124,
126, 129, 131, 159

lactic acidosis, 9, 122, 124, 126, 131

light microscope, 18

light microscopy, 18

linear control model, 130

lineshape, 29, 75, 76, 110, 111

linewidth, 34, 42, 51, 110, 111

linewidth broadening, 37, 42, 51

Lipmann, 10, 12, 37, 158

Lohmann, 9, 37, 159

longitudinal relaxation, 70

Lorentzian, 34, 76

Lundsgaard, 9, 14, 159

—M—

magnet, 34, 43, 49, 50, 72, 73, 75, 110,
137

magnetic field, 34, 50, 74

magnetic moments, 33, 163

Mahler, 40

malate dehydrogenase, 27

malignant hyperthermia, 67, 161

Margaria, 24, 30, 150, 159

Marsh, 125, 159

material science, 33

maximal velocity, 21, 44

Mayer, 5

McCully, 43, 148, 159, 162, 168

mechanical efficiency, 21, 154

metabolic capacitor, 11, 41

metabolic networks, 11, 12, 25, 29, 34

methimazole, 2, 47 , 52, 139

Method of Natural Lineshapes, 51, 76

Meyer, v, 38, 40, 144, 152, 153, 157,
160, 161, 162

Meyerhof, 8

microfilament, 96

Mitchell, 25, 161

mitochondrial matrix, 26, 31, 52, 68

MMI, 2, 47 , 50, 52, 54, 55, 58, 61, 66,
142

model, 14, 22, 40, 46, 49, 59, 65, 72, 78,
116, 117, 130, 137, 152, 160

173

Modelling, 3, viii, 116

momentum, 123

Mommarets, 13

monoexponential, 3, 24, 30, 41, 46, 51,
58, 67, 114, 116, 118, 120, 124, 160

monoiodotyrosine, 139

motivation, 7, 15, 43

motor unit, 4, 26, 28, 32, 122, 127, 147,
152, 153

motorneuron, 27 , 66

multicomponent, 121, 122, 129, 152

muscle biopsies, 2, 7O

muscle fatigue, 4, 9, 50

myasthenia gravis, 7

myosin, 1, 12, 17, 21, 26, 28, 29, 31, 43,
49, 52, 66, 96, 121, 145, 164, 167

myosin ATPase, 1, 22, 26, 28, 31, 43,
66

myosin heavy chain, 29, 49, 52

myosin light chain, 29, 164

myosin regulatory light chain, 96

myothermal, 16, 23

—N—

NAD, 15, 25

Needham, 8, 149, 161

network, 18, 25

niobium, 33

nitrogen, 24, 35, 49

NMR, 1, 2, 3, vii, x, xi, 3, 32, 35, 43, 48,
53, 56, 64, 69, 71, 73, 75, 77, 79, 110,
112, 113, 122, 125, 126, 130, 132,
135, 137, 141, 144, 145, 146, 147,
148, 151, 152, 153, 155, 157, 160,
161, 162, 163, 166, 167

NNLS, 3, ix, x, 76, 77, 112, 114, 115,
116, 119, 124, 126, 131

Noda, 38, 157, 162

normality, 77

numerical methods, 33

_o_

02, 7, 29, 30, 40, 65, 132, 150, 163

02 consumption, 7, 30, 150

Odum, 40, 162

optimal length, 17

oxidative capacity, 2, vii, x, 2, 3, 23, 29,
31, 40, 45, 46, 51, 60, 61, 64, 69, 116,
117, 121, 123, 126, 130, 162

‘ 0' .flg‘!

 

oxidative metabolism, 15, 23, 39, 50,
61, 66, 78, 123, 148

oxidative phosphorylation, 14, 16, 21,
59, 130, 148, 153

oxygen consumption, 1, 3, 24, 29, 30,
38, 40, 45, 48, 73, 86, 123, 154, 157,
159, 164

_p_

Padykula, 18, 162, 166

Parnas, 8

parvalbumin, 20, 158

Passive tension, 49, 72

PCA, 3, x, 3, 75, 110, 111, 112, 126

PCr, 2, vii, viii, ix, x, 2, 3, 5, 6, 9, 12, 15,
20, 23, 29, 30, 35, 38, 45, 46, 49, 50,
51, 54, 55, 57, 58, 60, 61, 62, 63, 64,
69, 75, 76, 78, 80, 81, 82, 83, 84, 85,
86, 87, 88, 95, 110, 111, 112, 114,
115, 116, 117, 118, 119, 120, 121,
122, 124, 126, 130, 131

