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dissertation entitled

THE METABOLIC CORRELATES OF FATIGUE IN

8/6/90

SKELETAL MUSCLE
presented by

Gregory Rex Adams

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THE METABOLIC CORRELATES OF FATIGUE IN SKELETAL MUSCLE

By

GREGORY REX ADAMS

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Physiology
1990

ABSTRACT
THE METABOLIC CORRELATBS OF FATIGUE IN SKELETAL MUSCLE
By

Gregory Rex Adams

Changes in the intracellular concentrations of lactic acid, hydrogen ion (H+),
inorganic phosphate (Pi), and diprotonated Pi (I-12P041-) have been suggested as causes
of fatigue in skeletal muscle. Nuclear magnetic resonance spectroscopy (NMR) was
used to measure changes in these metabolites during muscle contraction. Fast-twitch
biceps and slow twitch soleus muscles were surgically isolated, arterially perfused and
removed from anesthetized cats. Individual muscles were mounted in an NMR probe
equipped with a force transducer. Intracellular pH and the concentrations of PCr,
ATP, Pi and H2P041- were calculated from 31P-NMR spectra. In one set of
experiments interleaved 1H-NMR spectra were collected and used to calculate lactic
acid concentrations.

In yivg fluffer Capacity Gated 31P-NMR spectra were acquired after 9 seconds of 5
Hz stimulation in cat muscles. Net PCr hydrolysis was associated with an intracellular
alkalinization of 0.08i0.01 (meaniSE, n=3) pH units in the biceps and 0.05 $0.003
(n=3) in the soleus. The net change in [H+] expected from PCr hydrolysis was
calculated from B = AH+ / ApH. Buffer capacity was also estimated from titration of
muscle homogenates. The contribution of Pi to total B of the homogenates was
subtracted to ascertain the non-Pi B for each muscle. The non-Pi B values were added
to the actual amount of Pi present in stimulated muscles to calculate a predicted B at pH
7. The apparent B calculated from PCr and pH changes in intact muscles and the
predicted B were in good agreement (3819 vs. 38, cat biceps, 21 :7 vs. 30, cat
soleus). The results indicate that changes in pH during the first few seconds of

contraction can be accounted for by proton consumption via net PCr hydrolysis.

1W The expected pH change resulting from 1H-
NMR measured changes in lactic acid was compared to the actual ApH measured by
31P-NMR. Lactic acid production did not appear sufficient to account for the total
ApH. Addition of albumin to solutions of lactic acid caused an attenuation of the lactic
acid signal indicating that some portion might be bound and therefore NMR invisible.
W Peak tetanic tension development was measured during
acidosis resulting either from hypercapnia (70% C02) or repetitive stimulation in
isolated perfused cat muscles. During repetitive stimulation both acidosis and increased
[HZPOJ'] were correlated with decreased force production. However, neither acidosis
nor increased [I-12P041-] resulting from hypercapnia was related to changes in tetanic
tension. These results show that neither acidosis nor increased [H2P0,1-] directly cause
muscle fatigue. The observed correlations between these parameters and force
development during normocapnia appear to be coincidental to some other effect of

repetitive stimulation.

To my parents, Marvin and Jean Adams

iv

ACKNOWLEDGEMENTS

First and foremost I wish to thank my mother and father and my brother
and his family for their support and understanding as I pursued this goal.
Absorbed in academic pursuits I have neglected you all but you never
complained, thank you.

To my mentor Dr. Ron Meyer my heart felt thanks. My training in your
laboratory combined both the independence needed to develop self confidence
and the guidance needed to become a scientist.

I would also like to thank my second mentor and dear friend, Dr. Jack
Krier. Thanks to you, the word philosophy in Ph.D. is probably appropriate in
my case.

Dr. Jill Fisher, guidance committee member, co-investigator, critic,
councilor and friend. I hope I can become as critical a thinker, serious a
scientist, and compassionate a human being as you have.

Dr. Frantz and Dr. Dillon, both had a hand in guiding my graduate
career long before joining my committee. In particular I would like to thank
you both for your contributions to my development as a teacher.

My interactions with Dr. Thomas Adams contributed greatly to my
development both as a scientist and person.

Thanks to Dr. Paul Desrochers who provided my indoctrination to
graduate life. Without your help I would not have survived the early years of
my program.

Thanks also to Tim Dennerll, Jean Folley, Jerry Lepar and Don Tillet,
fellow graduate students whose help and friendship was invaluable.

Dr. Mary Lynn Bajt your love and support contributed greatly to my
success. Both personally and professionally you kept my life exciting.

TABLE OF CONTENTS

List of Tables vii
List of Figures viii
General Introduction 1
Chapter 1 Muscle buffer capacity estimated from pH
changes during rest to work transitions 14
Introduction 15
Methods 17
Results 22
Discussion 40

Chapter 2 Measurement of lactate accumulation
in contracting skeletal muscle using

1H nuclear magnetic resonance. 43
Introduction 44
Methods 46
Results 48
Discussion 58
Chapter 3 Increased H+ and H2P041- do not cause
fatigue in skeletal muscle. 60
Introduction 61
Methods 62
Results 67
Discussion 88
Summary and Conclusions 92
Appendix 96

List of References 113

vi

Table 1.
Table 2.
Table 3.
Table 4.
Table 5.

LIST OF TABLES

Measurements in muscle homogenates
Metabolite contents of unstimulated muscle
Calculation of B from in viva measurements
pH and metabolite concentrations

Contraction characteristics

vii

27
28
39
71
74

Figure 1.

Figure 2.
Figure 3.

Figure 4.
Figure 5.
Figure 6.
Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 1 1.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

LIST OF FIGURES

Twitch force record illustrating protocol for
gated NMR data collection.

Titration of muscle homogenates.

Total and non-phosphate buffer capacity of
cat and rat skeletal muscles.

31P-NMR spectra of homogenates from cat soleus
and biceps muscles.

31P-NMR spectra collected using protocol
illustrated in Figure 1.

Gated 31P-NMR spectra from rat gastrocnemius
muscle.

pH changes during and after stimulation in
cat and rat muscles.

1H-NMR SSECHO spectra from an isolated
perfused cat biceps muscle stimulated at
1 Hz for 6 min.

Changes in lactate and PCr during stimulation
and recovery in cat biceps muscles.

Intracellular pH measured by 3‘P-NMR and
calculated from lH-NMR measured change
in lactic acid.

Changes in lH-NMR measured lactic acid
peak area with addition of albumin.

The effect of perfusate equilibration
with increasing percentage of CO; on
intra and extracellular pH

31P-NMR spectra from cat biceps and soleus
muscles illustrating experimental protocol.

Sample force records from cat muscles
during 5% and 70% C02 perfusion.

Time course of force and metabolite changes

in cat biceps muscles during repetitive
tetanic stimulation

viii

20

24

26

30

33

36

38

50

52

54

57

70

73

76

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.

Figure 26.

Time course of force and metabolite changes
in cat soleus muscles during repetitive
tetanic stimulation

Relationship between peak tetanic force and pH,
in cat muscles during 5% or 70% C02 perfusion

Relationship between peak tetanic force and
[H2P041'] in cat muscles during 5% or 70% C02
perfusion.

Relationship between peak tetanic force and

Pi in cat muscles during 5% or 70% C02
perfusion.

Relationship between pH and time to relax from
90% to 10 % of peak tetanic force in cat muscles
during 5% or 70% C02 perfusion.

NMR probe for isolated perfused muscle
experiments.

Diagram of NMR coil and tuning
circuits.
Helmholtz NMR coil.

Detail of variable length muscle mount
with force transducer.

Wheatstone bridge with microfoil strain
gauges.

Calibration plot for Delrin force
transducer.

ix

78

8O

82

85

87

102

104

107

109

112

General Introduction

2
Most people recognize from personal experience that sustained physical activity

leads eventually to a decline in muscle performance. This fatigue has both neuro-
physiological and cellular causes. 0n the cellular level fatigue is generally thought to
result from the depletion of substrate for energy metabolism and/or the accumulation of
metabolites. The metabolites most commonly suggested as agents of fatigue are lactic
acid1 , hydrogen ion (Flt) and some form of inorganic phosphate (P,).

This dissertation focuses on the cellular biochemical factors which mediate
fatigue. Initial studies provide information on the relationship between changes in H+
production, lactate production and intracellular pH. Changes in isometric tension were
measured while metabolite concentrations were experimentally altered. Nuclear
magnetic resonance spectroscopy (NMR) was used to measure concentration changes in
fatigue related metabolites (i.e., H+ , Pi, and lactic acid) before, during and after
fatiguing muscle contraction.

The remainder of this introduction will review concepts related to muscle
metabolism and fatigue. The format of the sections on specific metabolites is intended
to present current doctrine and to discuss possible limitations of the underlying
research. The final sections introduce the specific questions asked, and the methods
and technology used, in the studies presented in this dissertation. This general
methodological introduction is intended to delineate the attributes of the present studies
which address the research limitations discussed in the preceding historical sections.
MuxfiMetamLism Many of the biochemical reactions related to muscle contraction
were identified during the first half of this century. A major goal had been to identify
the ”primary reaction” linking the liberation of chemical energy to the mechanieal
generation of force. One of the earliest studies showed that muscle contraction resulted
in the production of lactic acid. This observation lead to the theory that lactic acid was
the link between chemical energy and mechanical force production for muscles (54,56).

 

I At physiological pH lactic acid is essentially 100% ionized. The term lactate refers to the anionic form
of lactic acid and the two are often used interchangeably.

3
This so—called "lactate theory of contraction” required redefinition during the early

1920' s when it was discovered that of phosphorylated compounds are hydrolyzed
during muscle contraction (54,99). Hydrolysis of phosphocreatine (PCr) was identified
as the immediate reaction associated with contraction. The lactate producing pathway
was, however, still considered to be the primary provider of chemical energy for
muscular contraction (54). Although, processes involved in oxidative metabolism were
well known at this time, they were thought to play a role only during recovery from
contraction as an adjunct to lactate production of chemical energy (99).

The discovery of the hydrolysis of adenosine triphosphate (ATP) and the
regeneration of ATP at the expense of PCr was the last major refinement of the theory
of energy transduction in muscle (99,100). Current theory holds that the transduction
of chemical free energy into mechanical work is accomplished by hydrolysis of ATP by
the actomyosin ATPase2 during the crossbridge cycle. The resultant adenosine
diphosphate (ADP) is rephosphorylated by donation of Pi from PCr via the creatine
kinase reaction. In the presence of oxygen, the energy for regeneration of PCr and
ATP is supplied by oxidation of carbohydrate and lipid resulting in production of water
and C02. When there is insufficient oxygen to support energy demands via oxidation,
the excess demand is met by the break down of carbohydrate with the production of
lactic acid.

Fatigue The etiology of muscle fatigue has been the focus of investigation for
more than one hundred years (41). Despite continuous investigation, a definitive
explanation of the mechanisms underlying contractile failure has yet to be established.

