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dissertation entitled

MORPHOLOGICAL, BIOCHEMICAL, AND CONTRACTILE
PROPERTIES OF INNERVATED AND DENERVATED
STRIATED, SPHINCTERIC MUSCLES

presented by

Richard D. Kustasz

has been accepted towards fulﬁllment
of the requirements for

 

Ph.D . degree in m

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Major professor

Donald B. Jump, Ph.D.

Date June 27, 1996

 

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MORPHOLOGICAL, BIOCHEMICAL, AND CONTRACTILE
PROPERTIES or INNERVATED AND DENERVATED
STRIATED, SPHINCTERIC MUSCLES
By

Richard D. Kustasz

A DISSERTATION
Submitted to
Michigan State University
in partial ﬁtlﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology

1996

ABSTRACT
MORPHOLOGICAL, BIOCHEMICAL, AND CONTRACTILE
PROPERTIES OF INNERVATED AND DENERVATED
STRIATED, SPHINCTERIC MUSCLES
By

Richard D. Kustasz

The external anal sphincter (EAS) is a striated muscle innervated by the pudendal
nerves. Its ﬁmction is to maintain fecal continence by sealing the anal canal. Denervation
of the EAS muscle is the most common etiology for fecal incontinence. Changes in the
morphological, biochemical, and contractile properties of the chronically denervated EAS
muscle in the cat were studied to investigate the unique ﬁanction that this stn'ated muscle
provides compared with other striated limb and trunk muscles.

To compare the results of the denervated EAS muscles with innervated EAS
muscles, the biochemical classiﬁcation of its ﬁber type composition was investigated. The
cat EAS is composed of 66, 23, and 11% myosin heavy chain (MHC) isoform types IIB,
HA, and 1, respectively. MHC type IIB isoforms compose fast glycolytic muscle ﬁbers.
Myosin light chain isoform composition also characterized the EAS muscle to be a
predominantly fast glycolytic muscle.

Denervation of the EAS muscle resulted in a decrease in MHC type IIB content
and an increase in MHC type IIA content with no change in MHC type I content. The
denervated cat extensor digitorum longus (EDL) muscle has a similar isoform transition,
however, it has a progressive increase in MHC type I isoform content. This atypical MHC

isoform transition in the denervated EAS muscle is proposed to be because of its unique

embryologic origin and mechanical environment compared with those of the EDL muscle.

Atrophy of both the denervated EAS and EDL muscles was shown by their
decreases in wet weights and muscle protein contents. The muscle ﬁber cross-sectional
areas of the denervated EAS also decreased. Denervated EAS muscles had smaller ratios
between the percent decrease in the muscle protein content and the percent decrease in
muscle wet weight than did the denervated EDL muscles, presumably because of an
increase in denervated EAS connective tissue protein content.

Denervated EAS muscles had increased contraction and one-half relaxation times,
as well as increased fatigue resistances. These changes are consistent with the transition in
ﬁber type composition ﬁom predominantly fast glycolytic to fast oxidative-glycolytic. The
passive length-tension curves for the denervated EAS muscles were steeper, showing
increases in passive tensions at all muscle lengths. Passive tension is generated by the
connective tissue elements in a muscle. The increased passive tension in the denervated
EAS muscles is another indication that its connective tissue protein content increased. It
is proposed that the increase in passive tension assists the denervated EAS muscles in
maintaining some degree of fecal continence by providing the muscle with a greater

magnitude of passive force to resist the leakage of fecal material.

ACKNOWLEDGMENTS

My sincere gratitude goes to the Department of Physiology, whose support
throughout my undergraduate, medical, and postgraduate years at Michigan State
University has been remarkable. I am also very grateful to have had such a supportive
guidance committee: Dr. Donald Jump, for his leadership and insight into my progression
through my postgraduate years, Dr. William Helferich, for his willingness to teach
molecular biological techniques and for the use of his laboratory and for his camaraderie,
Dr. Seth Hootman, for his advice in many matters and for his guidance in teaching
physiology, Dr. Walter Esselman, for his advice concerning technical dilemmas, and most
of all to Dr. Thomas Adams, for his time, patience, concern, and wisdom, that have
inspired me during my years on campus.

Even greater gratitude goes to my former mentor, the late Dr. Jacob Krier. Dr.
Krier had a passion for knowledge that was unsurpassed, and I will never forget it. I wish
he were here today to share the data he made possible. Thanks to Dr. Krier for the
opportunity to learn from a great Physiologist.

My greatest gratitude goes to my family, for their love and support. Thanks to

Robin, Lauren, and Quinton - who keep me going. Thanks to my Mother and Father, for

everything.

iv

TABLE OF CONTENTS

LIST OF FIGURES ....................................................................................................... viii
KEY TO ABBREVIATIONS .......................................................................................... x
I. INTRODUCTION ..................................................................................................... l
A. External anal sphincter and fecal continence .......................................................... 2

B. Myoﬁbtillar proteins and muscle ﬁber types .......................................................... 4

C. The function of MHC and MLC subunits .............................................................. 9

D. Factors that regulate MHC and MLC isoform content ......................................... 10

E. The external anal sphincter and denervation ........................................................ 12

F. Rationale and speciﬁc aims ................................................................................. 14

11. MATERIALS AND METHODS .............................................................................. 16
A. Materials ............................................................................................................. 16

B. Surgical procedures ............................................................................................. 16

1. Chronic denervation ................................................................................... .1 6

2. Muscle excision .......................................................................................... 17

C. Morphological measurements .............................................................................. 18

1. Cross-sectional area detemIination .............................................................. l 8

2. Muscle protein content determination ......................................................... 19

3. Muscle wet weight determination ................................................................ 19

4. Staining for motor neurons and motor end plates ........................................ 20

D. Myoﬁbrillar protein extraction ............................................................................ 20

E. Myoﬁbrillar protein yield ..................................................................................... 21

F. MLC isoform analysis ......................................................................................... 22

G. MHC isoform analysis ......................................................................................... 23

H. Staining, destaining and quantiﬁcation of SDS gels ............................................. 24

I. Contractile measurements ................................................................................... 25

1. Muscle strip preparation ............................................................................. 25

2. Construction of the length-tension curve .................................................... 26

3. Determination of twitch contractile properties ............................................ 27

V

4. Determination of fatigability ....................................................................... 27

5. Determination of muscle strip force per cross-sectional area ....................... 28

J. Statistical analyses .............................................................................................. 28
III. RESULTS ............................................................................................................... 29
A. Morphological data ............................................................................................. 29

1. Muscle ﬁber cross-sectional areas ............................................................... 29

2. Muscle protein contents ............................................................................. 32

3. Muscle wet weights .................................................................................... 33

4. Motor neuron and motor end plate staining ................................................ 34

B. Biochemical data ................................................................................................. 36

1. MLC distribution of striated, sphincteric muscles ........................................ 36

2. MHC distribution of striated, sphincteric muscles ....................................... 37

3. MLC distribution of innervated and denervated EAS muscles ..................... 39

4. MHC distribution of innervated and denervated EDL muscles ..................... 4 1

5. MHC distribution of innervated and denervated EAS muscles ..................... 44

C. Contractile data ................................................................................................... 4 8

1. Transmural electrical ﬁeld stimulation with neuromuscular block ................ .48

2. Length-tension relation ............................................................................... 49

3. Twitch contractile data ............................................................................... 54

4. Fatigue data ............................................................................................... .57

5. Muscle force per cross-sectional area data .................................................. 59

IV. DISCUSSION ........................................................................................................ 60
A Morphologic properties ....................................................................................... 60

B. Biochemical data of striated, sphincteric muscles ................................................. 63

1. MLC compositions ..................................................................................... 63

2. MHC compositions ..................................................................................... 64

C. Biochemical data of denervated external anal sphincter muscles ........................... 66

1. MHC isoform transitions ............................................................................ 67

2. MLC isoform transitions ............................................................................. 69

D. Contractile data ................................................................................................... 72

1. Twitch contractile data ............................................................................... 73

2. Fatigue data ................................................................................................ 7 5

3. Muscle force per cross-sectional area data .................................................. 76

4. Length-tension relationship ......................................................................... 77

V. SUMMARY AND CONCLUSIONS ...................................................................... 80
APPENDIX ................................................................................................................... 83
BIBLIOGRAPHY .......................................................................................................... 85

LIST OF TABLES

Table l. Myosin heavy and light chain isoforrns of cat striated muscles ........................... 6
Table 2. Muscle wet weights and protein contents of innervated

and denervated cat muscles ............................................................................. 32
Table 3. Fast myosin light chain ratios for cat striated, sphincteric muscles ................... 37

Table 4. Fast myosin light chain ratios for innervated and denervated cat
external anal sphincter muscles ........................................................................ 4 1

Table 5. Contractile data for innervated and denervated cat external
anal sphincter muscles ..................................................................................... 56

LIST OF FIGURES

Figure 1. Drawing of the cat external anal sphincter muscle .......................................... 3

Figure 2. Schematic representation of skeletal muscle isomyosins and
their subunit compositions ............................................................................. 5

Figure 3. Photomicrographs showing H&E stain of cross-sections of
innervated and denervated cat external anal sphincter muscle
ﬁber bundles ................................................................................................ 30

Figure 4. Cross-sectional traces of innervated and denervated cat
external anal sphincter muscle ﬁber bundles ................................................. 31

Figure 5. Distribution of cross-sectional areas of innervated and
denervated cat external anal sphincter striated muscle ﬁbers ......................... 31

Figure 6. Photomicrographs showing staining of motor neurons and
motor end plates in innervated and denervated cat external
anal sphincter muscles .................................................................................. 35

Figure 7. Histogram showing myosin light chain isoform content of
cat striated, sphincteric muscles ................................................................... 36

Figure 8. SDS-polyacrylamide gel electrophoretic separation of cat
striated, sphincteric muscle myosin heavy chain isoforms .............................. 38

Figure 9. Histogram showing myosin heavy chain isoform content of
cat striated, sphincteric muscles ................................................................... 39

Figure 10. SDS-polyacrylamide gel electrophoretic separation of cat
innervated and denervated external anal sphincter muscle
myosin light chain isofonns .......................................................................... 40

Figure l 1. Histogram showing myosin light chain isoform content of
cat innervated and denervated external anal sphincter muscles ...................... 40

viii

Figure 12. Myosin heavy chain analysis of innervated and denervated
cat EDL muscles .......................................................................................... 42

Figure 13. Histogram showing myosin heavy chain isoform content of
cat innervated and denervated EDL muscles ................................................. 4 4

Figure 14. Myosin heavy chain analysis of innervated and denervated
cat EAS muscles .......................................................................................... 46

Figure 15. Histogram showing myosin heavy chain isoform content of
cat innervated and denervated EAS muscles ................................................. 47

Figure 16. Pen recording of electrical ﬁeld stimulation of a control cat
external anal sphincter muscle strip before and after
addition of d-tubocurarine ............................................................................ 48

Figure 17. Pen recording traces of active and passive isometric tensions
generated by a 60 day denervated cat external anal sphincter
muscle during one millimeter incremental stretches ....................................... 50

Figure 18. Plots of the relationship between isometric tensions and muscle
lengths for innervated and denervated cat external anal sphincter
muscles ........................................................................................................ 5 1

Figure 19. Passive length-tension relationships of innervated and denervated
cat external anal sphincter muscles ............................................................... 53

Figure 20. Twitch contractile responses of innervated and denervated cat
external anal sphincter muscles ..................................................................... 55

Figure 21. Fatigue responses of innervated and denervated cat external
anal sphincter muscles .................................................................................. 58