PCr breakdown, 9, 20, 30

PCr hydrolysis, vii, 6, 9, 13, 17, 20, 23,
30, 38, 44, 51, 55, 58, 88

PCr recovery, 2, viii, x, 3, 39, 45, 46,
58, 60, 61, 64, 70, 112, 114, 115, 116,
117, 118, 119, 120, 121, 122, 127,
130, 131

PCr/Tc, 66

PCrss, 44, 86, 87

pentobarbital, 48, 71

PEP, 11

peripheral vascular disease, 43

Peter, 25, 145, 163

Pette, 127, 145, 153, 158, 163

pH, 2, ix, x, 2, 26, 28, 36, 39, 45, 50, 51,
54, 55, 58, 59, 61, 62, 63, 64, 66, 69,
73, 75, 76, 88, 89, 90, 91, 92, 93, 94,
95, 121, 122, 123, 124, 132, 144, 147,
152, 157, 158, 160, 161, 163, 164,
166, 168

pH heterogeneity, 69, 168

pH inhomogeneity, 42

pH optima, 123

phosphagen, 9, 15, 44, 46, 150, 166

phosphate acceptor, 14

phosphocreatine, 2, vii, viii, 4, 5, 15,
21, 44, 46, 51, 69, 126, 147, 151, 157,
160, 161, 162, 165, 166, 167, 168

174

phosphofructokinase, 27 , 38, 124

phosphorus, 1, 35, 56, 69, 74, 79, 145,
149, 157, 160

phosphoryl, 11, 15, 37, 43

photorespiration, 40

photosynthesis, 40, 163

photosynthetic efficiency, 23

Pi, 2, 6, 9, 25, 36, 39, 45, 51, 54, 55, 67,
69, 76, 88, 95, 122, 125, 127

Pi peak splitting, 42, 69, 127

Pi/PCr, 39

Piiper, 24, 30, 163

plankton (232), 40

plant, 7, 33

plantaris, 96

plants, 23

Plexiglas, 135

poultry, 26

power, 2, 36, 40, 45, 50, 73, 75, 110,
126, 157

Principal Component Analysis, 3, viii,
3, 71, 75, 110, 111, 130

proton, 25, 33, 50, 55, 69, 73, 88, 121,
131, 162

psoas, 14

pyridine nucleotides, 13, 25, 157

pyruvate, 31, 161

_Q_
Q10, 20
Q02, 1, 30, 32, 65, 159
quadriceps, 125, 150, 153, 166
quantum, 32

—R—

rabbit, 14, 26, 153, 158

Radda, 41, 144, 147, 155, 157, 163, 167

radiofrequency, 50, 74

rat, 2, v, 3, 18, 37 , 38, 46, 47, 48, 64, 69,
71, 72, 73, 75, 79, 86, 110, 111, 112,
113, 114, 115, 120, 122, 123, 124,
136, 140, 141, 142, 144, 145, 149,
150, 152, 155, 156, 157, 160, 163,
164, 165, 166, 167, 168, 169

rate constant, 2, x, 39, 45, 46, 51, 59,
60, 64, 130

recovery, 2, viii, x, 3, 6, 8, 17, 23, 30,
38, 45, 46, 51, 55, 58, 60, 61, 63, 64,
70, 73, 74, 75, 76, 77, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 106, 112, 113, 114, 115,
116, 117, 118, 119, 120, 121, 122,
124, 126, 130, 131, 137, 144, 146,
149, 153, 154, 156, 157, 158, 161,
163, 168, 169