In order to discuss fatigue it is necessary to first present a precise definition of
it. This definition must include specific information on both intensity and duration of
contractile activity. Based on these criteria two distinct forms of cellular fatigue with

differing etiologies are commonly recognized. "Low intensity" fatigue is the

 

2 The contractile proteins actin and myosin form the cross bridge complex which has enzymatic activity.

4
depression of muscle performance resulting from prolonged, sub-maximal contraction

as seen in endurance running. This form of fatigue is generally attributed to depletion
of substrate for energy metabolism (8,49). ”High intensity” fatigue results from short
duration, near maximal contractions that might be encountered in heavy resistance
training. Under these conditions muscle cells often retain significant stores of substrate
for energy metabolism. This high intensity fatigue is thought to result from the
accumulation of metabolites or some toxin within muscle cells (45). The work
presented in this dissertation is concerned with the high intensity form of fatigue.

The immediate substrates and products of the actomyosin ATPase are ATP,
ADP, Pi, H+ and Mg2+ . As such these ions and compounds have become the focus of
much of the research on the mechanisms of fatigue. In addition, the development of
fatigue is often accompanied by an accumulation of lactic acid.

W One of the earliest, and longest lived, hypotheses is that
production and/or accumulation of lactic acid causes fatigue (38,56). From the
beginning of this century to present time, many investigators have reported a direct
correlation between lactate accumulation and depression of force (9,37 ,38,49,55, 103).

Direct measurement of lactate has traditionally involved homogenization of
tissue with subsequent biochemical assay (38,48). Metabolite measurements obtained
by this method often include significant discrepancies due to breakdown of metabolites,
release of bound molecules and loss of cellular and subcellular compartmental integrity
(3,30,31,86,120).

Some experimental results have called into question the role of lactic acid
accumulation in fatigue. Intracellular accumulations of 15-30 mM lactate are ofien
associated with fatigue (48). In contrast, Mainwood and Alward have produced fatigue
in superfused rat diaphragm when the biochemieally measured lactate concentration was
only 7mM (78). Following glucose loading, produced by preincubation with insan
and glucose, this preparation accumulated 17.5 mmol/ g lactate yet exhibited the same

5
degree of fatigue as seen with 7 mmol/g (78). These results demonstrate an apparent

dissociation of lactate accumulation and fatigue (11,78). However, superfused muscle
preparations, such as those used by Mainwood and Renaud, depend on diffusion for
delivery of substrate and removal of waste products. Regions of the muscle more than
2-3 cell layers thick may be functionally impaired by insufficient diffusion especially
during the metabolic stress of vigorous contraction. Thus the results of this study may
reflect some effect on oxidative metabolism.

Much of the current dogma on lactate production during exercise and/or
ischemia is based on measurement of blood lactate levels (61,62,134). This indirect
method of lactate assessment also suffers from several drawbacks which tend to
confound interpretation of results (14,63,123). First the lactate content of venous
blood will be the sum of the contribution and extraction of that metabolite by all the
tissue traversed since the blood entered the exchange vessels (14,44,51,62,111,112).
Second there is some latency in the appearance of lactate in the blood following its
production and accumulation in cells (51). In addition, there have been studies
showing that significant amounts of lactate are recovered as glycogen or oxidized in
both the working muscles and adjacent non-working muscles (62,92).

Chase and Kushmerick studied the effect of 50mM lactate on contraction in
skinned rabbit psoas muscle fibers independent of pH (21). A skinned muscle fiber
consists of intact myofilaments without the sarcolemma and sarcoplasm
(21,26,33,36,8l,96). The investigator supplies an artificial sarcoplasm, allowing for
controlled addition of metabolites inorder to study contractile behavior. When pH was
held constant at either 7.1 or 6.0, 50mM lactate per se had no effect on force
production. This suggests that the often-observed correlation between lactic acid
accumulation and fatigue may result from the concomitant production of H+ rather than
some effect of lactate.

6
W In 1880, Gaskel reported that acidification of cardiac muscle

caused a decrease in the force of contraction (41). Subsequently, many studies have
reported a direct relationship between changes in intracellular pH (pHi) and the
contractile properties of striated muscle (21,26,30,33,36,50,82,85,96,102).

The earliest measurements of pH at fatigue were made in minced muscle (40).
This technique results in a loss of cellular integrity and the possibility of continued or
accelerated hydrolysis of substrate and measurements may not reflect in vivo conditions
(97). The addition of a quick freezing step, often while muscles are in situ and
contracting, has helped to reduce the variability resulting from continued metabolic
activity in homogenized muscle samples (40).

Early methods of pH measurement in intact muscle cells involved the use of pH
sensitive dyes (97,117). It is difficult calibrate these dyes which limits their usefulness
(80,97). A later method involved following the distribution of weak acids such as
radioactive DMO (5,5-dimethyl-2,4-oxazolidine-dione-2-14C). This technique is
difficult to implement, destructive to the tissue, and it lacks temporal resolution
(e.g., ~1hr.in skeletal muscle) (80,97,117). In addition, recent evidence indicates that
DMO measurements represent some average intracellular pH, heavily weighted toward
the mitochondrial pH (1). Intracellular pH can also be measured using pH sensitive
microelectrodes (2,3,117). This technique requires the penetration of single muscle
cells limiting measurements to surface fibers (117). In addition, cell movement
precludes the use of this method during muscle contraction (17,80).

Despite limitations in absolute quantification of pI-Ii at fatigue, the consensus of
the majority of studies to date is that decreases in pI-Ii and force are directly correlated
(21,26,30,33,36,48,50,82,85,96,102).

Several mechanisms have been proposed which may account for pH effects on

contraction. They stem from two more general biochemical observations. First,

7
changes in hydrogen ion concentration ([H+]3), in the physiologicaly relevant range,

will alter the state of ionogenic groups at the active site of enzymes, thereby
modulating their reactivity (122). Second, protein conformation is also sensitive to
[11*], providing further opportunity for alterations in function (122). With specific
regard to muscle fatigue, H+ modulation of proteins reportedly results in: (i.) an
inhibition of glycolysis (118), (ii.) the disruption of excitation-contraction coupling
(36,39,60,90,97), (iii.) a decrease in the calcium (Ca2+) sensitivity of actomyosin
formation (21,26,33,34,36,39,65,82,91), and (iiii.) the alteration of Ca2+ uptake (60)
or release by the sarcoplasmic reticulum (77,90).

In summary, contracting skeletal muscle produces lactic acid when energy
demands exceed the capacity for oxidative regeneration of adenosine triphosphate. At
physiological pH and temperature, lactic acid (pK = 3.7) dissociates producing lactate
anion and hydrogen cations. Coupled with the aforementioned effects of H+ on force,
these observations provide the basis for the most widely held theory of fatigue resulting
from high intensity contractile activity: i.e. , an increase in intracellular hydrogen ion
concentration [H+] , resulting from lactic acid production, is responsible for decreases
in both maximum tension development and maximum velocity of shortening
(10,21 ,34,36,81 ,82, 102,1 10,).

W In biological systems H+ production is seldom linearly related to the
resulting change in p11,. Some portion of the H+ produced will be buffered by
chemical binding to Pi and ionogenic groups on proteins and peptides (108,122). The
change in pH resulting from a given increase in H+ is a function of the buffer capacity
(B) of the cells.

In order to assess the effects of changes in [H+] on contraction both the change
in H+ and the buffer capacity of a given muscle must be known. This parameter
(e.g.,B) is of particular importance when changes in pH are calculated from the

 

3 The brackets [] indicate concentration.

8
expected H+ production resulting from other metabolic changes such as increases in

lactate (59,105).

The conventional method for measuring buffer capacity has been to homogenize
the muscle and titrate the crude homogenate. These titrations have been conducted
under widely varying conditions of temperature, presence of metabolic inhibitors, and
C02 content. This has lead to substantial variation (i.e., from 10 to 116 Slykes) in
buffer capacity values reported in the literature (3,4,13,18,28).
We Contracting muscle can accumulate 25-35 mmol/g of
inorganic phosphate (P,) (30,86,115). This metabolite seems to have a paradoxieal role
in contracting muscle. There is evidence that an increase in P, is necessary to activate
energy metabolism (22,26,67) but will inhibit force generation (26,52,53,65).

In 1978 Dawson et al. noted an apparent correlation between increased
intracellular [P,] and decreased force (30). Subsequently, Hibberd et al. demonstrated
that elevated P, decreased maximum tension development in skinned rabbit psoas fibers
(53). The P, dependence of force generation has been demonstrated in many studies
since that time (5,35,65). However, within this body of literature there is variation in
the characteristics of reported P, effects. In the intact muscle studies reported by
Dawson, the major effect of P, on force is seen when concentrations increase above
20mM, below that level only slight depression of force is noted (29,30). However, in
studies employing the skinned fiber model, P, appears to depress force as the
concentration increases to 15 mM at which point force levels off (5,26,65).

In Dawson's experiments with intact muscle, P, was increased by contractile
activity (29). This contractile activity also decreased the intracellular pH. In the
physiological range of pH, inorganic phosphate will exist as I-IP042- or H2P041- with a
pK of 6.75 at 37°C. The proportion of H2P041' present can be calculated from total P,,
pH and pK. When Dawson et al. plotted [HZPOfl'] against force a highly linear

relationship was observed suggesting the possibility of a cause and effect relationship

9
(29). This observation has received support in subsequent studies of intact muscles

(89,127) and skinned muscle fibers (91). Based on these findings it has been
hypothesized that decreases in pH, indirectly cause fatigue by increasing the
concentration of H2P041' which directly interferes with the force producing step in the
crossbridge cycle (91).

In contrast, several recent skinned fiber studies have failed to find a pH
dependence of P, mediated decreases in force (21 ,25,41,65). Despite these
contradictory findings the H2P04I- theory is entering the dogma of fatigue mechanisms.
Sumary lactic acid accumulation during contraction is thought to eause a decrease
in intracellular pH. This decreased pH appears to cause fatigue, either directly or
indirectly, by alteration in the concentration of H2P04I'.

Each component of this theory suffers from some methodological limitation
and/or inconsistent experimental finding. Results from muscle homogenates tend to
over- or underestimate metabolite concentrations. Blood lactate levels represent a
spatial and temporal composite of metabolic events upstream from the sample site.
Most of the available pH measurement techniques lack temporal resolution.

Isolated, superfused muscle preparations have been used for data collection.
However, insufficient diffusional movement during the high metabolic activity
associated with contraction might lead to significant cell to cell metabolic heterogeneity
within a muscle.

Studies of skinned fibers allow for the control of more variables than either
homogenized or intact muscles. Unfortunately, the physiological relevance of data
collected from a preparation lacking both sarcoplasm and a cell membrane is open to
question. In particular, the substitution of a homogeneous buffer solution for the
complex gel-sol sarcoplasm may significantly alter access of metabolites to the
contractile proteins. This may explain the discrepancy in P, effects seen in intact vs.
skinned fibers (29,30,and present Chapter 3 vs. 5 ,26,65).