ANOVA
DENER
DU

EAS
EDL
EDTA
ESO
EUS

MLle
MLClsa
MLClsb
MLC2f
MLCZS
MLC3f

PAGE
PEC
SDS

KEY TO ABBREVIATIONS

One-way analysis of variance
Denervated

Distal urethra

External anal sphincter

Extensor digitonrm longus
Ethylenediarninetetraacetic acid
Esophagus

External urethral sphincter

Fast glycolytic

Fast oxidative-glycolytic
Hematoxylin and eosin
Innervated

Optimal length

Slack length at which passive tension is ﬁrst observed
Myosin heavy chain

Myosin light chain

A fast MLC isoform

A slow MLC isoform

A slow MLC isoform

A fast MLC isoform

A slow MLC isoform

A fast MLC isoform

Number of samples

Optimal tension

Polyacrylamide gel electrophoresis
Parallel elastic component
Sodium-dodecyl-sulfate
Standard error

Slow oxidative

Soleus

One-half relaxation time
N,N,N',N’-tetramethylethylenediamine
Tromethamine

MHC type I, the SO isoform
MHC type IIA, the FOG isoform
MHC type IIB, the PG isoform

L INTRODUCTION

The study of gastrointestinal motility began in 1896, when Walter B. Cannon
demonstrated esophageal peristalsis with the newly discovered Roentgen rays (Brooks et
a1. , 1975). It has evolved over the next one-hundred years from the radiographic
observance of the movement of a bolus to the study of the molecular arrangement of the
muscle cells that serve to propagate it. Molecular biological study techniques over the
past few decades have investigated the smooth muscle cell myoﬁbrillar proteins that
provide gastrointestinal motility. These techniques have developed to the point that they
can differentiate, for example, mRNA transcripts for two isoforms of actin in intestinal
smooth muscle cells that differ only by three of 375 amino acids (V andekerckhove and
Weber, 1979). The present study shows for the ﬁrst time the biochemical composition of
striated, sphincteric muscles in the alimentary and urogenital tracts.

Striated, sphincteric muscles, like the external anal sphincter (EAS), the external
urethral sphincter (EUS), and the upper esophageal sphincter, are arranged circumfer-
entially around hollow organs. They have striated muscle ﬁbers, like those in limb and
trunk muscles, that contract to close the lumen they surround and relax to allow passage
of lumenal contents. These muscles are innervated by somatic motoneurons and, like limb
and trunk muscles, undergo speciﬁc changes in their morphological, biochemical, and

contractile properties when denervated.

2

The EAS maintains fecal continence by closing the distal end of the anal canal.
Injury to the EAS muscle or its innervation leads to fecal incontinence. The most common
etiology for fecal incontinence is denervation of the EAS (Bartolo et al., 1994; Neill et a1. ,
1981). Trauma during childbirth is a common cause of EAS denervation. The pudendal
nerves that innervate the EAS are ﬁxed ﬁrmly in the pudendal canals. Delivery of the
fetus stretches the pudendal nerves, often causing partial, sometimes total, denervation of
the EAS and fecal incontinence (Bartolo et al., 1994). In fact, at three months after
childbirth, 19% of post-partum women remain fecally incontinent (Tetzschner et a1. ,
1995). Chronic straining during defecation, neuropathies, diabetes, and senescence also
produce EAS denervation. Seven percent of the elderly experience fecal incontinence at
least weekly (Bartolo et al., 1994). The current study presents an animal model to
investigate the morphological, biochemical, and contractile properties of the EAS

associated with denervation.

A. External anal sphincter and fecal continence

The EAS muscle lies circumferentially around the distal end of the anal canal
(Figure 1). It is under involuntary and voluntary control and ﬁrnctions to maintain fecal
continence. Distension of the smooth muscle of the rectum and anal canal initiates an
involuntary viscero-somatic reﬂex that contracts the EAS, thereby closing the distal
portion of the anal canal. Sensation of rectal fullness is a signal to initiate voluntary
contraction of the EAS. Afferent impulses ﬁom the rectum and anal canal are transmitted

via the pelvic nerve. The eﬁ‘erent motor outﬂow reaches the EAS via the pudendal nerves

(Figure 1).

I External anal sphincter

   

Anal canal
-I.. pudendal n.

 

Candad .. -—~ Orad

R. pudendal n.

 

 

Ventral

Figure 1. Drawing of the cat external anal sphincter muscle. It surrounds the distal anal
canal and is innervated by the right and left (R and L.) pudendal nerves (n.). It is typically
1 cm in width, 1 mm in thiclmess, and 6 cm in circumference.

The pudendal nerves are somatic motoneurons whose cell bodies are at the sacral
spinal cord level (82-83) in Onuf's nucleus (Krier, 1989). Dendritic branches from the
cell bodies receive input ﬁ'om the pelvic nerve and ﬁ'om descending supraspinal ﬁbers
(Nadelhaﬂ et al., 1980). Fibers originating in the cerebral motor cortex are involved in
voluntary contractions of the EAS, whereas impulses originating ﬁ'om the rectum and anal
canal initiate involuntary contractions (Krier, 1989).

Rectal distension is the primary stimulus for defecation. With sensation of rectal

4

fullness, the urge to defecate can be suppressed voluntarily by descending inputs to the
motoneurons that mediate contractions of the EAS, puborectalis, and levator ani muscles
(Krier, 1989). Descending inputs to the sacral spinal cord also initiate the principal
reﬂexes that underlie defecation. They activate parasympathetic efferent ﬁbers in the
pelvic nerve to initiate smooth muscle contraction of the rectum to propel the fecal
material anally. They also inhibit the excitatory input to the EAS, puborectalis, and
levator ani muscles to open the anal canal for fecal passage (Krier, 1989).

Intact pudendal nerves are essential in suppressing the urge to defecate.
Tetzschner er a]. (1995) have shown the risk for fecal incontinence increases with
decreased pudendal nerve ﬁrnction. They found a signiﬁcant increase in pudendal nerve
terminal motor latencies, a measure of conduction velocity, in post-partum women with
fecal incontinence, compared with a group of post-partum women without fecal
incontinence and with a group of control women. They concluded that the increased
latency was due to partial denervation of the EAS. The increased latency delays the

involuntary and voluntary contractions of the EAS, thereby causing fecal incontinence.

B. Myol‘rbrillar proteins and muscle ﬁber types

Striated muscle ﬁbers contain myoﬁbrillar proteins that make up the contractile
unit, the sarcomere. They include actin, myosin, troponins I, T, and C, trOpomyosin, C-
protein, desrnin, vimentin, a-actinin, myomesin, titin, and nebulin (Swynghedauw, 1986).
Myosin is the most abundantly expressed contractile protein, accounting for 60% of the

total myoﬁbrillar protein mass (Tsika et al., 1987).

5

The myosin molecule is a 460 kD hexarneric myoﬁbrillar protein composed of two

myosin heavy chain (MHC) subunits and four myosin light chain (MLC) subunits (Swyn-

 

 

Skeletci Muscle isomyosins

 

 

 

 

 

 

 

 

 

 

,. (A A SLCI
5'“: ﬂ 0 SLC:
Type] MHC
"~— (5
lm Q
Typol. MHC
"“3 {Z '
x FLCI
o FLC:
f (I: D FLC;
I‘ll; %
fin. {D
____/
Tunis MHC

Figure 2. Schematic representation of skeletal
muscle isomyosins and their subunit
compositions. ‘sz’ compose slow oxidative
ﬁbers, ‘Im’ compose fast oxidative-glycolytic
ﬁbers, and ‘ﬁnm’ compose fast glycolytic ﬁbers.
Abbreviations: MHC, myosin heavy chain; FLC,
fast light chain; SLC, slow light chain.
SLC,=MLCls, SLC2=MLC23, FLCl =MLC1f,
FLC2=MLC2f, FLC3=MLC3£ Figure is copied
from Tsika et al., (1987), with written
permission (see Appendix).

 

 

ghedauw, 1986) (Figure 2). There
are many isoforms of MHC and
MLC subunits. The number of
MHC isoforms varies among
species (Pin et al., 1994). Rat
limb and trunk muscles display six
different MHC isofomrs during
development. They are the
embryonic and neonatal isoforms
(Gunning and Hardeman, 1991), as
well as four adult isoforms - MHC
type I, the slow isoform present in
slow oxidative ﬁbers, and three
fast isoforms: MHC type IIA,
present in fast oxidative-glycolytic
ﬁbers; MHC type IIX, present in

intermediate ﬁbers; and MHC type

IIB, present in fast glycolytic ﬁbers (Bar and Pette, 1988; Schiafﬁno and Reggiani, 1994).
Cat limb and trunk muscles contain only three adult MHC isoform types: 1, HA, and IIB
(Table 1). Type 11X is not expressed (Talmadge et al., 1994). Figure 2 illustrates that the

two MHC subunits of a myosin molecule always exist as a homodimer and never as a

heterodimer (Tsika et al., 1987).

Table l. Myosin heavy and light chain isoforms of cat striated muscles.

 

 
  

 
  
 

Fiber
type

MHC isoforms MLC isoforms

      

 

 

 

FOG IIA (201.5) "(25-0)
Fast-twitch muscles 21‘ (18.0)
FG IIB (201.0) 3f (16.0)
lsa (27.5)
Slow-twitch muscles SO I (200.1) lsb (26.5)
2s (19.0)

 

 

 

Molecular weights (Weeds, 1976) are listed in parentheses (kD). Abbreviations: MHC,
myosin heavy chain; MLC, myosin light chain; FG, fast glycolytic; FOG, fast oxidative-
glycolytic; 80, slow oxidative; ‘t‘, fast; ‘8’, slow. The Arabic numbers denoting the MLC
isoforms indicate the order of distance migrated on a SDS-polyacrylamide gel (increased
distance with increased number).

In addition to the six MHC isoforms in rat limb and trunk muscles, there are two
tissue speciﬁc isoforms, the extraocular MHC isoform, found only in the adult extraocular
muscle and the superfast MHC isoform, found only in jaw-closing muscles (Gunning and
Hardeman, 1991). All eight MHC isoforms are encoded by distinct genes. The gene for
MHC type I is located on human chromosome 14, whereas the genes for the
developmental, fast, and tissue speciﬁc MHC isoforms are clustered on human

chromosome 17 (Schiafﬁno and Reggiani, 1994).

7

Typically, each muscle ﬁber expresses only a single MHC isoform, but some
muscles contain muscle ﬁbers that co-express two MHC isoforms, each as a homodimer
(Schiamno and Reggiani, 1994). Co-expression is commonly found in ﬁbers undergoing
ﬁber type transformation (Talmadge et al., 1993b). Fibers that co-express MHC isoforms
do so inaspeciﬁc manner. They co-express eitherMHCtypesIand IIAortypesIIAand
IIB. This is because ﬁbers undergoing ﬁber type transformation follow an obligatory
pathway of MHC gene expression in the sequence of MHC types IHIIAHIIB (Betto et
al., 1986; Schiafﬁno and Reggiani, 1994).

Each myosin molecule contains four MLC subunits, two on the amino-terminal
head of each MHC subunit (Schiaﬂino and Reggiani, 1994) (Figure 2). There are three
slow MLC isoforms, denoted by ‘s’, and three fast MLC isoforms, denoted by ‘f (Table
1). Slow-twitch muscle ﬁbers (slow oxidative, ‘Sm,’ in Figure 2) contain two of either
MLC 1 sa or MLClsb and two MLC2s. Fast-twitch muscle ﬁbers that are fast oxidative-
glycolytic (‘Im’ in Figure 2) contain one each of MLle and MLCls and two MLC2f.
Fast-twitch muscle ﬁbers that are fast glycolytic (‘ﬁn,,3’ in Figure 2) contain any
combination of two MLle and/or MLC3f and two MLC2f (Schiafﬁno and Reggiani,
1994; Swynghedauw, 1986; Thomason et al., 1986; Tsika eta1., 1987b).