recovery kinetics, 2, 39, 120, 121, 122,
168

recruitment, 1, 32, 43, 69, 127, 149

rectal thermistor, 49, 72

rectus abdominus, 14

Red, 26, 28, 136

red blood cells, 33

relaxation time, 28, 29, 54, 70, 106,
155

resonance stabilization, 11

resonant frequency, 34

respiratory chain, 13, 148

Rupp, 13, 161

_s_

saddle shaped transceiver coil, 135

Sahlin, 25, 120, 148, 153, 164

Sandberg, 17 , 165

Sandow, 18, 165

sarcoplasmic reticulum, 18, 158, 169

sartorius, 14, 20, 147, 157, 158

sciatic nerve, 4, 48, 72

SDH, 26, 28, 127

second law of thermodynamics, 5

Shepherd, 48, 148, 165

shortening, 17, 20, 23, 156, 157, 166

SigmaPlot, 51

signal processing, 32

Size Principle of motor unit
recruitment, 32

Sliding Filament Theory, 14, 17

slow twitch muscle, 31

SO, 2, 32, 136

sodium azide, 24

soleus, 20, 67, 96, 120, 123, 152, 163

spatial distribution, 37, 127, 133

spectroscopy, 2, vii, 1, 32, 46, 69, 137,
144, 145, 147, 158, 160, 161, 162, 169

SR, 18, 22, 29, 43, 127

stackplots, x, 56, 79

staining, 1, 26, 28, 127

Stainsby, 30, 166

staircase phenomenon, 96

175

steam engine, 5, 20

Subbarow, 6, 151

submaximal, 2, vii, x, 3, 45, 46, 50, 55,
56, 57, 58, 60, 61, 66, 78, 116, 118,
119, 120, 124, 130

submaximal stimulation, 2, vii, 3, 45,
55, 56, 57, 58, 60, 61, 66, 120

succinate dehydrogenase, 26, 127

superconducting, 33, 50, 74, 110

superconducts, 33

supramaximal, 3, x, 4, 49, 64, 66, 72,
106, 120, 124, 132

surface coil, 49, 72, 73, 132

—T—
T1, 70

T2, 70, 152

tau, 115, 116, 117, 119, 127
Teflon, 16

temperature, 5, 13, 16, 44, 49, 71, 72,
137

Terjung, 32, 144, 150, 155, 159, 161,
164, 167

tetanic, 18, 21, 44, 54, 152, 155, 166

tetanic force, 54

thermochemistry, 15

thermodynamic efficiency, 15, 21, 23

thermodynamics, 5, 169

thermopiles, 16

Thunberg, 7, 167

thyroglobulin, 139

Thyroid Binding Globulin, 139

thyroid hormone, 27 , 47, 139, 155, 158,
165

time constant, viii, 3, 38, 39, 45, 46, 47,
58, 70, 76, 77, 112, 114, 116, 119,
120, 123, 126, 130, 131

time to peak tension, 28, 29

titanium, 33

tortoise, 13

TPT, 106

TR, 50, 55, 56, 74, 137

Trained, 47, 50, 52, 53, 54, 55, 58, 61,
65

training, 2, 27, 31, 40, 47, 129, 132,
140, 141, 149, 151, 154, 156, 167

transient analysis, viii, 39, 69, 126

transmission cable, 40

transverse relaxation, 70

troponin, 20, 29, 153

TTI, 30

Tukey, 33, 51, 77, 148

twitch, 2, x, 14, 20, 21, 31, 38, 44, 46,
49, 50, 52, 54, 58, 61, 62, 66, 70, 72,
74, 86, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 108, 122, 144,
145, 147, 149, 152, 153, 155, 158,
160, 166

twitch force, x, 49, 52, 54, 61, 62, 67 ,
72, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 108

Type I, 1, 2, 26, 28, 42, 52, 70, 120, 127,
131

Type II, 1, 2, 26, 28, 42, 52, 70, 131

Type 11a, 52

Type IIb, 52
—-U—
unexplained enthalpy, 20
_v__

velocity, 13, 21, 44, 48, 121, 166
velocity of shortening, 21
V02max, 2, 1, 2, 31, 48

_w_

Walter, 124, 168

Watt, 22

Weber, 13, 168

White, 26, 28, 136

Wilkie, 21, 147, 149, 157, 169

Williams, 13, 148, 154

Winegrad, 18, 169

Woledge, 20, 149, 169

work, 5, 7, 9, 13, 15, 21, 31, 38, 39, 67,
70, 144, 148, 151, 152, 155, 156, 165,
169

wrist flexor, 39, 161

—Y—
Young, 8, 153

176

"llllllllllllllllf