10
A more global problem which applies to most studies results from use of

muscles without regard for fiber type composition. Changes seen in these studies may
be a function of sampling from a nonrepresentative population and/or averaging across
a heterogeneous population. For example, a given stimulation pattern may
preferentially activate a particular fiber type. A measured decrease in force production
from this protocol would result from fatigue of selected fibers. Metabolic
measurements from the whole muscle would not accurately reflect the state of just the
activated fibers.

It is clear that much of the research on the metabolic basis of fatigue has been
collected under conditions of severe methodological limitation. Newer technologies
hold the promise of data collection from preparations which more closely mimic in vivo
physiological conditions.

W NMR is a non-destructive technique which can
be used to collect data from muscles in vivo‘. This method allows for multiple
collections of data from the same muscle, eliminating experimental variability resulting
from muscle to muscle (i.e. , animal to animal) differences. The non-destructive
collection of data preserves the internal structure of the muscle cells avoiding the
physical mixing of compartment contents and/or a loss of compartmented function seen
in earlier studies.

Standard NMR collections have temporal resolution of seconds or minutes
eliminating much of the averaging seen with previous methods. In addition, gating
techniques, such as those detailed in chapter one, can reduce the time for data
collection into the millisecond range.

A major limitation of NMR as a tool in biological studies is its relative
insensitivity. While other forms of spectroscopy (e.g. , UV light or fluoresence) can be
used to detect metabolite concentrations in the “M (106M) range, biological NMR is

 

4 The term in vivo is used here and henceforth to mean 'chemical processes occurring within cells, etc.,
as distinguished from those occurring in cell free extracts'. -Stedman's Medical Dictionary 23 Ed.

1 1
limited to detecting mM (103 M) concentrations. This results in an inability to detect

metabolites such as ADP or adenosine monophosphate which have unbound
concentrations in the uM range.

WGIP-NMR) 31P-NMR collections provide data on the intracellular
concentrations of phosphorous containing compounds and ions such as ATP, PCr and
P,. The chemical shift of P, can be used to calculate the intracellular pH of muscles
while a pH sensitive extracellular marker, phenylphosphonate, can be used to calculate
extracellular pH (86). The intracellular concentration of Mg2+ can be calculated from
the chemical shift of ATP (46,69). As noted earlier, the immediate substrates and
products of the actomyosin ATPase are ATP, ADP, P,, H+ and Mg“. The similarity
between the list of parameters amenable to measurement by 31P-NMR and those of
interest in contractile reactions indicates the suitability of this technique for fatigue
studies.

W As the name implies, proton NMR (lH-NMR) has the potential to
measure any compound containing hydrogen. Biological samples contain many such
compounds in concentrations sufficient to be measured by NMR (i.e. >0.5mM).
However, the high concentrations of protons in water, proteins and lipids tend to
obscure signals from most other compounds. Recent advances in techniques for the
subtraction or suppression of these interfering signals have made quantifieation of
concentrations of metabolites such as lactate possible (47 ,49,66,67 ,1 19, 125). The
ability to measure lactate in a living muscle with good temporal resolution promises to
overcome many of the limitations of previous methods.

W15 The muscle models and techniques chosen for the studies presented in
the following chapters were selected in an effort to avoid the limitations which have
plagued similar research in the past. The primary models for these studies are the
isolated perfused cat soleus and biceps muscles. The cat soleus consists of greater than
92% slow twitch, oxidative fibers, the biceps contains 71% fast twitch, glycolytic,

12
24% fast twitch, oxidative, and 5% slow twitch, oxidative fibers (86). Studies

employing these two muscles provide a good comparison of effects of various
experimental perturbations in the two primary fiber types (fast vs slow). The relatively
pure fiber type of these muscles allows the assumption that the changes seen result from
uniform effects on the majority muscle fiber population rather than a small minority
population.

Controlled perfusion allows matching of flow to the metabolic needs of the
muscle both at rest and during exercise. The metabolic state of the muscle can be
constantly monitored by 31P-NMR. These two factors allow collection of data from
muscles relatively free from the impairment seen in superfused muscle preparations.

The aim of this dissertation has been the collection of both 1H and 31P-NMR
data from intact, isolated perfused muscles in an attempt to further characterize
metabolic and functional changes as they relate to the development of fatigue.

Chapter 1 presents data from experiments designed to accurately measure buffer
capacity in viva. The results from these experiments provide information which will
allow the calculation of buffer capacity as it changes during contraction. This
parameter can then be used to assess the effects of other metabolic reactions such as
lactate production on intracellular pH.

The experiments detailed in Chapter 2 deal with in viva measurement of lactate.
The aim of this work was to calculate the change in pH, expected for a measured
accumulation of lactate. This calculation included dynamic buffer capacity values
provided by the results from Chapter 1. The pH, calculated from lactate measurements
was compared to the empirically measured pH, (“P-NMR) to determine if lactate
production could account for the changes seen.

In the final chapter intracellular [H’r] and [I-12P041-] were altered independent of

contractile activity by hypercapnia. The question asked in this work was whether the

13
reported correlations between increases in these metabolites and the development of

fatigue represent a cause and effect relationship.

Chapter 1

Muscle buffer capacity estimated from pH changes
during rest-to-work transitions.

14

15
INTRODUCTION

One essential characteristic of muscle cells that must be known, in order to
measure the effects of changes in H+ production on contraction, is buffer capacity (B).
In biological systems H+ production is seldom linearly related to the resulting change
in pH. The difference arises from the binding of 11+ to P, and ionogenic groups on
proteins and peptides (108,122). Thus a change in pH resulting from a given increase
in H+ is a function of B.

The conventional method for measuring buffer capacity has been to homogenize
the muscle and titrate the crude homogenate. These titrations have been conducted
under widely varying conditions of temperature, CO2 content, and metabolic inhibition
(12,18,93). This has lead to wide variation in buffer capacity values reported in the
literature (3,4,13,18,28).

31P-NMR can be used to measure intracellular pH (pH,) in intact skeletal
muscle. Given conditions where a known amount of H+ is produced or consumed, and
the concurrent change in pH, measured by 31P-NMR, the apparent B could be
calculated.

The intracellular pH of skeletal muscle becomes transiently alkaline within
seconds after the onset of a series of twitch contractions (86,107,113). In many recent
studies using phosphorus NMR for pH measurements (19,70,86,107,ll3), both the
magnitude and time course of the initial alkalinization suggest that it is at least partly
due to proton consumption associated with net phosphocreatine (PCr) hydrolysis :

ATP+H20 —> ADP+P,+a,H+
PCr + ADP + H+ <

 

> creatine + ATP

 

 

PCr+H20+aPH+ > Cr+P,,
where the coefficient a, = (l-a.) is 0.4 at pH 7 and increases at lower pH (42,70).

Assuming an intracellular buffer capacity of 40 Slykes for mammalian skeletal muscle

l6
(12,18,93), net hydrolysis of 10 pmol/g PCr at pH 7 should produce a maximum

alkalinization of 0.10 pH units. This small alkalinization is consistent with what is
typically observed in NMR studies, suggesting that PCr hydrolysis might be entirely
responsible for the transient alkalinization.

In contrast, Connett (24) recently reported a much larger transient alkalinization
(0.75 pH units) after 5 seconds of twitch contraction in dog gracilis muscle. pH was
estimated by chemical assay of metabolites and the calculation of H+ using in vitra
equilibrium constants for 5 enzymes. An alkalinization of 0.75 pH units is too large to
account for by net PCr hydrolysis alone. The alkalinization calculated in Connent's
study was reversed after 15 seconds. This early alkalinization would have been missed
by NMR studies in which data accumulation was averaged over the first 15 or more
seconds of a contraction series (71,85).

The purpose of this study was to exploit this early alkalinization to measure B in
intact skeletal muscle. To do this it was necessary to determine whether or not the
transient alkalinization observed during the first few seconds of a series of contractions
can be quantitatively accounted for by net PCr hydrolysis. This was accomplished by
comparing the buffer capacities (B, Slykes = AHflApH) of three different mammalian
muscles calculated from titrations of muscle homogenates in vitra with their buffer
capacities estimated from the observed alkalinization during brief series of contractions.
The latter was calculated assuming that net PCr hydrolysis was the only significant
metabolic reaction effecting pH, i.e., A[H+] = up it A[PCr], and hence:

I. = orp x A[PCr] / ApH.

PCr and pH were measured at discrete times by gating acquisition of phosphorus NMR

seans to specific times during and after repeated bursts of 5 Hz contractions.

17
The results support the assumption that net PCr hydrolysis is the only significant

metabolic reaction affecting pH during this time period. The H+ consumption of this
reaction is known and thus an in viva B can be calculated from the observed changes in
pH and PCr.

METHODS
Whistles.

These studies were performed on three animal muscle preparations described
previously: the superficial 2-3 mm (predominantly fast-twitch glycolytic) portion of the
rat gastrocnemius muscle in situ (70,85), and the isolated, arterially perfused cat biceps
brachii (fast-twitch) and soleus (slow-twitch) muscles (71,86).

Male Sprague-Dawley rats (300-350 g) were anesthetized with sodium
pentobarbital (50mg/kg, IP), and an IP catheter inserted to allow delivery of further
anesthetic as needed. The sciatic nerve was dissected free, cut, and placed within a
bipolar platinum electrode. Rats were mounted in a specially designed NMR probe
with the Achilles tendon attached to a force transducer as described previously (85).
Stimulation voltage and muscle length were adjusted to produce maximum twitch force.

Male or female cats weighing 34 kg were sedated with Ketamine (11 mg/kg,
1M) and anesthetized with sodium pentobarbital (30 mg/kg, iv.). The biceps (7.5 i0.5
g, SE, n=3) or soleus muscles (4.3 $0.5 g, SE, n=3) were vascularly isolated,
excised, and perfused at constant flow (0.2—0.4 ml/min/g) via the arteries with a 20%
suspension of sheep red blood cells in Krebs-Henseleit (1.2mM KH2P04) solution
containing 3.5% BSA, 5mmol glucose, 0.15mM sodium pyruvate, and 30mg/l
papaverine HCl (86). Perfusion pressure was 90—1 10 Torr. Following muscle
dissection, the cats were killed with pentobarbital. Perfused muscles were attached to a
force transducer and platinum stimulation electrodes in a specially designed 7.4 cm
diameter NMR probe (See Appendix). Each muscle was repeatedly lengthened and

18
shortened while stimulated with supramaximal voltage twitches until the length for

maximum force production (L) was estabilshed. Stimulation voltage, Lo, and
temperature (37i2°) were monitored and maintained at optimal levels during the
experiments.