Each MLC is encoded by a distinct gene, with two exceptions. The MLle and 3f
isoforms originate from a single gene, but are transcribed by alternative promoters and
have alternative splicing of the ﬁrst exon (Barton and Buckingham, 1985). The MLC l sa
and lsb isoforms are from similar transcripts but are post-translationally modiﬁed into
distinct slow isoforms (Gunning and Hardeman, 1991). Unlike all but one of the MHC

genes that are clustered on one human chromosome, each MLC gene is located on a

8

separate one (Schiaﬁino and Reggiani, 1994).

Physiologically deﬁned muscle ﬁber types are characterized by their protein
isoform proﬁle (Talmadge et al., 1993b). As shown in Figure 2, MHC and MLC subunits
exist as different isoform combinations in diﬁ‘erent muscle ﬁber types. Other myoﬁbrillar
proteins that have different isoforms in diﬁ‘erent muscle ﬁber types include troponins C
and I and tropomyosin (Talmadge et al., 1993b). There are nineteen fast and seven slow
troponin T isoforms (Hartner et al., 1989). Even non-myoﬁbrillar proteins show ﬁber
type speciﬁc distributions. For example, the relative amount of parvalbunrin, a calcium
binding protein, is greatest in fast glycolytic ﬁbers, intermediate in fast oxidative-glycolytic
ﬁbers, and smallest in slow oxidative ﬁbers (Schmitt and Pette, 1991). Other non-
myoﬁbrillar proteins that are not equally expressed in different muscle ﬁber types include
GLUT-4 (the insulin sensitive glucose transporter), glycolytic enzymes, and sarcoplasmic
ATPase enzymes (Talmadge et al., 1993b). Because striated muscle ﬁbers are
multinucleated, all myonuclei within each ﬁber must be coordinated to express the same
myoﬁbrillar isoforms and the same relative amounts of non-myoﬁbrillar proteins
(Talmadge et al., 1993b).

Because different muscle ﬁber types express different myoﬁbrillar protein isoforms
and varying amounts of other speciﬁc proteins, the histochemical identiﬁcation of muscle
ﬁber types by staining for these proteins has been made (Peter et al., 1972). Fibers high in
succinate dehydrogenase (SDH) activity and low in myosin adenosine triphosphatase
(ATPase) activity are classiﬁed as slow oxidative (80). Those with high SDH and
ATPase activity are fast oxidative-glycolytic (FOG) and the ﬁbers with high ATPase and

low SDH activity are fast glycolytic (F G).

9

A major emphasis in muscle research is to relate ﬁber type inducing agents to the
molecular regulation of isoform expression. Multiple mechanisms regulate the expression
of these isoforms at the transcriptional and translational levels (Gunning and Hardeman,
1991). Two muscle regulatory factors (transcription factors) have been related to
differential muscle gene expression. MyoD mRNA has been found to be more abundant in
fast-twitch muscle ﬁbers (those that express MHC types IIA and/or IIB) and myogenin
mRN A more abundant in slow-twitch muscle ﬁbers (those that express MHC type I)

(Weintraub et al., 1991).

C. The function of MHC and MLC subunits

The myosin molecule is the “thick” ﬁlament of the sarcomere. It interacts with the
“thin” ﬁlament, actin, to provide the structure for which the ﬁlaments slide past each other
to produce a muscle contraction (Alberts et al., 1989). The contraction of a muscle is
characterized by three properties: speed of contraction (“contraction time” or “velocity of
shortening”), speed of relaxation (“one-half relaxation time”), and resistance to fatigue
(Krier and Adams, 1990).

The MHC isoforms differ ﬁom each other functionally in their contribution to a
ﬁber's velocity of shortening and resistance to fatigue (Hoh and Hughes, 1988; Reiser et
al., 1988; Schiaﬁino and Reggiani, 1994; Swynghedauw, 1986; Thomason et al., 1986).
SO ﬁbers contain MHC type I and have a slow shortening velocity (long contraction time)
and are resistant to fatigue. Fibers that contain MHC type IIA (FOG ﬁbers) have a fast
shortening velocity and are also resistant to fatigue. Fibers with MHC type IIB (F G

ﬁbers) have the fastest shortening velocity and are susceptible to fatigue (Betto et al.,

10

1986; Peter et al., 1972; Reiser etal., 1985). The three MHC isoforms are responsible for
the variances in the velocities of shortening and, in part, the resistances to fatigue of the
three muscle ﬁber types because they each contain different ATPase activities (Reiser et
al., 1985; Schiaﬂino and Reggiani, 1994; Sweeney et al., 1986; Wagner, 1981).

The MLC isoform distribution also correlates with a muscle ﬁber's contractile
properties (Greaser et al., 1988; Schiaﬁ'rno and Reggiani, 1994; Weeds and Pope, 1971).
As shown in Figure 2, there is a preferential association of MLC isoforms with MHC
isoforms (Bottinelli etal., 1994; Tsika et al., 1987). This light chain-to-heavy chain
isoform relationship deﬁnes different velocities of shortening and endurance that are
required of a muscle ﬁber's different functions. For example, when a fast-twitch muscle
ﬁber's MHC type IIB isoform composition is constant, velocity of shortening is related to
the relative content of MLC3f. The velocity of shortening is faster in the ﬁber with a
greater abundance of MLC3f (Bottinelli et al., 1994a; Greaser et al., 1988; Moss et al.,
1990). It has been proposed that the MLC isoform composition of the myosin molecule
ﬁne tunes the contraction velocities within the ranges predetermined by the MHC isoforms

(Wada and Pette, 1993).

D. Factors that regulate MHC and MLC isoform content

The MHC and MLC isoform content of a muscle ﬁber is regulated by
developmental, neuronal, mechanical and hormonal factors (Gunning and Hardeman,
1991). Numerous experiments have been performed to investigate their effects on the

expression of MHC and MLC isoforms and the resultant transition in muscle ﬁber types.

11

Developmentally, it appears that myoblasts in the embryo are committed to express a
speciﬁc MHC isoform (Dirnario et al., 1993). Innervation of the newly formed myoﬁber
induces a MHC isoform transition, depending on the pattern of ﬁring by the motoneuron
(T almadge et a1. , 1993b). The myoﬁber exhibits a MHC isoform transition with the onset
of innervation even when the motoneuron’s electrical activity is abolished by spinal cord
transection above and below its exit from the spinal cord and bilateral deafferentation
between the two sectioned sites (Pierotti et al., 1991). This suggests the presence of a
neurotrophic inﬂuence between the motoneuron and the muscle ﬁbers with which it
contacts (Talmadge et al., 1993b).

Speciﬁc changes occur when an adult limb or trunk muscle has its neural input
altered. Denervation of a fast-twitch muscle results in a speciﬁc MHC isoform transition.
There is a switch in the predominance of the MHC type IIB isoform to MHC type IIA
when the predominantly FG adult rat extensor digitorum longus muscle is denervated
(Midrio et al., 1988). Chronic electrical stimulation, weight training, endurance training,
chronic stretch, synergistic muscle removal, phosphocreatine depletion, limb suspension,
limb immobilization, spaceﬂight, spinal transection, and hypo- and hyperthyroidism also
produce speciﬁc MHC isoform transitions (T almadge et al., 1993b).

A pattern of MHC isoform and ﬁber type transition has emerged ﬁom these
experiments. It appears that the transitions induced by equal perturbations of muscles of
the same ﬁber type composition are generically similar (Matsuda et al., 1984). For
example, hypothyroidism results in a shift of MHC isoform content to the left in the
isoform transition sequence (IHIIAHIIB). Thus, a predominantly SO muscle will

become more SO by increasing its number of SO ﬁbers, thereby increasing its MHC type I

12

content. On the other hand, a predominantly FG muscle (MHC type IIB) will become less
PG and more FOG (MHC type 11A) and SO (MHC type I) (Izumo et al., 1986).

Likewise, denervation of predominantly FG muscles has been shown consistently to shift
MHC isoform content to the left in the isoform transition sequence. There is a decrease in
MHC type IIB and an increase in MHC types HA and I isoform content. Concomitant
with the shift of MHC isoforms toward the slower types is a slowing of the contractile
properties and an increased resistance to fatigue (Midrio et al., 1988; Tyc and Vrbova,
1995). This is consistent with the fact that the MHC isoforms differ ﬁ'om each other
ﬁrnctionally in their contribution to a ﬁber's velocity of shortening and resistance to

fatigue.

E. The external anal sphincter and denervation

Denervation of the EAS is the most common etiology for fecal incontinence.
Although many studies have shown that denervated limb and trunk muscles undergo
characteristic changes in their myosin isoform content with chronic denervation (Carraro
et al., 1981; Gauthier and Hobbs, 1982; Ishiura et al., 1981; Matsuda et al., 1984; Midrio
et al., 1988; Tyc and Vrbova, 1995), the myosin isoform content of the denervated EAS
has not been studied. Because transitions induced by equal perturbations of muscles of the
same ﬁber type composition are generically similar (Matsuda et al., 1984), it would be
assumed that the EAS undergoes similar changes to denervation that occur in a limb or
trunk muscle of similar ﬁber type composition. The present study questions this
assumption because the EAS, although striated, is unique. It differs from limb and trunk

muscles in its reﬂex arc, morphology, and contractile properties.

13

The EAS, like limb and trunk muscles, is innervated by a somatic motoneuron.
Unlike limb or trunk muscles, though, the motoneuron is part of a viscero-somatic reﬂex
arc (Krier, 1985). This fact, and because the EAS is virtually devoid of intraﬁrsal muscle
ﬁbers (Borghi et al., 1991; Krier et al., 1988), suggests its activation is more likely ﬁom
mucosa] mechanoreceptor stimulation than it is ﬁom static or dynamic length changes in
the muscle itself (Krier and Adams, 1990).

The morphology of the cat EAS is different ﬁom that of limb and trunk muscles.
Unlike a limb or trunk muscle that is ﬁxed to bones by tendons, the EAS is arranged
circumferentially around the distal end of the anal canal. It has large amounts of
connective tissue separating muscle fascicles, and even the muscle ﬁbers themselves (Krier
et al., 1989). Limb and trunk muscles have relatively small amounts of connective tissue
between muscle fascicles and none between muscle ﬁbers. Muscle ﬁber size is also a
distinguishing feature of the cat EAS. The mean cross-sectional area of cat limb and trunk
muscle ﬁbers is 12-14 times larger than that of cat EAS muscle ﬁbers (Krier et al., 1989).

Contractile properties of the cat EAS are different from those of limb and trunk
muscles. The passive length-tension curve of the EAS has a steeper slope (Krier and
Adams, 1990), which resembles that of the other type of striated muscle, cardiac muscle.
Cardiac muscle is like striated, sphincteric muscle in that it surrounds a hollow organ
without a ﬁxed insertion.

These distinctive properties of the EAS enable it to function in maintaining fecal
continence (Krier et al., 1988; Krier et al., 1989). Its viscero—somatic reﬂex arc allows
information from the viscera (the rectum and anal canal) to regulate its contraction related

to a fecal mass located 3-20 cm orad to the sphincter. The circumferential orientation

14
permits it to guard the anal oriﬁce by generating a centriﬁcally directed contraction, unlike
the linear forces developed by limb and trunk muscles. Its steeper passive length-tension
curve protects the EAS ﬁom being overstretched beyond the optimal length for force
generation, unlike a limb or trunk muscle that is protected from overstretch by their bony

insertions and by opposing muscles.