Prior to muscle stimulation, fully-relaxed phosphorus NMR spectra (162 MHz,
7000 Hz sweep width, 2K complex data, 90 degree nominal pulse width, interpulse
interval 15 sec) were acquired from each muscle. Muscles were then stimulated with 6
(rat) or 10 (cat) second trains at 5 Hz, with 4.5 min between each train. Acquisitions
of phosphorus NMR spectra were gated to specific times during and after the trains of
stimulation by triggering the stimulator from the spectrometer's computer (Aspect 3000
of a Bruker AM400). Scans were acquired at 5 (rat) or 9 (cat) seconds into the train,
and at 20, 60, 90, 120, 180, and 240 seconds after the trains. After an additional 15
second delay, the cycle was typically repeated to a total of 8 scans per spectrum (see
Figure 1). It should be emphasized that although spectra are the average from several
scans, the effective time resolution of the gated spectra is equal to the acquisition time
per scan, i.e., 145 ms. Free induction decay (FID) data sets were zero-filled to 4K and
multiplied by an exponential corresponding to 25 Hz linebroadening before Fourier
transformation. The minimum interpulse delay used in this protocol was 20 seconds,
therefore the spectra were fully-relaxed (86) and no saturation corrections are
necessary. PCr, ATP and P, peaks were integrated by an iterative Lorentzian fitting
routine’ (87), and integrals scaled to pmol/g assuming pre-stimulation ATP levels of

7.2, 7.0 and 3.8 pmol/g for rat gastrocnemius (80), and cat biceps and soleus,
respectively (86).

 

5This routineminimizes theemrbetweenacurvefittothedataandtheequation fortheideal
Lorentzian line shape (y= ll l-xz) at the designated peak location (87).

19

Figure 1. Twitch force record from rat gastrocnemius muscle illustrating protocol for
gated NMR data collection at 5 seconds after the initiation of 5 Hz stimulation and at
the indicated intervals during recovery. The cycle of stimulation and recovery was
repeated eight times with each NMR collection being added to the appropriate computer

memory block.

20

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21
Because of the relatively lower signal-to-noise ratio and a tendency toward non-

Lorentzian lineshape of the P, peak in spectra acquired during stimulation, the inorganic
phosphate content of muscles during stimulation was estimated from the biochemically
determined P, content of resting muscle plus the P, expected from the observed PCr
hydrolysis. Intracellular pH was estimated from the chemical shift of the inorganic
phosphate peak as described previously (86). The apparent buffer capacity in intact
muscle was calculated according to equation 1, assuming (1,, = 0.4 near pH 7.
Wm.

Animals were anesthetized as above and the muscles of interest dissected free.
In each case, a 0.5 (rat) or 1 g (cat) sample roughly corresponding to the area in the
sensitive volume of the NMR coils was removed and homogenized in 20 ml of 0.9%
NaCl/g muscle. In order to avoid the possibility of variable metabolic changes, and in
particular variable hydrolysis of phosphate metabolites, all homogenates were incubated
at 37°C for 45 min before titration. A 1 ml portion of each homogenate was then
frozen in liquid nitrogen and extracted in perchloric acid for measurement of inorganic
phosphate (86) and protein (75) content. In addition, two perchlorate extracts of each
muscle type were examined by phosphorus NMR (162MHz, 4K complex data, 8000Hz
sweep width, 45 degree pulse, 1 s pulse interval, 800-2400 scans) in a standard
broadband probe. The remaining homogenate was acidified to pH 6 with HCl and
titrated at 37°C to pH 8 with 0.2 N NaOH in 10 ul steps. The resulting mean titration
curves were fit to a fourth order polynomial, from which the slope (total B, Slykes)

over the pH range 6-8 was computed by differentiation.

22

RESULTS
MW.

Titration curves of the homogenates of each muscle type appear in Figure 2.
These curves were remarkably reproducible within a muscle type. For example, the
pH after addition of 30 pmol of base to rat homogenates initially adjusted to pH 6 was
7.02 10.04 (SE, n=6). The total buffer capacity over the range pH 6-8 computed
from the slopes of polynomial fits to the mean titration data appear in Figure 3 (open
symbols). At pH 7 the total buffer capacity of the fast-twitch muscles (rat
gastrocnemius and cat biceps) are similar, while that of the soleus is somewhat less
(Table 1). These results appear to be consistent with previous studies which reported
buffer capacities in highly glycolytic muscles of around 50-60 Slykes, with somewhat
lower capacities in red muscle (12,18).

However, all of the homogenates contained very high levels of free inorganic
phosphate (Table 1) which was not present in intact, unstimulated muscle (Table 2),
and therefore represents hydrolysis of PCr, ATP and other phosphate metabolites. This
was confirmed by examination of 31P-NMR spectra of perchlorate extracts of the
homogenates after incubation (Figure 4). In soleus extracts, P, was the only peak
resolved in the spectra. In biceps muscles, one additional peak with area 20-25% of
the area of the P, peak was resolved. This peak resonated coincident with IMP added to
the extract.

23

Figure 2. Titration of muscle homogenates from pH 6 to 8 in 0.2 uM steps with NaOH
(mean :1; SE, n=6 for each muscle type).

El RAT GASTROCNEMIUS

A CAT BICEPS
0 CAT SOLEUS

24

HOPN II“

 

8.0

7.5

7.0

6.5

6.0

25

Figure 3 Total buffer capacity (open symbols) calculated from slope of mean titration
curves in Figure 2, and non-phosphate buffer capacity (closed symbols), calculated by
subtracting contribution of P, (Table l) to total buffer capacity, of cat and rat skeletal

muscle.

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Figure 4 31P-NMR spectra of homogenate extracts from cat soleus (A) and biceps
(B) muscles. The major peak in both A and B is P,. In spectrum C IMP was added to

the biceps extract to verify that the minor peak seen in B was IMP.

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31
The pK,I of inorganic phosphate titrated under conditions identical to those used

for the homogenates (i.e. , muscle specific concentration, 37°C) was 6.75. Considering
the high levels of inorganic phosphate in the homogenates (Table 1), P, must make a
major contribution to the total buffer capacity of the homogenates in the range pH 6.5-
7.0. The solid curves in Figure 3 are the buffer capacity of the homogenates after
subtracting the calculated contribution due to the inorganic phosphate present, i.e. , the
non-phosphate buffer capacity of the homogenates (Table 1). After this correction, the
apparent correlation of buffer capacity with muscle fiber type is lost, as the rat muscle
has the lowest buffer capacity while the two cat muscles are not markedly different.

,3 f ' p.1i§x1 l at m o in in ., th n t 0 ti Luci.

Most muscles in these studies performed 8 serial trains of contraction with a
total of 36 min between the first and last bouts. There was no significant decrease in
peak twitch force over the course of the experiment in any muscle (e. g., in rat muscle
mean peak force was 2.22 iO.3g/g (SE) body wt. during the first train and
2.32 iO.3g/g during the last train). In one experiment on a large cat biceps, sufficient
S/N was obtained to acquire useful spectra in a single scan (Figure 5a), allowing
complete data collection during and after a single 10 second train of stimuli. The
results from this muscle were similar to that obtained from other muscles in which
spectra were averaged over 8 cycles of stimulation (Figure 5b). These results
demonstrate that the response to a brief train of stimuli is not altered by application of

previous trains separated by a 4.5 min rest period.

32

Figure 5. Sets of phosphorus NMR spectra acquired by protocol of Figure 1. Series
A is from a large biceps muscle from which the S/N was sufficient to obtain good

spectra with a single scan. Series B is representative of 8 scan spectra.

34
Figure 6 is a set of representative spectra from a rat experiment demonstrating

the downfield (alkaline) shift of the phosphate peak after 5 seconds of 5 Hz stimulation
relative to the spectrum acquired 4.25 min later. There was a significant increase in
pH (Figure 7) during the stimulation train which was reversed during the subsequent
recovery period (Figure 7). The pH in the last spectrum of the recovery period
(6.92i0.04, 70310.01, and 7.061001 for rat gastrocnemius, and cat biceps and
soleus, respectively) was not significantly different from pH before any stimulation.
The changes in pH during stimulation were coincident with significant decreases in PCr
in all muscle types (Table 3). The apparent buffer capacities (Table 3) calculated from
the changes in PCr and pH using equation I lie intermediate between the total buffer
capacities and the non-phosphate buffer capacities of the muscle homogenates.
However, if the estimated P, content in the muscles after 5 (rat) or 9 (cat) seconds of
stimulation is added to the non—phosphate buffer capacity, then the predicted buffer
capacities (Table 3) from the homogenate data agree with the apparent buffer capacities
calculated from PCr and pH changes in the intact muscles.

35

Figure 6 Series of gated phosphorus NMR spectra (each 8 scans) from rat
gastrocnemius collected as described in methods and in Figure 1. The zero time
spectrum is identical to the 240 sec spectrum, and is plotted twice so that the alkaline

shift of Pi is apparent. Bar on time scale represents 6 second contraction.

r
C
P

 

 

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37

Figure 7. pH changes relative to the final recovery spectrum during and after
muscle stimulation at 5 Hz for 6 (rat) or 10 seconds. * = significantly different from

final recovery spectrum (p<0.05)

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40
DISCUSSION

The major conclusion to be drawn from this study is that the transient
alkalinization which occurs at the onset of stimulation in mammalian skeletal muscles
can be quantitatively accounted for by proton consumption due to net PCr hydrolysis.
If there were another quantitatively significant source or sink for protons during the
first few seconds of contraction, then the buffer capacity calculated from the transient
alkalinization accompanying PCr hydrolysis would differ from that predicted from
titration of the homogenates. In fact, these two estimates of buffer capacity are similar
in all three muscle types. Thus, it appears unlikely that there is a quantitatively
significant shift of protons either extracellularly, or into subcellular organelles at the
onset of muscle stimulation (24).

The fact that the observed pH changes can be fully accounted for by PCr
hydrolysis also suggests that little accumulation of lactic acid had occurred in these
muscles during the first few seconds of contraction. Accumulation of lactic acid during
the stimulation would oppose the alkalinizing effect of PCr hydrolysis, and thus result
in higher buffer capacities calculated via equation 1. Although little lactic acid
formation would be expected in the highly aerobic soleus muscle (15,86), stimulation
of rat or cat fast-twitch muscle at 5 Hz for longer periods is well-known to result in
lactic acid accumulation (87,88). Apparently, full activation of glycolysis is not
achieved in these muscles until sometime after the first 5-10 seconds of stimulation.

The alkalinization observed in this study at 5 and 9 seconds is much less than
that calculated by Connett (24) from metabolic data in dog gracilis muscle after 5
seconds. In that study an alkalinization from pH 7.05 to 7.8 was calculated with no
significant change in PCr. Even assuming a PCr change of 10 umol/g, the buffer
capacity of dog muscle would have to be less than 6 Slykes if this alkalinization were
due to PCr hydrolysis. It seems unlikely that the buffer capacity of dog muscle would
be dramatically lower than that of rat and cat muscles (12,18). A more likely

41
explanation for the high pH calculated in Connett' s study after 5 seconds is that one or

more of the glycolytic reactions used to calculate pH was not near equilibrium at that
time. On the other hand, the pH calculated for unstimulated muscle in Connett's study
is very similar to that observed in this and previous phosphorus NMR studies,
suggesting that these reactions are near equilibrium in muscle at rest. The agreement
between pH measurements calculated from metabolic equilibria and phosphorus NMR
in unstimulated muscle suggests that there is no complicating subcellular
compartmentation of the metabolites used in the calculations (i.e., PCr, creatine, P,,
lactate, pyruvate, dihydroxyacetone phosphate and 3- phosphoglycerate (24)).