F. Rationale and specific aims

The objectives of the present research are to determine the morphological,
biochemical, and contractile properties of the cat EAS muscle before and after its
denervation. Properties of the innervated EAS will be compared with other innervated,
striated, sphincteric muscles (viz. external urethral sphincter and upper esophageal
sphincter). They will also be compared with properties of a fast-twitch limb muscle (cat
extensor digitorum longus [EDL]) and slow-twitch limb muscle (cat soleus).

The cat EAS has been classiﬁed as a fast-twitch muscle based on its histological
and mechanical properties (Krier et al., 1988). Limb and trunk muscles have also been
classiﬁed by both properties, but have additionally been classiﬁed biochemically according
to their myosin isoform content. Because the EAS has not been biochemically classiﬁed,
this study tests the hypothesis that the EAS consists of myosin isoforms representative of
fast-twitch muscle. Since other striated, sphincteric muscles have a similar ﬁrnction as the
EAS, it is proposed they consist of similar myosin isoform proﬁles.

When a limb or trunk muscle is denervated, its muscle ﬁbers undergo speciﬁc
changes in their morphological, biochemical, and contractile properties. The unique

characteristics of the cat EAS muscle may cause it to respond differently to denervation

15

than would a limb or trunk muscle of similar ﬁber type composition. Because the cat EAS
has been histochemically classiﬁed as a predominantly fast glycolytic muscle and
demonstrates a high speed of contraction and relaxation (Krier et al., 1988), its
denervation changes can be compared with changes in a denervated fast glycolytic limb
muscle. The cat EDL is predominantly fast glycolytic with a similar high speed of
contraction (Ariano etal., 1973; Midrio et al., 1988; Tyc and Vrbova, 1995). This study
tests the hypothesis that the EAS will undergo atrophic changes similar to those of the
denervated EDL, but will show unique changes in its biochemical properties that relate to
its distinctive ﬁrnction of sealing the anal canal.

The speciﬁc aims of the present study are to test the hypotheses that:

1. Denervation of the cat external anal sphincter striated muscle leads to decreases in
muscle ﬁber cross-sectional area, muscle wet weight, and muscle protein content

just as for a denervated limb or trunk muscle.

2. The myosin isoform content of the cat external anal sphincter and other striated,

sphincteric muscles is representative of fast-twitch muscle.

3. Denervation of cat external anal sphincter striated muscle leads to changes in
myosin isoform content that are unlike those induced by denervation of a fast-

twitch limb or trunk muscle.

4. Changes in contractile properties of denervated cat external anal sphincter striated

muscle are related to changes in myosin isoform content.

II. MATERIALS AND METHODS

A. Materials

Adult female cats (3-4 kg) were legally obtained from county licensed, official
animal pounds. They were housed in bilevel cages (4 ft’) in the University Laboratory
Animal Research (ULAR) building. Daily light cycle was 12 hours room light, 12 hours
dark. Temperature was maintained at 72°C and humidity at 45%. Litter boxes were
changed daily. Dry cat food and ﬂesh water were replenished daily. The cats were
inspected and handled daily by the present investigator and the ULAR staﬂ‘. Cats were
euthanized humanely and painlessly while under anesthesia (sodium pentobarbital [35

mg/kg, IP]) by intracardiac injection of 20 mg/kg sodium pentobarbital.

B. Surgical procedures

1. Chronic denervation

Adult female cats (3-4 kg) were anesthetized with sodium pentobarbital (35
mykg, IP). An intravenous catheter was inserted into the right brachial vein to administer
5% dextrose and lactated Ringers to maintain electrolyte balance and for delivering
supplemental sodium pentobarbital (10 mg/kg, IV). Bilateral pudendal nerve trunks and

16

17

their branches to the EAS muscle were isolated by skin incision and blunt dissection in the
sciatic notch region. Tibial nerve trunks and their branches to the EDL muscles were
similarly isolated in the lateral legs below each knee. Bilateral pudendal nerves and the
tibial nerve branches to the right EDL were sectioned 5-10 mm from their entry into their
respective muscle. Five mm segments of nerve were removed to prevent reinnervation
(Gauthier and Hobbs, 1982; Tyc and Vrbova, 1995). The tibial nerve branches to the left
EDL were isolated, but not sectioned, to serve as sham-operated controls. The muscle
fascia and then the skin incisions of each region were sutured with 3-0 ethilon silk and the

cats were allowed to recover.

2. Muscle excision

After 10, 30, or 60 days, cats were again anesthetized with sodium pentobarbital
(35 mg/kg, IP) and catheterized intravenously as previously described. The EAS was
isolated by skin incision in the sciatic notch and perineal regions. Striated muscle was
separated ﬁom the underlying serosal surface of the anal canal by blunt dissection. The
sphincter was cut perpendicular to its long axis on its ventral surface to permit its removal.
The external urethral sphincter (BUS) muscle and distal urethra were isolated by a midline
abdominal incision and blunt dissection extending from the umbilicus to the pubic
symphysis. A cannula was inserted into the urethra to facilitate muscle dissection. The
proximal one-third of the esophagus, which is composed of skeletal muscle (Floyd and
Morrison, 1975), was excised through a skin incision and blunt dissection in the thoracic
cavity and neck region. Bilateral EDL muscles were similarly isolated in the lateral leg

below each knee. The EAS, EUS, distal urethra, proximal esophagus, and bilateral EDL

18

muscles were excised and placed in a modiﬁed Krebs solution (21°C). The solution was
continuously aerated with 95% O2 and 5% CO2 and contained (mM): 117 NaCl, 4.7 KCl,
2.5 CaCl,, 1.2 MgClz, 1.2 NaHzPO“ 25 NaHCO, and 11 glucose. Cats were then
euthanized by intracardiac injection of sodium pentobarbital (20 mg/kg).

The excised muscles were cleaned under a dissecting microscope while bathed in
the Krebs solution. Adherent fascia, tendons, adipose, glandular and connective tissues
were removed. Muscles were then placed either in formalin or citric acid for use in
morphological procedures, ﬂown in liquid nitrogen for biochemical procedures, or

mounted in an organ bath for contractile measurements.

C. Morphological measurements

1. WW

Excised EAS muscle was laid ﬂat and pinned, without inducing stretch, to the
sylgard ﬂoor of an organ bath containing aerated Krebs solution (21°C). This solution
was replaced with 10% formalin in 0.1 M phosphate buffered saline to ﬁx the tissue.
Tissue was embedded in para-plast plus (American Scientiﬁc). Ten pm sections were
obtained at room temperature, transferred to glass slides, and then stained with
hematoxylin and eosin.

Measurements of muscle ﬁber cross-sectional areas were made from microscope
tracings of cross-sectional muscle ﬁber bundle ﬁelds at a ﬁnal magniﬁcation of 250x. Five
randomly chosen areas of muscle ﬁber bundles were measured: one from the mid-dorsal

region, one each ﬁom the left and right dorso-lateral regions, and one each ﬁ'om the left

l9

and right ventro-lateral regions. A total of 500 muscle ﬁbers was sampled for each
innervated and denervated muscle. Cross-sectional areas were calculated by scanning the
traces using a computer-assisted integration technique (Sigma-Scan, Jandel Scientiﬁc).
Sixty day denervated muscle ﬁber cross-sectional areas were not measured because of the
high degree of atrophy, which rendered the muscle too thin to make accurate cross-

sectional slides.

2. Musele protein content determination

Muscle protein content was determined as described by Babij and Booth (1988a).
Muscles (25 mg) were placed in 0.4 ml of cold, double deionized water in a Corex tube
and homogenized. One ml of perchloric acid (PCA) (2.5% (v/v) at 4°C) was added,
vortexed and placed on ice for 10 minutes. Muscle samples were then centrifuged at
34,858 X g for 15 minutes at 4°C. The supernatant was discarded and 1.0 ml PCA (1.0%
(v/v) at 4°C) added. The pellet was broken and the suspension vortexed. The suspension
was centrifuged at 34,858 X g for 15 minutes at 4°C. The supernatant was then discarded
and 0.8 ml of potassium-hydroxide (0.3 N) added, pellet broken, vortexed and placed in a
37°C water bath for two hours with occasional mixing. Muscle protein concentration was
determined ﬁom the alkaline digest using the biuret method as described below (Gomall et

aL,1949)

3. Mgsele wet weight determinetien
Innervated and denervated muscle wet weights were measured using ﬁozen muscle

samples. Muscle wet weights are used as an index of muscle atrophy after denervation

20
(Midrio er al., 1988).

4. ainin formo rneur us m r n it

Motor neuron and end plate staining was performed by the “gold impregnation
method” as described by Zinn and Morin (1962). Excised EAS muscle was cleaned in
aerated Krebs solution and then ﬁxed in a petri dish containing 0.001 M citric acid for 10
min followed by rinsing with double deionized water ﬁve times. It was then immersed in a
1.0% (w/v) aqueous gold chloride solution for one hour followed by ﬁve rinses with
double deionized water. The muscle was then placed in 33% (v/v) formic acid for 24
hours in the dark. It was then rinsed ﬁve times with double deionized water and placed
ﬂat, in glycerol, on a glass slide. Muscle ﬁbers were teased apart and mounted under a

cover slip in glycerol jelly.

D. Myofibrillar protein extraction

Myoﬁbrillar proteins were extracted as described by Adams et a]. (1994). Frozen
muscle (300mg) was minced with scissors in 4 ml of sucrose extraction solution (250 mM
sucrose, 100 mM KCl, and 5 mM EDTA-Naz). The sample was then homogenized with a
Polytron homogenizer for 10 seconds, then centriﬁrged at 1,086 X g for 10 minutes at
7°C. Supernatant was discarded and the sucrose extraction procedure was repeated
twice. The resultant pellet was then resuspended in 4 ml of triton X-100 solution (175
mM KCl, 2 mM EDTA-Nag and 0.5% (v/v) triton X-100), vortexed, and centrifuged at

1,086 X g for 10 minutes at 7 °C. The supernatant was discarded and this procedure was

21

repeated twice. The resultant pellet was resuspended in 4 ml KCl (150 mM), vortexed,
centrifuged at 1,086 X g for 10 minutes at 7°C, supematant discarded and this procedure
was repeated twice. The washed myoﬁbrillar pellet was then suspended in 1 ml KCl (150
mM) and the total myoﬁbrillar protein concentration of each muscle sample was

determined for subsequent use in myosin light and heavy chain isoform analyses.

E. Myof'rbrillar protein yield

Myoﬁbrillar protein yield was detemrined by the biuret method (Gomall et al.,
1949). Different concentrations of bovine serum albumin (BSA) (0.00, 0.25, 0.50, 0.75,
and 1.00 jig/pl) made from stock solution (0.5% (w/v) BSA, ﬁaction V, in 150 mM KCl)
were used to plot the relation between BSA concentration and absorbance at 540 nm
(standard curve). These data were ﬁt to a ﬁrst-order polynomial of the form:

y=bx +a
where 'y’ is the absorbance at 540 nm, 'x' is BSA concentration, 'a' is the y-axis intercept,
and 'b' is the slope of the line. Correlation coeﬂicients (r) were calculated and ranged ﬁ'om
0.9880 to 0.9996.