Titration of muscle homogenates is the conventional method for measuring
muscle buffer capacity. Unfortunately, these titrations have been conducted under
widely varying conditions of temperature, presence of metabolic inhibitors, and C02
content. The most quantitatively significant reactions likely to occur in muscle
homogenates are lactate production and hydrolysis of high energy phosphates. Of
these, the former is of no concern, since neither glycogen nor lactic acid contribute
significantly to buffering above pH 6. Our results also indicate that accumulation of
phosphorylated glycolytic intermediates is also of little concern, inasmuch as significant
levels of these were not observed in spectra of homogenate extracts. The conversion of
ATP (pK6.5, (42)) to IMP and/or AMP (pK6.2, (86)) observed in homogenates of fast
muscle would reduce their buffer capacities compared to intact muscles at pH 7 by only
1.0 Slyke, assuming 7 umol ATP converted/g muscle (19,86), and ignoring the effect
of the P, liberated. However, as our results demonstrate, the generation of inorganic
phosphate from ATP and PCr in the homogenates has a major impact on the measured
buffer capacity. For example, in the rat muscle, 68% of the buffering capacity of the
homogenates at pH 7 was due to P,, most of which is not present in the intact muscle.
Thus, it seems quite possible that variable hydrolysis of high energy phosphates at some

time during preparation of the homogenates is responsible for much of the variation in

42
buffer capacities reported using the titration method (12,18,93). In this study the

muscle homogenates were incubated at 37 °C, without metabolic inhibitors before
titration. The high levels of phosphate measured after incubation (Table 1) were due to
the near complete hydrolysis of PCr and ATP. Titration of these homogenates was
extremely reproducible (Figure 2), and after correction for phosphate content, the
results are in good agreement with the estimate from intact muscles.

Our results have two additional implications. First, because the non-phosphate
buffer capacity, particularly of the rat muscle, is relatively low, the phosphate released
by PCr hydrolysis during muscle stimulation contributes significantly to intracellular
buffer capacity. This may be a physiologically important role of the creatine kinase
reaction in highly glycolytic fast-twitch muscles (12,93,101). Second, the agreement
between buffer capacities predicted from homogenate titrations vs. calculated from the
intact muscle data confirms that COzlbicarbonate is not quantitatively important for
intracellular buffering in muscle, as the homogenates were titrated at ambient C02
levels (12,68). Similarly, this agreement confirms that extracellular fluid does not
make a significant contribution to buffer the capacity of muscle homogenates. As
reviewed by others (93), the major contributors to the non-phosphate buffer capacity

around pH 7 are probably proteins and peptides such as anserine and carnosine.

Chapter 2

Measurement of Lactate Accumulation in Contracting Skeletal Muscle
Using 1H Nuclear Magnetic Resonance Spectroscopy

43

44
INTRODUCTION

Contracting skeletal muscle produces lactic acid when energy demands
exceed the capacity for oxidative regeneration of adenosine triphosphate (ATP).
At physiological pH lactic acid dissociates producing lactate and hydrogen ions
(11*). Intracellular acidification resulting from this process is considered a
primary cause of decreased force production seen in all muscle types during
periods of prolonged, intense, contractile activity.

Many of the current concepts relating lactate accumulation to specific
metabolic states have been formulated from data collected by measuring lactate
levels in blood (61,62,134). However, blood will contain lactate contributed
from all the tissues upstream from the collection site. In addition, the
intracellular accumulation of lactate and its subsequent appearance in the blood
are not temporally linear events. An alternative to this approach has been to
freeze muscles and assay muscle homogenates for lactate concentrations. This
method is hampered by the continued metabolism during both the freezing and
assay procedures (3,6,31,86,120).

The use of nuclear magnetic resonance (NMR) spectroscopy with
biological samples is non-destructive and non-invasive allowing serial
collections of metabolic data (7,125). To date, most of the notable
contributions of this technology in intact cells has resulted from measurements
of phosphorous (MP) and to a lesser extent carbon (13C), nuclei.

Proton NMR (III) has much greater sensitivity than other biologically
applicable nuclei (e.g. , 16 times the sensitivity of 31P—NMR). The chemical
shift range of 1H is much smaller than that of 31P-NMR (10ppm vs. 30ppm) and
the number of 1H containing compounds present in NMR detectable

concentrations is many times greater than those containing 31P. These factors

45
lead to significant overlap in resonances. In particular, lH spectra from intact

cells are dominated by signals arising from water and lipid protons.

Cell water is present in concentrations approaching 40mM. The signal
from water saturates the digitizing capacity of spectrometers preventing
resolution of signals from compounds present in 5 to 10 mM concentrations.

Several strategies have been employed to negate the signals arising from
cell water. Selective excitation techniques avoid perturbation of the resting
magnetization of water (7,58,125). Saturation or inversion recovery procedures
minimize the magnetization of water during the observation period (7,58,119).
A third approach, Spin-echo, takes advantage of the fast decay of the cell water
signal relative to that of many metabolites ( 126). Spin-echo techniques refocus
a decaying NMR signal. If the refocusing pulse occurs after the decay of the
water signal subsequent acquisitions will contain information only from
compounds with slower decaying signals.

The suppression of both water and lipid proton signals generally requires
a combination of techniques. Spectral editing techniques have been used in
muscle to observe the lactate methyl group which is otherwise obscured by the
signals from the lipids present. In addition to spin-echo collections to suppress
the water signal, compounds with spin coupled resonances are measured by
collecting first a coupled then a decoupled spectrum. The decoupled spectrum
will contain a peak which is 180° out of phase with the same peak in the
undecoupled spectrum. Subtraction of the two spectra will leave just the peak
which was out of phase. If the out of phase peak was obscured by lipid
resonances it will now be resolved. Using this technique Williams et al were
able to resolve a lactate peak as it increased during ischemia in rat muscles

(126).

46
Recently, Meyer developed a pulse sequence which suppresses both

water and lipid protons without the need for spectral editing (83). This
technique employs spin-echo collections formed by continuous, frequency
selective, pulsing of the spectral region of interest. Water protons are not
excited and lipid proton signals have decayed before data collections are made.
This steady state echo (SSECHO) method has been used to measure lactate
formation in skeletal muscle (83) and heart (64).

In this study, interleaved SSECHO IH and 31P-NMR collections were
employed to quantitatively compare changes in pH and lactate in intact,
contracting skeletal muscles. The results indicate that some portion of
intracellular lactate may be bound and therefore not measurable using NMR.

METHODS
Musclepreparation Male or female cats were sedated with Ketamine (11
mg/Kg, IP) and anesthetized with pentobarbital (30 mg/Kg, IP). Anesthesia was
maintained throughout the procedure by iv. administration of pentobarbital,
following muscle excision euthanasia was achieved by an overdose of
anesthetic.

Biceps muscles were dissected free and vascularly isolated, maintaining
the arterial and venous flow of the muscle. The artery was then eannulated and
perfusion begun at 0.2 to 0.4 mllmin/g. Perfusion pressure ranged from 90-130
Torr. The perfusate consisted of 116mM NaCl, 25mM NaHC03, 4.6mM KCl,
1.2mM KH2P04, 2.5mM CaClz, 1.2mM MgSO.” 5mM glucose, 0.15mM
sodium pyruvate, 3.5 % albumin, 30ug/ ml papaverine chloride, and a 20%
suspension of sheep red blood cells. The perfusate was passed through multiple
windings of Silastic tubing in a sealed Plexiglass chamber to allow equilibration

with 95% 02/5 % C02. Following excision each muscle was wrapped in

47
parafilrn and mounted in the center of a Helmholz NMR coil in a specially de-

signed NMR probe (see Appendix). The tendons of the muscle were attached to
a variable length muscle mount / force transducer. Platinum stimulating
electrodes were attached at each end of the muscle. Stimulation voltage and
muscle length were adjusted to produce maximum twitch force. Muscle
temperature was monitored by a thermistor and maintained at 37 12°C by
circulating water through copper tubing in the probe.

W The NMR probe used in these studies was equipped with a
single Helmholz coil and isolated circuitry with both 31P and ‘H channels.

The NMR probe was inserted in the 7 .4cm bore of a 9.4 Tesla, Oxford
Instruments magnet. The resonant frequency of the coil was tuned to both
400MHz (1H) and 162MHz (31P) on separate circuits. Magnetic field
homogeneity was shimmed on the proton circuit using the signal from muscle
water, proton line width was less than 50 Hz. 31P-NMR collection parameters
were: Sweep width 7000Hz, 2K data set, 2.85 sec pulse interval, 16 scans per
spectrum. Proton spectra result from the collection of 640 negative echo FID's
with a 0.1 3 pulse interval (83).

The acquired data sets for each spectrum were multiplied by an
exponential function equivalent to 25Hz line broadening prior to Fourier
transformation. The resultant spectral peaks were integrated by an iterative
Lorentzian line fitting routine (87). Intracellular pH (pH,) was calculated from
the chemical shift of P, relative to that of phosphocreatine (PCr).
W Muscle stability (temperature, pH,P,,PCr,ATP) was
monitored for a minimum of sixty minutes under control perfusion conditions.
Muscles were then stimulated at le for 6 min. 1H- and 31P-NMR collections

were interleaved. Each collection required approximately one minute.

48
Calculations The effects of the increases in lactate on pH were calculated

from:
I. pH = A[Lactate] - A[PCr] x or, / B,
where orp is the proton change resulting from net A[PCr] and B, is the apparent
buffer capacity (See Chapter 1) calculated from:

B, = non-phosphate B + [P,]
The nonphosphate B and A,” were corrected for pH using the 31P—NMR
measured value. Equation 1. assumes a 1:1 production of H+ to lactate. At
each time point the [P,] was calculated from the decrease in [PCr]. The B, term
takes into account the changing contribution of P, to buffer capacity.
W SSECHO 1H-NMR spectra (1280 scans) were collected on a
0.9% NaCl solution containing 20mM Lactic acid. Sucessive aliquots of 0.1ml
35% BSA ( ~0.039g) were added and spectra collected.

RESULTS

Figure 8 presents representative 1H-NMR spectra from an isolated
perfused cat biceps muscle before, during and after le stimulation. The
increase in peak area at 1.36 PPM was assumed to be a build up of lactate
resulting from contraction. The integrated areas of the lactate peaks were
normalized to known resting levels. The changes in concentration of lactate and
PCr are presented in Figure 9. Lactate increased and PCr decreased
substantially as a result of contraction.

The open circles in Figure 10 represent the change in intracellular pH
from two cat biceps muscles calculated from the observed change in chemical
shift of the P, peak. The calculated effects of the observed increase in lactate on
pH are plotted as closed symbols in Figure 10.

49

Figure 8. 1H-NMR SSECHO spectra from an isolated perfused cat biceps
muscle stimulated at 1 Hz for 6 min. The peak at 1.36 PPM is assumed to be

lactate.

m
_.||l.lJ

was a a A

 

51

Figure 9. Changes in lactate (open symbols) and PCr (closed symbols) during

1H2 stimulation and recovery in two cat biceps muscles.

52

AEEV eEc.