The different concentrations of BSA were made by adding 200 pl of biuret color
reagent (100 mM Na2CO3, 600 mM sodium citrate, and 70 mM CuSO,) and 2 ml of
NaOH (6% w/v) to various volumes (0.0, 0.2, 0.4, 0.6, and 0.8 ml) of the BSA stock
solution. A sample with 0.1 ml KCl (150 mM) instead of BSA served as the 0.00 jig/pl
BSA standard. Muscle sample solutions were made with 0.1 ml of myoﬁbrillar sample in

place of BSA The ﬁnal volume of each BSA standard and myoﬁbrillar sample was

22

brought to 4 ml by the addition of double deionized water. The absorbances were
measured and the values were calculated from the BSA standard curve. These values
were multiplied by 40 (dilution factor) to give the myoﬁbrillar protein concentration of
each muscle sample (pg/pl). Samples were brought to a ﬁnal protein concentration of ﬁve
jig/pl with the appropriate volume of protein sample and the addition of one-third part

100 mM Na,P20-, and two-third parts electrophoretic grade glycerol and stored at -20°C.

F. MLC isoform analysis

Myoﬁbrillar protein samples were electrophoresed under denaturing conditions to
determine MLC isoform content of muscles, as described by Laemmli (1970). The
sodium-dodecyl-sulfate (SDS) polyacrylamide separating gels were composed of 15%
(w/v) acrylamide, 0.075% (w/v) bis (200:1 acrylamide to bis ratio), 1.5 M Tris (pH 8.8),
0.2% (w/v) SDS, and 0.05% (w/v) ammonium persulfate (APS). A 15% separating gel
maximizes the separation of the low molecular weight (14-27 kD) myosin light chains
(Tsika et al., 1987). Oxygen was removed ﬁ'om the solution by a water aspirator degaser
for 10 minutes to facilitate polymerization (Greaser et al., 1983). This was followed by
the addition of 0.0004% (v/v) N,N,N',N-tetramethylethylenediamine (TEMED), and this
solution was poured in a 16 x 18 x 0.15 cm casting stand. After the separating gel
hardened (one hour), a polyacrylamide stacking gel composed of 3% (w/v) acrylamide,
0.15% (w/v) bis (20:1 acrylamide to his ratio), 0.125 M Tris (pH 6.8), 0.1% (w/v) SDS,
and 0.05% (w/v) APS was made. This solution was degased for 10 minutes followed by

addition of TEMED (0,0004% v/v). This solution was poured on top of the separating

23

gel with a 15 lane separating comb in place. After hardening (one hour), gels were placed
in a Hoefer SE 600 buffer tank. Buffer was added to the upper and lower reservoirs (0.05
M Tris, 0.384 M glycine, and 0.1% (w/v) SDS).

Aliquots of each myoﬁbrillar sample (15 pl = 75 pg) were mixed with 35 pl of
protein denaturing buffer (62.5 mM Tris-base, 20% (v/v) glycerol, 5% (v/v) 2-
mercaptoethanol, 2.3% (w/v) SDS, and 0.05% (w/v) bromophenol blue). After boiling for
two minutes, 75 pg (50 pl) of each sample and a standard of low molecular weight
proteins (14-70 kD, Dalton Mark VII-L, Sigma) were loaded into wells. The gels were
electrophoresed at constant voltage (100 V) for 16 hours at 4°C with constant stirring of

the lower buﬁ‘er by a magnetic stir bar.

G. MHC isoform analysis

Myoﬁbrillar protein samples were electrophoresed under denaturing conditions to
determine MHC isoform content of muscles, as described by Talmadge and Roy (1993 a).
The SDS polyacrylamide separating gels were composed of 8.0% (w/v) acrylamide,
0.16% (w/v) bis (50:1 acrylamide to bis ratio), 30.0% (v/v) glycerol, 0.2 M Tris (pH 8.8),
0.1 M (w/v) glycine, 0.4% (w/v) SDS, and 0.1% (w/v) ammonium persulfate (APS).
Oxygen was removed from the solution by a water aspirator degaser for 10 minutes to
facilitate polymerization (Greaser et al., 1983). This was followed by the addition of
0.05% (v/v) TEMED, and this solution was poured in a 16 x 18 x 0.075 cm casting stand.
After the separating gel hardened (one hour), a poly-acrylamide stacking gel composed of

4.0% (w/v) acrylamide, 0.08% (w/v) bis (50:1 acrylamide to bis ratio), 30.0% (v/v)

24

glycerol, 70 mM Tris (pH 6.7), 4.0 mM (w/v) EDTA, 0.4% (w/v) SDS, and 0.1% (w/v)
APS was made. This solution was degased for 10 rrrinutes followed by addition of
TEMED (0.05% v/v). This solution was poured on top of the separating gel with a 20
lane separating comb in place. After hardening (one hour), gels were placed in a Hoefer
SE 600 buffer tank. The upper running buffer consisted of 0.1 M (w/v) Tris-base, 150
mM (w/v) glycine, and 0.1% (w/v) SDS. The lower running buffer consisted of 50 mM
(w/v) Tris-base, 75 mM (w/v) glycine, and 0.05% (w/v) SDS.

Aliquots of each myoﬁbrillar sample (1 pi = 5 pg) were mixed with 39 pl of
protein denaturing buﬁ‘er (62.5 mM Tris-base, 20% (v/v) glycerol, 5% (v/v) 2-
mercaptoethanol, 2.3% (w/v) SDS, and 0.05% (w/v) bromophenol blue). After boiling for
two urinates, 1 pg (8 pl) of each sample and a standard of high molecular weight proteins
(30-200 kD, SDS-6H, Sigma) were loaded into wells. The gels were electrophoresed at
constant voltage (275 V) for 24 hours at 4°C with constant stirring of the lower buffer by

a magnetic stir bar.

H. Staining, destaining and quantification of SDS gels

After each timed run, the gels were placed in staining solution (0.05% (w/v)
Coomassie Brilliant Blue R-250, 50% (v/v) methanol, and 9.2% (v/v) acetic acid) and
gently swirled for two hours on a rotating plate. They were then placed in destaining
solution (10% (v/v) methanol and 7.5% (v/v) acetic acid) overnight. DisPO-plugs were
placed in the destaining trays to absorb free Coomassie Blue. The appropriate bands on

the gels were than quantiﬁed by densitometry (Hoefer GS3 65W electrophoresis data

25

system 3 .01). The intensity of each isoform's band was represented as the percentage of
area (Gaussian integrated) under the peak generated by that band compared with the

summated peak areas of all the muscle's MHC or MLC isoform bands.

L Contractile measurements

1. W

The EAS was isolated as described in the surgical procedure. The sphincter was
laid ﬂat and pinned to the sylgard ﬂoor of an organ bath containing aerated Krebs solution
(21°C). Adherent connective tissue and blood vessels were removed. Two 1cm strips, 2-
3 mm wide, were cut out of the dorsal aspect of the muscle, parallel to the long axes of
the ﬁbers (Krier et al., 1989).

A one gram weight was hung on a force transducer (Grass model FT-O3C) and its
deﬂection was recorded on a pen recorder (Gould model 2400) for calibration. Pen
recorder paper speed was maintained at 0.10 mnI/sec throughout the experiment, unless
noted otherwise. The prepared strips were then mounted vertically in the long axis of the
ﬁbers under isometric conditions in heated 90 ml organ baths ﬁlled with aerated Krebs
solution (37°C). Two organ baths were used, one for each strip. One end of each strip
was attached to the force transducer by a silk thread. The force transducer was connected
to a micrometer (Newport Corporation, model 420) to enable incremental stretching of
the strips. The other end of each strip was attached to a rigid ﬁame by another silk thread.
Strips were allowed to equilibrate at slack length for one hour after which their lengths

were measured.

26

Transmural electrical ﬁeld stimulation was applied using two 6 x 50 mm platinum
electrodes mounted parallel to the strips. Current was supplied by a constant-current
ampliﬁer whose voltage and ﬁ'equency were controlled by a stimulus function generator
(Grass model S-88). The voltage intensity that produced the maximal twitch response was
determined by increasing the voltage until no further increase in the amplitude of the
response was elicited. Strips were then transmurally stimulated at supramaximal intensity
(10-12 V), using square wave pulses of 1.5 ms duration throughout the experiment to
produce a consistent contractile response.

To show that the amplitude of twitch contraction produced by transmural electrical
ﬁeld stimulation had no neural contribution, the neuromuscular blocking agent, d-
tubocurarine was used. Three min into an 18 min period of stimulation at 0.05 Hz, d-
tubocurarine was added to the bath (200 pM). During the ensuing 15 min, if the twitch
contractile amplitude did not decrease, then a neural contribution could be ruled out

(Parlani et al., 1992).

2. Qeeetmgien ef the length-tensien eerve

Passive, active, and total isometric tensions were recorded by the pen recorder at a
paper speed of 0.25 mrn/sec. Muscle strips were stretched in increments of one
millimeter. Starting at “slack length” (no stretch) and then one min after each stretch,
strips were stimulated at 0.3 Hz for a one min period. Passive tension was determined by
the mean of the amplitudes measured at one and two min after the stretch. Active tension
was determined by the mean of the twitch contractile response amplitudes generated

during the one min of stimulation. Total tension was determined by the sum of the means

27

of the passive and active tensions. The stretched length that produced the maximal twitch
contractile response was considered L0 (optimal length) and the tension produced at L,
was considered Po (optimal tension). The relationship between tension and length was

plotted where tension was expressed as a ﬁ'action of Po, and length was expressed as a

ﬁaction of L0.

shamammmsammm

Single twitches were evoked in muscle strips that were stretched to their
determined optimal length (L0) to measure contraction time and one-half relaxation time
(Tm). Contraction time is the measure of time from the onset of stimulation to peak
twitch force. T1,2 is the measure of time from peak twitch force to one-half peak twitch
force. These times were recorded by stimulating the muscle strips with single pulses at
supramaximal intensity using square wave pulses of 1.5 ms duration. Pen recorder paper
speed was increased to 200 mm/sec to expand responses for enhanced time resolution.
Three twitch contractions were elicited per strip and their contraction and Tl,2 times were

averaged.

4. Determinetien ef fatigabilig

To determine their fatigability, muscle strips were stimulated at supramaximal
intensity using square wave pulses of 1.5 ms duration at 20 Hz to produce tetanus. The
pen recorder paper speed was set at two mm/sec. Peak tetanic tension and the tension
after 30 sec of tetanic stimulation were measured and a fatigue index was calculated as

follows:

28

M tetanic tension) —.(tetan.ic team—W4 x 100
(peak tetaruc tensron)

 

 

The fatigue index represents the percent of tension lost because of muscle fatigue.

5. Determinatien pf mpsele strip feree pg mee-geetipnal arg

The cross-sectional area of the muscle strip was determined as described by
Herlihy and Murphy (1973). Muscle strip wet weight was determined after blotting to
remove excess moisture. This value was divided by the product of the muscle density
(1.050 g/ml) and the determined Lo. Muscle strip force (g) was determined by dividing the
amplitude of the twitch contraction evoked at Lo by the number of mm of deﬂection
generated by the one gram weight. Muscle tension for each muscle strip was expressed as

force per cross-sectional area (kg/cm’).

J. Statistical analyses

Data are represented as means :1: SE. Statistical analyses were performed by
Graphpad Instat software, version 1.11a (Graphpad Software). Statistical signiﬁcance for
diﬁ‘erences among mean values in all muscle groups was determined by one-way analysis
of variance (AN OVA). Statistical signiﬁcance for differences between pairs of groups was
determined by the AN OVA “post test” Bonferroni method. Differences were considered

signiﬁcant if the p values were less than 0.05.