 

 

em NN om mp m— .1 NF op m m N o
q q . a . . a q . w.t.h.l.. O
\ / mum
0.3 e o
.\.\ n 4 OP
" .0
o m \ . on
Gaol.- m .. mm
Bsoflolo " r 9
0.0.0.0.“. A

 

me

elosnur 6/e|ou.rrl

53

Figure 10. Intracellular pH calculated from the chemical shift of P, in 31P-NMR
spectra (open symbols). Closed symbols represent the change in pH expected from
a 1:1 production of H+ by lactate accumulation measured with lH-NMR using the
SSECHO sequence.

54

AEEV oEc.

 

 

 

¢N NN ON 0P 0F .3 NF OF 0 0 e N 0
been.
\0 n O/ r
o\00 n o
o o\\\ u /
u o -
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0.0

0.0

N0

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55
Spectra were collected from solutions of lactic acid, before (Figure 11A,

bottom spectrum) and after the addition of 3.5 % albumin (Figure 11A). In
Figure 11B the peak areas from 11A are plotted against the amount of albumin
present in the solution. Addition of albumin resulted in attenuation of the

lactate signal.

56

Figure 11 A. SSECHO 1H-NMR spectra of physiological saline solution
containing 20mM lactic acid (bottom spectrum) and increasing amounts of
albumin. Peak at as 1.3 PPM arises from the methyl protons of lactate. B. Plot

of integrated peak areas from spectra in A (r=0.92).

Peak Area

30

F
c:

 

57
+0.052q/ml

W;

+0.0399/ml
Albumin

+0.0269/ml
Albumin

A

+0.01 Jq/rnl
Albumin

 

t—V ' V I V U r ' r V U '7‘ l’ V T V T ' U I V U ' I t

4.0 2.0 1 .0 0.0 -1 .0

ppm

 

_J

0.01 0.02 0.03 0.04 0.05 0.06
Albumin g/ ml

58
DISCUSSION

Taken together, interleaved collection of 1H and 31P-NMR spectra from
contracting skeletal muscle provide information on changes in the concentrations
of PCr, P,, and lactate as well as pH,. The protocol employed in this study
resulted in accumulation of lactate and significant intracellular acidosis.
However, the observed lactate accumulation does not appear to be sufficient to
account for the all of the change in pH, (Figure 10). Hope et al. reported
similar results in lamb brain during hypoxia and ischemia (57). These authors
used spin echo collections to remove the contribution of lipid protons in the -
CH3 region. pH was measured by 31P-NMR. Hypoxia-ischemia resulted in
greater acidification than could be accounted for by the observed lactate
accumulation.

Chang et al. conducted parallel in viva and in virra 1H-NMR and
enzymatic determinations of lactate accumulation in hypoxia and ischernic rat
brains (20). Their results indicated that some of the in viva lactate was not
detectable.

We found that addition of albumin to a physiological salt solution
containing lactate eliminated the -CH3 lactate resonance from subsequent 1H-
NMR collections. This result leads us to speculate that some fraction of the
lactate generated during contractions may be bound to intracellular proteins thus
rendering it undetectable under our collection conditions.

Much of the utility of NMR in biological research stems from this
method's relative insensitivity to nuclei in immobile compounds. However, in
this instance the binding of some fraction of lactate anion to intracellular
proteins would invalidate the assumption of a 1:1 relationship between NMR
visible lactate and H+ production. This would lead to an underestimation of pH,

calculated from 1H-NMR measures of lactic acid accumulation. As result of

59
this limitation, traditional biochemical methods will have to be employed to

measure lactate accumulation. Such investigations will entail the rapid freeze
clamping, extraction and subsequent chemical assay of lactate concentrations.

A reasonable protocol might include the establishment of steady state
lactate and P, levels as measured by NMR. Muscles could then be rapidly
removed from the magnet and frozen. The chemical assay values could then be
compared to NMR measurements of these same metabolites in the extracts.
Comparison of the in viva and extract [P,] would provide an indication of the
degree of hydrolysis that took place during the freezing, and extraction
procedures. It is possible that some known relationship between the 1H-NMR
visible lactate fraction and the chemically measured value might be uncovered.
If so this would allow the use of 1H-NMR collections with a mathematical
correction to give an accurate in viva concentration. Until these options are
explored lactate concentrations measured by lH-NMR must be considered
unreliable.

Chapter 3

Increased H+ and mm; do not cause fatigue
in mammalian skeletal muscle.

61
INTRODUCTION

Repeated contraction of skeletal muscle at power outputs which cannot be
sustained by aerobic metabolism results in a progressive decrease in peak isometric
force (23,30,116). This decrease in force, or fatigue, is associated with decreased rates
of ATP utilization (30).

Muscle fatigue at high power outputs might be of adaptive value, because it
would assure the preservation of some energy reserve for maintenance of other cell
functions (29). In this context, the idea that muscle force is directly regulated by
specific metabolites linked to cellular energetics is attractive, and for many years
attempts have been made to link muscle fatigue to changes in various metabolites (32).

Studies of skinned muscle fibers suggest that changes in hydrogen ion
concentration ([II+]) may modulate muscle force in viva. Acidic pH is reported to
decrease the calcium sensitivity of force activation (33,36), and the peak force and the
maximum velocity of shortening during maximum calcium activation (21,25,82). The
conductance of the ryanodine-sensitive channels responsible for calcium release from
the sarcoplasmic reticulum is also strongly dependent on pH (77).

Considering these diverse effects, one might expect force in intact muscle to be
profoundly depressed by intracellular acidosis. However, studies of intact muscles
(30,80,89) and isolated fibers (72,124) have not established a consistent correlation
between pH and force.

In the few studies which used hypercapnia to decrease pH, independent of
contractile activity, force did significantly decline (79,95,102). However, the
metabolic state of the superfused muscles in these studies was either not examined, or
was clearly compromised (95,102).

Increased [P,] also decreases peak force in mammalian skinned fiber
preparations (21,26,65). Intact skeletal muscle is known to accumulate 25-35 mmol/g

62
of inorganic phosphate (P,) during sustained contraction (30,86,115). There have been

several reports of a correlation between increased intracellular [P,] and decreased force
(5,30,35) in intact muscles.

In intact muscle studies the major effect of P, on force is seen when
concentrations increase above 20mM, below that level only slight depression of force is
noted (29,30). In contrast, skinned fiber preparations are sensitive to increasing [P,] up
to ~15mM at which point force levels off (5,26,65).

Dawson (29) and Wilkie (94) have suggested that the effects of both pH and P,
reflect direct inhibition of cross-bridge formation by the diprotonated form of P,.

While this proposal was supported by the results of Nosek, et al (91), other skinned
fiber studies have not found a significant interaction between the effects of pH and P,
on force (21,26,65). Several NMR studies of human muscle have found a good
correlation between diprotonated phosphate (HzPO4') and force during a fatiguing
series of voluntary contractions in normal subjects (16, 17 ,89, 127).

The purpose of this study was to examine the relationships between intracellular
pH and/or diprotonated phosphate vs. force in perfused cat fast- and slow-twitch
muscles. Intracellular pH was altered both by hypercapnia, and by fatiguing series of
contractions. Results indicate that increases in H+ and H2P041- can be produced with

no decrease in peak tetanic tension development.

METHODS

W. Adult male cats were sedated with Ketamine-HCI (11mg/kg, ip)
and anesthetized with pentobarbital (30 mg/kg, ip), with subsequent iv doses of
pentobarbital administered as indicated. Biceps brachii or soleus muscles were
vascularly isolated and dissected free as described previously (86). The artery was
cannulated (PESO) and perfused at 0.2-0.4mein/ g (perfusion pressure 80-100 Torr)

63
with a 20% suspension of sheep red blood cells in bicarbonate buffered Krebs-Henseleit

solution containing 3.5% bovine serum albumin, 5mM glucose, 0.15mM Na pyruvate,
30ug/ml papaverine HCl, long/ml gentamycin sulfate, and 10mM Na
phenylphosphonate (PPA), a phosphorus NMR detectable indicator of extracellular pH
(86). Perfusate was initially equilibrated with 5 %C02/95%O2 at 37 °C in a silastic
tube oxygenator.

The perfused muscles were wrapped in Parafilm and mounted in a 1.5 cm
diameter Helmholtz configuration coil within a custom-made 7.4 cm diameter NMR
probe (see Appendix). The distal tendon was clamped to a fixed support under the coil
and the proximal tendon (or in the case of the soleus, the excised head of the tibia (86))
was clamped to a custom-made cantilever beam force transducer mounted on an
adjustable frame at the top of the probe. Probe temperature was monitored by a
thermistor and maintained at 37 12°C by circulating water through copper tubing in the
probe. Muscles were stimulated directly via platinum wire electrodes wound around the
tendons. Muscle length was adjusted to produce maximum isometric twitch tension in
response to a single supramaximal pulse (SO-70V, 1 ms). Addition of 50uM
succinylcholine, a neuromuscular blocker, to the perfusate had no effect on peak twitch
or tetanic force.

W. The probe was inserted in the 7.5 cm bore, 9.4T magnet of a Bruker
AM400 spectrometer, and tuned to phosphorus (162 MHz) or proton (400 MHz)
resonance via two independent switch-selectable circuits. The magnet was shimmed on
the proton signal of muscle water to a linewidth of 40-60 Hz. The 90° pulse width for
phosphorus was estimated for each muscle at the start of the experiment. Phosphorus
NMR spectra (sweep width 10,000Hz, 2K data, 15 sec pulse interval, 4 or 8 scans,
corresponding to l or 2 min averages, for biceps and soleus, respectively) were
continuously acquired throughout the experiment. FID's were zero-filled to 4K and

multiplied by an exponential corresponding to 25Hz linebroadening prior to Fourier

64
transformation. Peaks were integrated by an iterative Lorentzian fitting routine (87).

Intracellular metabolite concentrations were estimated from peak integrals by assuming
P,+PCr concentrations of 38.1 and 26.7 mM for biceps and soleus muscles,
respectively (86). Intracellular (pH,) and extracellular pH (pH,) were estimated from
the chemical shifts (6, ppm) of inorganic phosphate and phenylphosphonate,
respectively, as follows (86):

pH, = 6.75 + log (0.89-6)/(5-3.19)

pHe = 7.02 + log (l3.9-6)/(6—ll.8)

The PCr peak (2.52 ppm) was used as a reference. Diprotonated phosphate content
was calculated from P, and intracellular pH assuming pK=6.75, and neglecting the
small contributions of P043" and H3PO4.

WM- Preliminary experiments demonstrated that graded increases in
CO2 content in the perfusate gas resulted in graded decreases in both intracellular and
extracellular pH (Figure 12A). There was a linear relationship between extracellular
and intracellular pH during hypercapnia (Figure 12B), but no significant decrease in
PCr or P, content during perfusion at CO2 contents up to 70%. Based on these results,
70%CO2 was chosen for the study.