III. RESULTS

A. Morphological data

1. Muscle ﬁber crees-mienal m

Cross-sectional areas of innervated and denervated EAS muscle ﬁbers were
measured from traces made ﬁom slides of serially cross-sectioned muscles. Figure 3
shows photomicrographs of cross-sections from an innervated EAS muscle (panel A) and
a 30 day denervated EAS muscle (panel B). Figure 4 is a sample of traces of ﬁber bundle
cross-sections ﬁom an innervated (panel A) and 30 day denervated (panel B) EAS muscle.
The myoﬁbers in panel A of Figures 3 and 4 are larger than those in panel B, suggesting
muscle ﬁber atrophy of the denervated muscle.

Figure 5 shows the distribution of muscle ﬁber cross-sectional areas in innervated
and denervated cat EAS muscles. Innervated muscle ﬁber areas ranged ﬁom 50 to 825
pm’. Denervated muscle ﬁber areas ranged from 25 to 600 pm’. The mean cross-
sectional areas (t SE) of the innervated, 10 day denervated and 30 day denervated EAS
muscle ﬁbers were 247 i 9, 172 :i: 3, and 138 :i: 6 pm”, respectively. The 10 day
denervated and 30 day denervated EAS muscle ﬁbers showed a 30 and 44% decrease in
mean cross-sectional areas, respectively, compared to control (p < 0.0001 among all three
groups (ANOVA) and p < 0.001 between each pair of groups (Bonferroni post tests)).

29

30

These data show that the denervated muscle ﬁbers undergo progressive atrophy.

 

Figure 3. Photomicrographs showing H&E stain of cross-sections of innervated and
denervated cat external anal sphincter (EAS) muscle ﬁber bundles. Panel A is from the
left dorso-lateral region of an innervated EAS muscle. Panel B is from the left dorso-
lateral region of a 30 day denervated EAS muscle. Both photomicrographs are magniﬁed
100X. Calibration bar in panel A is 50 pm and also refers to panel B.

 

Figure 4. Cross-sectional traces of innervated and denervated cat external anal sphincter
(EAS) muscle ﬁber bundles. Traces were drawn ﬁ'om slides containing cross-sectional
serial sections at 250X magniﬁcation. Panel A shows ﬁber bundles ﬁ'om an innervated
EAS and panel B ﬁ'om a 30 day denervated EAS. Calibration bar in panel A is 100 pm

and also refers to panel B.

 

 

100 -
D innervated (control) EAS
10 day dara'vated EAS
80 h — — 30 day denervatedEAS

 

 

it of ﬁbers within 25 pm’ groups
to cs
0 o
I I

RF
0 I 1II ’ l I I I I I
50-75 150-175 250-275 350-375 450-475 550-575 650-675 750-775
0-5 1m1§ 200-25 soc-325 400-45 500525 W 700-75 800-825
Fiber cross-sectional area (pm’)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5. Distribution of cross-sectional areas of innervated and denervated cat external
anal sphincter (EAS) striated muscle ﬁbers. The ordinate shows the mean number of
ﬁbers, of the 500 sampled per muscle, that were within 25 pm2 groupings. Three
innervated muscles (bars), three 10 day denervated muscles (solid line), and three 30 day
denervated muscles (dashed line) were studied.

 

32

2. W

Denervation of the EDL muscle induced a signiﬁcant change in protein content
among experimental and control groups (p < 0.0003, ANOVA). Compared with the
innervated control EDL, the 10 and 60 day denervated EDL muscles showed decreases in
protein contents of 29 and 40%, respectively (Table 2). The decrease in protein content
was signiﬁcant between control and 10 day denervated (p < 0.003) and between control
and 60 day denervated (p < 0.0006) EDL muscles, but not between 10 day and 60 day

denervated EDL muscles (Bonferroni post tests).

Table 2. Muscle wet weights and protein contents of innervated
and denervated cat muscles.

 

          
   
   
   

MUSCLE
PROTEIN
CONTENT

(mg/muscle)

MUSCLE
WET
WEIGHT

(0)

115.1: 6.5 81215.3 $2: 1.6 22011.8 18.0tO.7 16110.8 12.5:13

   

29%1' 13%1 27%1‘ 4396i“

 

23%.!" 20961 40%1‘ 50%1’

3.4 t 0.2 2.0 t 0.3 1.8 t 0.2 1.2 1 0.1 1.0 t 0.1

 

 

 

 

 

 

 

 

Values are mean :1: SE, n = number of muscles studied. Percent decrease compared with
innervated control muscle is indicated as ‘96 i ’. Abbreviations: EAS, external anal
sphincter; EDL, extensor digitorum longus; dener, denervated; inner, innervated. #, p <
0.003 vs. innervated EDL; &, p < 0.03 vs. innervated EAS; @, p < 0.03 vs. 10 day
denervated EAS; *, p < 0.02 vs. innervated EDL; S, p < 0.03 vs. innervated EAS.

33

Denervation of the EAS muscle induced a signiﬁcant change in protein content
among experimental and control groups (p < 0.004, ANOVA). Compared with the
innervated control EAS, the 10, 30, and 60 day denervated EAS muscles showed
decreases in protein contents of 18, 27, and 43%, respectively (Table 2). The decrease in
protein content was signiﬁcant between control and 30 day denervated (p < 0.03 ), control
and 60 day denervated (p < 0.003), and 10 day and 60 day denervated (p < 0.03) EAS
muscles, but not between control and 10 day denervated, 10 day and 30 day denervated,

or 30 day and 60 day denervated EAS muscles (Bonferroni post tests).

1ng

Denervation of the EDL muscle induced a signiﬁcant change in wet weight among
experimental and control groups (p < 0.002, ANOVA). Compared with the innervated
control EDL, the 10 and 60 day denervated EDL muscles showed decreases in mass of 23
and 36%, respectively (Table 2). The decrease in muscle mass was signiﬁcant between
control and 10 day denervated (p < 0.02) and between control and 60 day denervated (p <
0.002) EDL muscles, but not between 10 day and 60 day denervated EDL muscles
(Bonferroni post tests).

Denervation of the EAS muscle induced a signiﬁcant change in wet weight among
experimental and control groups (p < 0.02, ANOVA). Compared with the innervated
control EAS, the 10, 30, and 60 day denervated EAS muscles showed decreases in mass
of 20, 40, and 50%, respectively (Table 2). The decrease in muscle mass was signiﬁcant
between control and 30 day denervated (p < 0.03) and control and 60 day denervated (p <

0.01) EAS muscles, but not between control and 10 day denervated, 10 day and 30 day

34

denervated, 10 day and 60 day denervated, or 30 day and 60 day denervated EAS muscles

(Bonferroni post tests).

4. Moter neprpn apd meter end plate staining

Denervation of the EAS muscle resulted in the degeneration of motor neurons and
the disappearance of motor end plates. Figure 6 shows photomicrographs from innervated
control EAS (panel A) and ﬁ'om 10 day denervated EAS (panel B). Panel A shows a dark
stained motor neuron and its motor end plates forming synapses on muscle ﬁbers. Panel B
shows degeneration of the motor neuron’s axon and a total absence of motor end plates,

indicative of chronic denervation.

35

 

Figure 6. Photomicrographs showing staining of motor neurons and motor end plates in
innervated and denervated cat external anal sphincter (EAS) muscles. Panel A is ﬁom the
dorsal region of an innervated EAS muscle. Notice the motor neuron dividing into several
branches and terminating on single muscle ﬁbers as motor end plates (white arrows).
Panel B is ﬁ'om the dorsal region of a 10 day denervated EAS muscle. Notice the absence
of motor end plates and the degeneration of the motor neuron’s axon (white arrow). Both
photomicrographs are magniﬁed 400X. Bar in A is 20 pm and also refers to B.

36

B. Biochemical data

Figure 7 shows the percent distributions of each MLC isoform present in cat
striated, sphincteric muscles as measured by densitometry and compares them with the
distributions in the cat soleus (SOL) and EDL. A representative SDS-polyacrylamide gel
separation of the MLC isoforms of cat striated muscles is shown in Figure 10. The MLC
distribution of the SOL is solely Isa, lsb and 23. These MLC isoforms are found in S0
muscle ﬁbers. In contrast, the EDL has a predominance of MLle, 2f and 3f. These
MLC isoforms compose FG fast-twitch muscle ﬁbers. The EDL has a small content of
MLC 1 sa and lsb, indicating there are also FOG fast-twitch muscle ﬁbers present, but the
absence of MLC2s indicates the lack of SO ﬁbers. The cat EAS, EUS, distal urethra
(DU), and esophagus (ESO) have a distribution of MLC isoforms like the fast-twitch EDL

and unlike the slow-twitch SOL.

 

_ -SOL\EUS

[jam-DU
.EASEBSO

    

 

 

    

+11

2ttt§§RNRNNNSNNRNNNSSRNSN§§§§§§§§§
I9, V///////////////////////////////////////////A

% isoform content
8 8 8 8 8
I
H

..n
O
l
ZZZZZZZZZZ3ZZZ35%ﬁéﬂéﬁﬂﬁéﬁﬁﬁéﬁé£552222222

RFRFSS5atiasNNSSSSSSSS$§§§§§§§RRRRR§§

541222
lllllllllllll
4545
llllllllllllll

 

 

 

 

 

léﬂéﬁdﬂﬁﬂéééﬁé’
Illllll

IIIIIIIII
‘tttttttttttttttttt
IIIIIIIII
SSNNNNRRRSSSSSS

IIIIIIIIII
SNN§¥$S

O

 

 

‘—

3

"5

l

“t
on:
E

inc
5'

2:

Figure 7. Histogram showing myosin light chain (MLC) isoform content of cat striated,
sphincteric muscles. Percent isoform content was determined by densitometric scanning
of SDS gels. Ordinate represents the amount of each MLC isoform present, expressed as
a percentage (mean :1: SE) of the total MLC isoform content for each muscle.
Abbreviations: SOL, soleus (n = 4); EDL, extensor digitorum longus (n = 5); EAS,
external anal sphincter (n = 5); EUS, external urethral sphincter (n = 4); DU, distal urethra
(n = 4); ESO, esophagus (n = 5). 1f, 2f, and 3f are the fast MLC isoforms; lsa and lsb
are the slow MLC isoforms.

37

Table 3 shows the relative concentrations of MLC3f expressed by the ratio of
MLC isoforms: 3f / (1f + 3f). This ratio is indicative of the predominant ﬁber type content
of the muscle (Wada and Pette, 1993). A larger ratio is associated with a fast-twitch
muscle composed of predominantly FG ﬁbers (fastest contracting fast-twitch ﬁbers) and a
smaller ratio with a fast-twitch muscle composed of predominantly FOG ﬁbers (slowest
contracting fast-twitch ﬁbers). The EUS and DU have the largest ratio, indicating they are
the fastest muscles. The ESO has the smallest ratio, indicating it is the slowest of these

fast-twitch muscles.

Table 3. Fast myosin light chain ratios for cat striated, sphincteric muscles.

 

 

 

 

 

 

 

 

 

1f(%content) 37.8 :I: 3.2 32.1 t 1.7 37.2 t 2.8 35.0 :i: 3.5 35.4 :i: 1.9
31(%content) 10.0 a: 3.5 7.5 :I: 3.9 13.5 :I: 4.5 14.7 :t 4.1 7.0 :1: 3.8
3fl(1 f+31) 0.209 0.189 0.266 0.296 0.165

 

 

Values are mean percentage of total myosin light chain isoform content :1: SE, n =
number of muscles studied. Abbreviations: EDL, extensor digitorum longus; EAS,
external anal sphincter; EUS, external urethral sphincter; DU, distal urethra; ESO,
esophagus; 1f; MLle; 3f, MLC3f. A greater 3f7(1f+3f) ratio indicates a higher fast
glycolytic muscle ﬁber content.