Each muscle was perfused under control conditions (37°C, 5%C02 I95 9602 )
for a minimum of 1 hour to establish metabolic and mechanical stability. The perfusate
gas was then switched to 70%C02/30%O2 and phosphorus spectra monitored until
extracellular and intracellular pH approached a new steady-state, typically within 30
min. Mechanical response to several test twitches and tetani (0.5 sec @ 100 Hz for
biceps, 1.5 sec @ 20Hz for soleus) applied a minimum of 5 min apart were then

recorded.

65

Figure 12. A. The effect of perfusate equilibration with increasing percentages of
C02 (balance 02) on extracellular (open symbols) and intracellular (closed symbols) pH
in an unstimulated biceps muscle. Figure 12B shows the relationship between extra-

and intracellular pH in this muscle.

pH

pH.

 

 

 

 

6.4 ~

 

6.2
6.4

6.6

6.8

7.0
PHa

7.2

 

7.4

60

7.6

67
In order to increase P, content, the muscle was then repetitively stimulated at moderate

rates, e.g., 0.1 Hz for biceps and 1 Hz for soleus, for 3-5 min. These relatively mild
twitch stimulation regimens were selected on the basis of preliminary experiments
which showed that they resulted in significant PCr hydrolysis and Pi accumulation
without any additional metabolic acidification. Phosphorus spectra were continuously
monitored during this hypercapnic stimulation period and the stimulation was stopped
when PCr level was reduced by over 50% relative to prestimulation level. Additional
test tetani were recorded after the hypercapnic stimulation. The perfusate gas was then
returned to 5 %CO,/95 %O2 and phosphorus spectra monitored until intracellular pH
approached the initial 5 %CO2 level. Following several additional test contractions, the
muscles were repetitively stimulated at 2 (biceps) or 6 (soleus) tetani/min for 10-15
min. Flow to the soleus muscles was then stopped and these muscles were stimulated
again at 6 tetani/min until peak tetanic force had decreased by over 50%.

Values of p < 0.05 were considered significant for regression coefficients and

comparisons of means.

RESULTS

Figure 13 shows sample phosphorus spectra acquired from representative
muscles during the protocol. The effects of hypercapnic perfusion on pH and
phosphate metabolite levels are summarized in Table 4. The observed relationship
between intra- and extracellular pH was similar to that measured with recessed-tip pH-
sensitive microelectrodes (3). Intra- and extracellular pH decreased by over 0.5 pH
units in both muscle types during hypercapnic perfusion. There was no significant
change in PCr or P, contents in either muscle. Figure 14 shows sample isometric force
records from normal and hypercapnic perfusion. There was no significant effect of

hypercapnia on peak tetanic force in either muscle type (Table 5). However,

68
hypercapnia resulted in significant increases in both the rise and relaxation times of

tetani in the soleus, and of relaxation time in the biceps. The peak twitch/tetanus ratio
was significantly reduced in biceps, and a similar trend was evident in the soleus.

Figures 15 (biceps) and 16 (soleus) show the time course of changes in PCr, P,,
H2P041', ATP, pH, and peak tetanic force during the final periods of repetitive
stimulation during normocapnic perfusion. In biceps muscles there was a steady
decrease in PCr, intracellular pH, and force throughout the stimulation period. In
contrast, PCr and force rapidly approached steady-states in the perfused soleus muscle,
despite the 6—fold greater tetanus train rate and 18-fold greater duty cycle of contraction
applied to this muscle. This result is consistent with the higher aerobic capacity and
lower myosin ATPase activity in slow vs. fast-twitch muscle (5). However, during
ischemic stimulation PCr and force rapidly declined in soleus muscles, and pH
decreased to levels similar to that achieved during hypercapnia.

Considering only the data from the final repetitive stimulation periods, there
were significant correlations between both intracellular pH and diprotonated phosphate
levels vs. peak tetanic force in both muscle types (Figures 17,18 open symbols). For
example, in biceps and soleus muscles the correlation coefficients for force vs. pH
were 0.95 and 0.91 , respectively. However in both cases the relationships were very

different during hypercapnia.

69

Figure 13. Representative 31P—NMR spectra of biceps (left) and soleus (right) muscles
during the experimental protocols. Each spectrum is an average of 4 (biceps) or 8
(soleus) scans with 15s pulse interval. PPA designates the phenylphosphonate peak.
The vertical lines under Pi and PPA indicate the chemical shifts of these peaks at rest
with 5% C02.

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72

Figure 14. Sample force records of test tetani during 5% and 70% CO2 perfusion in
soleus (left) and biceps (right) muscle. Small deflections in the soleus record are

stimulus artifact.

73

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75

Figure 15. Time course of force and metabolite changes during final repetitive
tetanic stimulation (2 tetani/min) with 5% C02 perfusion in cat biceps muscles (xiSE,

n=4).

76

1.1 Soleus
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77

Figure 16. Time course of force and metabolite changes during final repetitive
tetanic stimulation (6 tetani/ min) with 5% C02 perfusion (open symbols) and no

perfusion (closed symbols) in cat soleus muscles (xiSE, n=4).

78

SP.
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Force (x max)
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79

Figure 17. Relationship between peak tetanic force and intracellular pH in cat biceps
(A) and soleus (B) during 5% CO2 (open symbols) and 70% (closed symbols) perfusion.

Pooled results from 4 muscles of each type.

P

FORCE (% max.)

FORCE ( 95 max)

1.1

1.0

r Biceps

P

 

80

 

T

 

6.2

4

6.4

g

6.6

6.8

Intracellular pH

7.0

 

81

Figure 18. Relationship between peak tetanic force and [HzPO4l‘] in cat biceps (A)
and soleus (B) during 5% C02 (triangles) or 70% (circles) perfusion. Filled circles are
results from test tetani recorded before P; was increased by stimulation. Open circles

are results following stimulation to increase PI. Pooled results from 4 muscles of each

type-

A.

FORCE (5% max.)

FORCE (% max.)

1.1

1.0

0.9

0.8

0.7

0.6

0.5

1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
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1 5 20

83
There was no significant correlation between intracellular pH or diprotonated phosphate

and peak tetanic force during hypercapnia in either muscle. In both muscles, the
relation between Pi content and force was biphasic (Figure 19), with little decrease in
force up to 20 mM, and a precipitous drop in force above 25 mM. Hypercapnia had
no obvious effect on this relationship. Finally, there was a significant correlation
between tetanus relaxation time and intracellular pH during both normal and

hypercapnic perfusion (Figure 20).

84

Figure 19. Relationship between peak tetanic force and P, in cat biceps (A) and
soleus (B) during 5% CO2 (open symbols) or 70% CO2 (closed symbols) perfusion.

Pooled results from 4 muscles of each type.

F orce (%max)

Force (%max)

1.1

1.0

0.9

0.8

0.7

0.6

0.5

1.1
1.0
0.9
0.8
0.7
0.6
0.5

0.4 -

0.3
0.2
0.1

’ 0.0

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85

 

 

 

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I- 00
O 5 10 15 20 25 30 35 40

86

Figure 20. Relationships between pH and time to relax from 90 to 10% of peak
tetanic force in cat biceps (A, r=0.63, slope=-0.17, p<0.05) and soleus (B, r=0.63,
slope=—O.48, p<0.05) muscles during 5% CO2 (open symbols) and 70% C02 (closed
symbols) perfusion. Pooled results from 4 muscles of each type.

0.6

0.5

0.4

0.3

TIme (sec)

0.2

0.1

0.0

6.1

1.4

1.2

1.0

0.8

0.6

‘I‘Ime (sec)

0.4

0.2

0.0

6.1

Q
o
(D
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l

 

r Soleus

r

 

87

L

pH

 

L j l

6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1

O 5% 002
C 70% 002

 

L L l I

6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1

88
DISCUSSION

The results of this study demonstrate that neither pH nor diprotonated
phosphate levels consistently correlate with peak tetanic force in either fast— or
slow-twitch muscles. There were significant correlations between either pHi or
[HzPO41-] and force only when these parameters were changed by contractile
activity. Independent manipulation of pI-Ii and [HZPO41'] had no effect on peak
force. The simplest interpretation of these results is that decreased peak tetanic
force, or fatigue, during repetitive stimulation is not directly due to either
increased hydrogen ion or H2P041', but to some other effect of contractile
activity.

An alternative explanation of these results might be that the intracellular
acidification induced by metabolic activity during repetitive contraction is
somehow fundamentally different from that induced by hypercapnia. It might
be argued that there are subcellular compartments in which pH is modified
differently by metabolic activity than by hypercapnia. The data from this study
provides no evidence for such compartmentation. The Pi observed by NMR,
which is used to estimate pH, is almost certainly in free solution within the
same compartment as the myofibrillar apparatus (5,86). In both muscle types a
single, narrow, Pi peak was observed when pHi was lowered by hypercapnia.
This peak increased without splitting during stimulation. Significant
accumulation of Pi in a compartment with higher pH would have produced a
second or split peak.

Measured functional responses also argue against compartmentation of
H+ . Reductions in pH due to hypercapnia did modify the rise and relaxation
times of cantractions in both muscle types. This correlation of pH and
relaxation time has been reported in other studies (16,30). Thus, the pH change
induced by hypercapnia did impact aspects of the activation process which are

89
presumably cytosolic. The changes observed are consistent with reported

inhibition of calcium release (77) and sequestration mechanisms (60), and
decreased maximum velocity of shortening (21), by acidosis.

Another conceivable explanation for the differing effect of hypercapnic
vs. metabolic acidosis is that lactate rather than hydrogen ion is the cause of the
decrease in force during repetitive stimulation. However, Chase and
Kushmerick have shown that lactate per se has no effect on force in skinned
fibers (21).

The results in both the cat biceps and soleus are consistent with reports
that Pi accumulation contributes to a decline in peak force (5,21,30,65).
Further testing of the role of Pi must await development of a method to
independently manipulate Pi levels in intact muscle.

Our results are difficult to reconcile with the consistent relationship
between pH and peak force seen in skinned fiber studies. (21,25). Ignoring the
additional effect of acidosis on calcium sensitivity (33,36), a pH change from 7
to 6.4 might be expected to decrease force by at least 20% in slow-twitch fibers,
and 30% in fast twitch fibers (21,25).

In skinned fiber preparations, force development is most sensitive to
increases in Pi from 1 to 15mM (26,65). In the current study there was no
relationship between force and Pi levels below 20mM P,. This result is similar
to that seen in previous studies of intact muscles (29,30). Assuming that cat
muscle is not uniquely resistant to acidosis and intracellular phosphate, our
results suggest that the contractile behavior of intact muscle cannot be directly
extrapolated from studies of skinned fibers.

In contrast to our results, hypercapnia caused significant reductions in
force in superfused frog (95) and rat muscles (102). However, in the latter case

hypercapnia was also associated with decreased PCr content, an effect attributed

90
to a shift in the equilibrium of the creatine kinase reaction (102). In the current

study hypercapnia had no significant effect on PCr levels provided that perfusate
flow was maintained sufficient for oxygen delivery to the tissue. The decreases
in both PCr and force observed in superfused muscles during hypercapnia may
reflect reduced oxygen availability rather than an pH effect.