Figure 8 is a representative SDS-polyacrylamide gel separation of the MHC
isoforms of cat striated, sphincteric muscles. Other myoﬁbrillar proteins are shown on

this gel, including actin, which appears equally intense in each lane, indicating equal

38
loading of proteins. Figure 9 shows the percent distribution of each MHC isoform for
each striated, sphincteric muscle as measured by densitometry. The cat soleus (not
shown) has 100% MHC type I (the SO MHC isoform). In contrast, the EDL has only
2.9% MHC type I, but has 68.5% MHC type IIB, the MHC isoform found in FG muscle
ﬁbers. The cat EAS, EUS, DU, and ESO also show a predominance of MHC type IIB,

indicating that they are composed of predominantly FG muscle ﬁbers.

 

Figure 8. SDS-polyacrylamide gel electrophoretic separation of cat striated, sphincteric
muscle myosin heavy chain (MHC) isoforms. Muscle samples were run in an 8%
polyacrylamide gel to separate the high molecular weight MHC isoforms. Abbreviations:
eso, esophagus; ed], extensor digitorum longus; eus, external urethral sphincter; eas,
extemal anal sphincter; du, distal urethra; t, titin; n, nebulin; IIA, MHC type IIA; IIB,
MHC type IIB; I, MHC type I; aa, a-actinin; v, vimentin; d, desmin; act, actin.

39

90-

      

        

//

76///

% isoform content

 

 

 

 

 

 

 

Iii] \li 1.
l l

EAS EUS ' DU ESO
-MHCtypeI l:IMI-1CrypeIIA MHCtypeIIB

  

 

 

 

 

 

Figure 9. Histogram showing myosin heavy chain (MHC) isoform content of cat
striated, sphincteric muscles. Percent isoform content was determined by densitometric
scanning of SDS gels. Ordinate represents the amount of each MHC isoform present,
expressed as a percentage (mean :1: SE) of the total MHC isoform content for each
muscle. Abbreviations: EDL, extensor digitorum longus (n = 4); EAS, external anal
sphincter (n = 4); EUS, external urethral sphincter (n = 4); DU, distal urethra (n = 4);
ESO, esophagus (n = 4). MHC type I is the slow oxidative isoform, HA is the fast
oxidative-glycolytic isoform and IIB is the fast glycolytic isoform.

3. MLC dietributien pf innervetﬁ epd denervgtﬂ EAS mpglee

Figure 10 is a representative SDS-polyacrylamide gel separation of the MLC
isoforms of cat innervated and denervated EAS muscles. Figure 11 compares the MLC
isoform contents of the innervated and denervated EAS muscle groups. There are no
statistically signiﬁcant differences in the MLC isoform distributions among the four

groups (ANOVA), nor between pairs of groups (Bonferroni post tests).

40

std 60d 30d 10d CEAS EDL SOL

    

29— , --...
24—
m
u

20 --*- -—-— -—
- -..._. _d

c ”-4-. _3f

Figure 10. SDS-polyacrylamide gel electrophoretic separation of cat innervated and
denervated external anal sphincter (EAS) muscle myosin light chain (MLC) isoforms.
Muscle samples were run in a 15% polyacrylamide gel to separate the low molecular
weight MLC isoforms. In this gel, EDL and SOL lanes were loaded with twice the
amount of protein sample to enhance the MLC isoform bands. CEAS lane was
underloaded. Abbreviations: SOL, soleus; EDL, extensor digitorum longus; CEAS,
innervated (control) EAS; 10d, 10 day denervated EAS; 30d, 30 day denervated EAS;
60d, 60 day denervated EAS; std, molecular weight standards lane; ti, troponin I; tc,
troponin C; Isa, lsb, and 2s, slow MLC isoforms; 1f, 2f, and 3f, fast MLC isoforms.

1

Figure 11. Histogram showing myosin light chain (MLC) isoform content of cat
innervated and denervated external anal sphincter (EAS) muscles. Percent isoform
content was detemrined by densitometric scanning of SDS gels. Ordinate represents the
amount of each MLC isoform present, expressed as a percentage (mean :t SE) of the total
MLC isoform content for each muscle. Innervated (control) external anal sphincter (n =
5), 10 day denervated EAS (n = 4), 30 day denervated EAS (n = 4), and 60 day
denervated EAS muscles (n = 4). 1f, 2f, and 3f are the fast MLC isoforms; 1sa and lsb
are the slow MLC isoforms.

 

.WEAS Ewdaydenorvatod
[:deaydonervatod mmwdaydenorvated

 

 

 

% isoform content
N
O

 

   

r

 

 

k)

41

Table 4 shows the relative concentrations of MLC3f of the innervated and
denervated EAS muscle groups as expressed by the ratio of MLC isoforms: 3f / (1f + 3f).
The innervated and denervated EAS muscles showed no statistically signiﬁcant
differences in relative MLC3f concentrations among all four groups (AN OVA), or

between pairs of groups (Bonferroni post tests).

Table 4. Fast myosin light chain ratios for innervated and denervated
cat external anal sphincter muscles.

 

 

11' (%content) 32.1 t 1.7 29.9 :i: 2.8 28.5 :1: 2.2 30.2 1: 3.6

 

31 (%content) 7.5 :1: 3.9 6.6 rt 5.0 5.8 :i: 3.7 5.9 t 4.9
3f!(1f+30 0.189 0.181 0.169 0.163

 

 

 

 

 

 

 

Values are mean percentage of total myosin light chain isoform content :t SE, n =
number of muscles studied. Abbreviations: CEAS, control (innervated) external
anal sphincter; 10d EAS, 10 day denervated EAS; 30d EAS, 30 day denervated
EAS; 60d EAS, 60 day denervated EAS; 1f, MLle; 3f, MLC3f. A greater
3f7(1f+3f) ratio indicates a higher fast glycolytic muscle ﬁber content.

4. We:

Figure 12A is a representative SDS-polyacrylamide gel separation of the MHC
isoforms ﬁom the three cat EDL muscle groups. Figure 12B shows the densitometric
tracings of the MHC isoform bands ﬁom each group. MHC type I migrated the farthest
distance (lowest band in Figure 12A) and is represented by the peak labeled ‘1’ in Figure
12B. MHC type IIA migrated the least distance (highest band) and is represented by the

peak labeled ‘IIA’. MHC type IIB had an intermediate migration distance (middle band)

42
and is represented by the peak labeled ‘IIB’. The Gaussian integrated areas under each
peak were measured and reported as the percentage of the summated areas of all three

peaks for each muscle.

 

SOl. 60d l 0d CEDL

CEDL

 

I IIB IIA

Figure 12. Myosin heavy chain analysis of innervated and denervated cat EDL muscles.
A) SDS-polyacrylamide gel electrophoretic run of innervated EDL (CEDL), 10 day
denervated EDL (10d), and 60 day denervated EDL (60d). Isoforrns are labeled as
MHC type I (I), MHC type HA (HA), and MHC type IIB (IIB). The cat soleus (SOL)
contains only MHC type I and is shown for reference. B) Densitometric traces of the
MHC isoform bands ﬁ'om the three lanes containing EDL muscles in A. Traces are
superimposed to compare peak area changes (representing changes in isoform content).
Abbreviations as in A.

43

The percent content of each MHC isoform in innervated and denervated cat EDL
muscles is shown in Figure 13. Compared with the innervated (control) EDL muscles,
MHC type I ﬁber content progressively increased 300% after 10 days and 455% after 60
days of denervation (p < 0.0009 among all three groups, ANOVA). These are large
increases, but the total content of this isoform remained low compared with the other
two. The increase in MHC type I content was signiﬁcant between control and 10 day
denervated (p < 0.02), control and 60 day denervated (p < 0.001), and 10 day and 60 day
denervated (p < 0.04) EDL muscles (Bonferroni post tests). MHC type IIA ﬁbers also
showed a progressive increase compared with controls: 189% after 10 days and 237%
after 60 days of denervation (p < 0.003 among all three groups, AN OVA). The increase
in MHC type IIA content was signiﬁcant between control and 10 day denervated (p <
0.02) and between control and 60 day denervated (p < 0.003) EDL muscles, but not
between 10 day and 60 day denervated EDL muscles (Bonferroni post tests). MHC type
IIB ﬁber content progressively decreased 46 and 72% after 10 and 60 days denervation,
respectively, compared with controls (p < 0.0008 among all three groups, AN OVA).
The decrease in MHC type IIB content was signiﬁcant between control and 10 day
denervated (p < 0.01) and between control and 60 day denervated (p < 0.001) EDL
muscles, but not between 10 day and 60 day denervated EDL muscles (Bonferroni post
tests). MHC types I and HA increased progressively and MHC type IIB decreased

progressively with increasing duration of denervation.

H®

 

a)

o

I
H (93

 

0'1
0
l

0)
O
l

 

% isoform content
&
O
l

N
O
l

.x
O
l

 

 

 

 

 

 

   

 

 

1

 

 

 

CEDL lOdEDL 60dEDL
- MI-ICtypeI [:1 MHCtypeIIA MHCtypeIIB

Figure 13. Histogram showing myosin heavy chain (MI-1C) isoform content of cat
innervated and denervated EDL muscles. Percent isoform content was determined by
densitometric scanning of SDS gels. Ordinate represents the amount of each MHC
isoform present, expressed as a percentage (mean at SE) of the total MHC isoform
content for each muscle. Abbreviations: CEDL, innervated (control) extensor digitorum
longus (taken ﬁom sham operated contralateral leg of experimental cats, n = 4);
lOdEDL, 10 day denervated EDL (n = 4); 60dEDL, 60 day denervated EDL (n = 4).
MHC type I is the slow oxidative isoform, type [IA is the fast oxidative-glycolytic
isoform, and type IIB is the fast glycolytic isoform. 1‘, p < 0.02 vs. CEDL MHC type I;
#, p < 0.04 vs. lOdEDL MHC type I; @, p < 0.02 vs. CEDL MHC type IIA; &, p < 0.01
vs. CEDL MHC type IIB.

5. MHC dietributien pf innervgtﬂ end denervetﬁ EAS mpgles

Figure 14A is a representative SDS-polyacrylamide gel separation of the MHC
isoforms from the four cat EAS muscle groups. Figure 14B shows the densitometric
tracings of the MIIC isoform bands ﬁom each group. The percent content of each MHC

isoform is shown in Figure 15. Similar to the denervated cat EDL, the MHC type HA

4S

isoform content progressively increased 249, 262, and 306% after 10, 30, and 60 days of
denervation, respectively, compared with the innervated (control) EAS muscles (p <
0.0001 among all four groups, ANOVA). The increase in MHC type IIA content was
signiﬁcant between control and 10 day denervated (p < 0.0001), control and 30 day
denervated (p < 0.0001), control and 60 day denervated (p < 0.0001), 10 day and 60 day
denervated (p < 0.005), and 30 day and 60 day denervated (p < 0.02) EAS muscles, but
not between the 10 day and 30 day denervated EAS muscles (Bonferroni post tests).
Also similar to the denervated cat EDL, the MHC type IIB isoform content progressively
decreased 41, 57, and 71% after 10, 30, and 60 days denervation, respectively, compared
with controls (p < 0.0001 among all four groups, ANOVA). The decrease in MHC type
IIB content was signiﬁcant between control and 10 day denervated (p < 0.0002), control
and 30 day denervated (p < 0.0001), control and 60 day denervated (p < 0.0001), and 10
day and 60 day denervated (p < 0.005) EAS muscles, but not between 10 day and 30 day
denervated or 30 day and 60 day denervated EAS muscles (Bonferroni post tests). In
contrast to the denervated cat EDL, the denervated cat EAS muscles showed no
statistically signiﬁcant changes in MHC type I isoform contents among all four groups

(ANOVA), or between pairs of groups (Bonferroni post tests).