Ours is not the first study to show a dissociation between acidosis and
fatigue. The inverse of our results, i.e., fatigue in the absence of acidosis, has
been reported in several studies of patients with McArdle' s disease
(myophosphorylase deficiency) (17,73,74,98). Ross et al found no acidosis in a
McArdle's patient after 45 sec of ischemic exercise to exhaustion (98). Control
subjects sustained the exercise for several minutes, despite a decrease in
intracellular pH from 7.1 to 6.4. This clearly indicates that intracellular
acidosis is not an obligatory factor in muscle fatigue. The more rapid PCr
hydrolysis and greater Pi accumulation in exercised muscle of McArdles vs.
control subjects suggests that Pi accumulation is a factor in fatigue in these
patients (73).

The current results do not imply that pH plays no role in the more
general phenomenon of fatigue during exercise. Acidosis was associated with
changes in contraction times and twitch/tetanus ratio, which could have a
profound effect on muscle performance in an intact organism.

The current study was limited to short bursts of intense contractile
activity during hypercapnic acidosis. Prolonged repetitive stimulation may
result in decreased Ca2+ release uncovering an inhibitory effect of acidosis on
activation. In addition, decreased pH may inhibit glycolysis (110) or otherwise
decrease substrate supply for aerobic metabolism. This could indirectly limit
force production during a more prolonged series of contractions. Acidosis may

also delay recovery of force after a fatiguing series of contractions (114).

91
In summary, the results of this study show that peak force development

in intact cat muscle is relatively insensitive to decreased pH and increased

diprotonated Pi induced by hypercapnia. This indicates that neither acidosis nor
increased concentration of HZPO; represents an necessary condition for muscle
fatigue. The correlations between pH or diprotonated phosphate and peak force
observed in this and other studies of intact muscle are apparently coincidental to

some other effect of repetitive contraction.

Summary and Conclusions

92

93
Summary

Changes in the intracellular concentrations of lactic acid, H+, Pi,
[H2P041-] have been suggested as the causal factors in cellular level fatigue in
contracting skeletal muscle. The experiments described in this dissertation were
designed to characterize both changes in these metabolites and related processes
which modulate the effects of those changes.

In chapter 1 a working method of measuring intracellular buffer
capacities was established. An important concept developed in this chapter was-
the dynamic character of this parameter (8). Studies employing calculations
based on a single 8 value spanning a period of muscle contraction will not
accurately reflect the intracellular pH. Under these conditions the intrinsic B
and the current [Pi] must be used to calculate the effective 8 and pH. The
intrinsic 8 values for the muscles used in this study were identified for the entire
physiologically relevant range. These values can be used in future studies for
metabolic calculations.

The results reported in chapter 2 present a cautionary note for future
attempts at quantifying intracellular lactate using lH-NMR. Although the
equipment and pulse sequences used allow measurement of lactate, results
suggest that some fraction of this compound may be bound and therefore NMR
invisible. There have been many studies reporting lH-NMR measurements in
brain (47,57,121). If the same phenomenon occurs in this tissue the results of
these studies will be quantitatively in error .

The studies reported in chapter 3 were designed to assess the role of
changes in pH (i.e. , AH +) and diprotonated-Pi in the depression of maximum
force associated with fatigue. Changes in these two metabolites figure
prevalently in the current theories of cellular level fatigue. The results

presented here show that manipulation of pI-Ii and [I-IZPO41-] independent of

94
contraction does not produce a direct relationship with maximum force as seen

in previous studies. This suggests that the often reported correlation between
these metabolite changes and decreased maximum force production does not
indicate a cause and effect relationship. However, acidosis was associated with
changes in the characteristics of contractions (e.g. , relaxation time) which might
effect the development of force in the intact animal.

Conclusions

There is an extensive body of literature relating metabolic events to the
development of fatigue. However, much of this research has been conducted
either in vitro or in situ with indirect data collection. It is possible that some of
the variability and contradiction found in the fatigue literature results from
previously unavoidable methodological limitation.

The application of scientific method implies the ability to control or at
least measure as many system parameters as possible. However, this
requirement must be tempered by the need to observe processes under
conditions resembling those found in the native (i.e. , in situ) state. The
conceptual impetus for the studies presented in this dissertation was heavily
dependent on the availability of a relatively new technology, NMR.
Experiments using this technology were designed to assess the effects of
changes in metabolites in viva under controlled conditions closely resembling
those found in situ.

The combination of NMR technology and the isolated perfused muscle
preparation described in this dissertation has proven to be an effective means of
studying muscle metabolism. The experimental design for each study was
formulated to avoid perceived methodological limitations of earlier studies. The
isolated perfused muscle preparation is a reasonable compromise between poorly

controlled experiments in intact animals and highly controlled experiments in

95
less physiological preparations such as skinned fibers. The functional unit of a

muscle, the muscle cell, remains intact and in an environment which closely
resembles that in situ. In contrast to many investigations, the energetic and
functional state of the muscles in these studies were subject to frequent
assessment. Variability was avoided by collecting data under control,
experimental and recovery conditions in the same muscle and the temporal
resolution of the observations was well suited to the time course of the
metabolic and functional events.

The experiments reported here uncovered results which may have been
obscured by methodological limitations in previously published studies. The
results of these experiments add significantly to our understanding of the
intracellular response to an acid load and the way in which muscle function is
effected by it. Future metabolic studies will benefit from the refinements in
calculation of buffer capacity presented in chapter 1. The results presented in
chapter 2 will help to avoid erroneous conclusions based on similar research
methods. The observed dissociation of changes in pH and [H2P04‘-] from force
(chapter 3) will require revision of fatigue doctrine. This revision should
provoke further investigation of possible metabolic agents responsible for

muscle fatigue.

Appendix

96

97

In order to conduct experiments using isolated perfused cat muscles an
NMR probe was designed and constructed. Important design considerations
included:

Use of Non-ferric (i.e. , nonmagnetic) materials.
Both 1H and 31P frequency sensitivity.

Circuit tuning and matching while in magnet.
Accommodation of perfusion lines.

Muscle mounting and length adjustment.
Facilities for force measurement.

Muscle stimulation electrodes.

Temperature measurement and control.

The shell of the NMR probe was constructed from an aluminum tube cut
in half and provided with a base plate and surfaces for mounting the probe
contents. The bottom 1/4 of the probe consisted of a service module housing
the connectors for telemetry and input lines (Figure 21). All external
connections were passed through radio frequency (rt) chokes (cut off frequency
= 100MHz) in the service module. The rf chokes prevented unwanted signals
from reaching the NMR coil circuitry. Provision was made for incoming

perfusion lines and for the collection of venous out flow from the muscle.

98

Figure 21 . NMR probe for isolated perfused muscle experiments.

    
 
 
 
 
  
  

__ M' II
— "£11: llgurt

_— Force Transducer

— llelnlroltz Coil

7- ,1 \Circrlrting llater
Stunting Electrode:

 

Terperrture Probe
Effluent Drain

Tuning lode

 

 

g; _Mio Frequency
5 filter:

#0“ for Perfusion lines

L—Telnelry Connectors

lml

 

 

100
Various coil circuit designs were evaluated with respect to concurrent 1H

and 31P frequency sensitivity. Dual coils and single circuit dual tuned coils
failed to provide the necessary performance (27,43,76, 104,109). The final
design consisted of a single coil with supporting circuitry for tuning to both 400
MHz (1H) and 162 MHz (31P) (Figure 22). The two circuits were isolated by a
mechanical switch.

An Helmholtz configuration coil was constructed from 16 gauge, coated,
copper wire. Equation 1 was used to determine the optimal coil dimensions

(Figure 23).

(1) Size (mm) x 21r / 3 = X.

Where each horizontal leg = X, the front and rear gaps = X/2 and the vertical
legs = X x 1.1. A diameter of 20mm was used to ensure space for
accommodation of cat biceps muscles.

The electronic characteristics of each coil were determined from the
relationship between frequency, inductance and capacitance:
(2) f= 1/ 21 L95 x c'5
Where: L= coil inductance, C = total capacitance, f =frequency.
Equation 3 is the actual working equation derived from equation 2 . This
equation was used to determine L and intrinsic C (Cx) of the coil.
(3) C" = (1/ (21rF)2 ) / L - Cr
Where: C, = known (i.e. , added) capacitance and F = observed frequency.
Intrinsic capacitance and inductance the coil was determined by measuring F at
C, = 0 and at some C. and solving for the apparenth in each case. The
difference between these two Cx values divided by the known C,I provided L.

Equation 3 was then solved for the actual Cx.

101

Figure 22. Diagram of NMR coil and tuning circuits. The ‘H and 31P

circuits are isolated by a mechanical switch.

Coi

%/ Switch

102

 

 

 

_.rm:

 

 

 

 

 

 

 

 

103

Figure 23. A. Design template for Helmholtz NMR coil. B. Final coil

configuration.

104

 

.U -

 

 

 

A..:vx

 

 

 

 

 

 

 

 

o 0 .i.

 

105
The coil circuitry was mounted in a removable module in the probe

shell. All variable capacitors of the two coil circuits were mounted facing the
bottom of the probe. These capacitors were mated to spring loaded screw
drivers which extended through the bottom of the probe. These extensions
allowed frequency tuning and matching of both circuits while the probe was in
the magnet. A separate extension allowed operation of the frequency selection
switch while the probe was in the magnet.

A fixed muscle mount was attached to the coil module below the NMR
coil. The upper muscle mount was attached to a threaded rod which allowed for
changes in length with the muscle in place (Figure 24). The variable mount
could be adjusted from outside the magnet.

Force was measured as the stress on a cantilever beam made of a non-
magnetic polymer (Delrin, DuPont). Microfoil strain gauges (SR-4, BLH
Electronics) were attached to opposite faces of the beam (Figure 24). These
strain gauges made up two of the four legs of a Wheatstone bridge (Figure 25).
The bridge was balanced (i.e. , no current movement) with zero stress on the
beam. Stress on either face of the beam caused a change in resistance in the
two strain gauges. This change in resistance resulted in current movement
which was amplified and recorded on a strip chart recorder. This arrangement
represents a functional force transducer. The transducer was calibrated by
hanging various weights from the muscle mount and recording the output. The
Delrin based transducer used in the cat muscle experiments had a linear

response from 1 to 10 Kg (Figure 26).

106

Figure 24. Detail of variable length muscle mount with microfoil strain

gauges for force measurement.

107

 

Threaded
/ Rod

 

 

 

 

 

Foil
Strain Gauges

 

1

Muecle
Mount

108

Figure 25. Diagram of Wheatstone bridge with microfoil strain gauges as two

legs.

109

 

 

 

 

 

 

 

1 10
Stimulating electrodes consisted of 2cm lengths of platinum wire

attached to copper wire.

Temperature measurement was accomplished by placing a thermistor
(YSI) against the muscle. Temperature was controlled by circulating heated
water through a copper tube in the muscle compartment of the probe (Figure
21).

111

Figure 26. Calibration plot for Delrin force transducer. Pen deflection on the chart

recorder vs. weight attached to muscle mount (r=0.997).

112

NP

OF

30 £90;
0

 

1

l

 

or
m_.
ON
mm
on
mm
0.:

(mm) uogroauag

References

113

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