46

A 1
0‘ r, ‘ _
II. ‘ '
Ill}; b'ﬂ .
I’— ""' 'ﬂ
CEAS 10d sod 60d sor.

 

 

Figure 14. Myosin heavy chain analysis of innervated and denervated cat EAS muscles.
A) SDS-polyacrylamide gel electrophoretic analysis of myosin heavy chain isoforms of
innervated EAS (CEAS), 10 day denervated EAS (10d), 30 day denervated EAS (30d),
and 60 day denervated EAS (60d). Isoforrns are labeled as MHC type I (I), MHC type
HA (HA), and MHC type IIB (IIB). The cat soleus (SOL) contains only MHC type I
and is shown for reference. B) Densitometric traces of the MHC isoform bands from the
four lanes containing EAS muscles in A. Traces are superimposed to compare peak area
changes (representing changes in isoform content). Abbreviations as in A.

47

80' -%$
70— 17

60 E
50

T

40

l

30 #&

% isoform content

20?

10 -
0 j t [j
CEAS ' lOdEAS 30dEAS 60dEAS

- MHCtypeI l:l MHCtypeIIA MHCtypeIIB

 

 

 

 

 

 

/////////////////////////////////=k

  

 

 

 

 

 

 

 

 

 

Figure 15. Histogram showing myosin heavy chain (MHC) isoform content of cat
innervated and denervated EAS muscles. Percent isoform content was determined by
densitometric scanning of SDS gels. Ordinate represents the amount of each MHC
isoform present, expressed as a percentage (mean :1: SE) of the total MHC isoform
content for each muscle. Abbreviations: CEAS, innervated (control) external anal
sphincter (n = 6); lOdEAS, 10 day denervated EAS (n = 4); 30dEAS, 30 day denervated
EAS (n = 4); 60dEAS, 60 day denervated EAS (n = 4). MHC type I is the slow
oxidative isoform, type 11A is the fast oxidative-glycolytic isoform, and type IIB is the
fast glycolytic isoform. *, p < 0.0001 vs. CEAS MHC type IIA; %, p < 0.005 vs.
lOdEAS MHC type HA; 3, p < 0.02 vs. 30dEAS MHC type IIA; #, p < 0.0002 vs.
CEAS MHC type HB; &, p < 0.005 vs. lOdEAS MHC type IIB.

48

C. Contractile data

1. Tr mur l ' ﬁl imulainwithneurm l l k

To determine if there was any neural contribution to twitch contractile
amplitudes, EAS muscle strips were electrically stimulated at supramaximal intensity, 1.5
ms duration, at 0.05 Hz for 18 min. D-tubocurarine was added to the organ bath (200
pm) three min into the 18 min stimulation period. Figure 16 shows a representative
recording of an innervated (control) EAS muscle strip exposed to d-tubocuran'ne. The
amplitude of the twitch contractions remained constant throughout the 18 min
stimulation, indicating that there was no neural contribution to the electrically stimulated

twitch amplitude.

 

Figure 16. Pen recording of electrical ﬁeld stimulation of a control cat external anal
sphincter (EAS) muscle strip before and after addition of d-tubocurarine. The strip was
stimulated at 12 V, 1.5 ms duration at 0.05 Hz. Pen recorder paper speed was 0.10
min/sec. D-tubocurarine was added to the organ bath (200 pm) three min into the 18
min stimulation period (arrow). The ensuing 15 min of stimulation shows no change in
twitch contractile amplitude.

49

2. Length-teneien reletien

To plot the relationship between the length and isometric tension of the
innervated and denervated EAS muscles, strips were incrementally stretched at one
millimeter intervals and electrically stimulated for a one min period beginning one min
after stretch initiation. Figure 17 shows the resulting active and passive tensions
generated by a 60 day denervated EAS muscle strip. Panel A shows the active tensions
(twitch contractions) produced at “slack length” (no stretch) and at one min after the ﬁrst
one millimeter stretch. Panels B—G Show the resulting passive tensions (height of
baseline) and active tensions generated after additional one millimeter stretches. Passive
tensions were measured at the one and two min marks alter the stretch and averaged.
The passive tension progressively increased with increased stretch. The active tensions
were measured by taking the mean of all twitch amplitudes evoked during the one rrrin
stimulation period. The active tension progressively increased until panel D, then
progressively decreased with increased stretch.

The plotted relationships between length and isometric tension of the innervated
and denervated EAS muscle strips are Shown in Figure 18. Plot A is from innervated
(control) EAS muscles (n = 4), plot B is ﬁ'om 10 day denervated EAS muscles (11 = 4),
and plot C is from 60 day denervated EAS muscles (n = 4). The abscissas represent the
muscle length expressed as a fraction of L,. The ordinates represent the tension
generated by the muscle expressed as a ﬁaction of P,. Total tensions (dashed lines) were
determined by the sum of the active and passive tensions. In all three panels, the active
tensions (solid lines) progressively increase until the muscle length (L) equals L, (LIL, =

1), where they begin to decrease progressively. Also in all three panels, the passive

50
tensions (dotted lines) show a progressive, nonlinear increase as the muscle strip length is

"mum..- munmmm Jill

WWW

c d

AIIWWM

e f 9

Figure 17. Pen recording traces of active and passive isometric tensions generated by a
60 day denervated cat external anal sphincter muscle during one millimeter incremental
stretches. Pen recorder paper speed was 0.25 mm/sec. The muscle strip was stimulated
at 10 V, 1.5 ms duration, at 0.3 Hz for generation of active tension. Panel A shows
active tension at “slack length” (no stretch) and active and passive tensions at one mm
stretch. The following panels show active and passive tensions generated after additional
one mm stretches. The passive tension shows an increase after each stretch, whereas the
active tension increases to a maximum of 17.5 mm in panel D and then progressively
declines.

51

4.0— A

Tension (PIPo)

 

 

 

0.6 0.8 1.0 1.2 1.4

Tension (P/Po)

 

 

 

 

1.4
Length (L/Lo)
4.0 i" C y] :-l-
8 3.2 '- / .':
Q ' 7:1
9, 2.4 — 7/
.5 “ / I
g 1.6 - 7
cu . :
l.—
l
1.4

 

Length (L/Lo)

Figure 18. Plots of the
relationship between isometric
tensions and muscle lengths for
innervated and denervated cat
external anal sphincter (EAS)
muscles. Plot A is from
innervated (control) EAS muscles
(n = 4), plot B is ﬁ'om 10 day
denervated EAS muscles (n = 4),
and plot C is ﬁom 60 day
denervated EAS muscles (n = 4).
The abscissas represent the
muscle length expressed as a
ﬁ'action of L,. The ordinates
represent the tension generated by
the muscle expressed as a fraction
of P,. Active tension is
represented by a solid line,
passive tension by a dotted line,
and total tension by a dashed line.
Standard error bars only point
downward for the active and
passive tensions and only upward
for the total tensions to avoid

error bar overlap.

 

— active tension
-------- passive tension
——- total tension

 

 

 

52

Figure 19 shows the passive tensions from the innervated and denervated EAS
muscle groups. The passive tension curves in the two denervated muscle groups are
steeper than that of the innervated (control) EAS muscle group. The mean (:1: SE) passive
tension (P/P,) exhibited by the control EAS muscles at L, was 0.22 :h 0.03 (which is 22%
the magnitude of the maximal active tension). The mean passive tensions exhibited by the
10 day and 60 day denervated EAS muscles at L, were 0.99 :i: 0.10 (which is 99% the
magnitude of the maximal active tension) and 1.12 i 0.20 (which is 112% the magnitude
of the maximal active tension), respectively. This is an increase of 4.5 and 5.1 times the
mean passive tension of the control EAS at L, (p < 0.002 among all three groups,

AN OVA). The increase was signiﬁcant between control and 10 day denervated (p <
0.006) and between control and 60 day denervated (p < 0.003) EAS muscles, but not

between 10 day and 60 day denervated EAS muscles (Bonferroni post tests).

53

 

3.6 _ "" Innervated (control)
—— 10d denervated
"""" 60d denervated

 

 

3.2 -

 

2.8 r

2.4 -

2.0 -

1.6 —

Tension (PlPo)

1.2 -

 

 

 

l l l l l l l l l l l

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6
Length (ULo)

Figure 19. Passive length-tension relationships of innervated and denervated cat external
anal sphincter (EAS) muscles. The passive length-tension curves ﬁ'om the three plots in
Figure 18 are plotted together to illustrate the degree of change in passive tensions
between innervated and denervated groups. The abscissa is the muscle length expressed
as a ﬁ‘action of L,. The ordinate is the tension generated by the muscle expressed as a
ﬁaction of P,. The innervated (control) EAS is represented by the dashed line, the 10 day
denervated EAS by the solid line, and the 60 day denervated EAS by the dotted line.
Standard error bars only point downward for the 10 day denervated and upward for the 60
day denervated EAS muscle groups to avoid error bar overlap.

54
3. Twiteh mntraetile date

Twitch contractile properties were measured by stimulating muscle strips stretched
to their optimal length (L,) with single pulses at supramaximal intensity at 1.5 ms duration.
Pen recorder paper speed was 200 mm/sec. Figure 20 shows traces ﬁ'om each of the three
EAS muscle groups tested. Table 5 shows the mean (:|: SE) twitch contraction and one-
half relaxation times (T m) for each of the three muscle groups.

Contraction times ranged ﬁom 35-38 ms for innervated, 44-55 ms for 10 day
denervated, and 45-55 ms for 60 day denervated EAS muscles. Compared with the
innervated EAS muscles, there was a 38 and 36% increase in contraction times of the 10
day and 60 day denervated EAS muscles, respectively (p < 0.003 among all three groups,
AN OVA). The increases in contraction times were signiﬁcant between innervated and 10
day denervated (p < 0.004) and between innervated and 60 day denervated (p < 0.006)
EAS muscles, but not between the 10 day and 60 day denervated EAS muscles
(Bonferroni post tests).

Tl,2 ranged ﬁom 23-28 ms for innervated, 38-50 ms for 10 day denervated, and
43-50 ms for 60 day denervated EAS muscles. Compared with innervated EAS muscles,
there was an 84 and 76% increase in Tm of the 10 day and 60 day denervated EAS
muscles, respectively (p < 0.0003 among all three groups, ANOVA). The increases in T1,2
were signiﬁcant between innervated and 10 day denervated (p < 0.0009) and between
innervated and 60 day denervated (p < 0.002) EAS muscles, but not between the 10 day

and 60 day denervated EAS muscles (Bonferroni post tests).

55

 

 

 

Figure 20. Twitch contractile responses of innervated and denervated cat external anal
sphincter (EAS) muscles. Panel A is ﬁ'om an innervated EAS, B is from a 10 day
denervated EAS, and C is from a 60 day denervated EAS. Muscles were stimulated by
single pulses at supramaximal intensities (10-12 V) at 1.5 ms duration. Pen recorder paper
speed was 200 mm/sec. Vertical calibration bar in panel A equals 2 g and also applies to
panels B and C. Horizontal calibration bar in panel A equals 50 ms and also applies to
panels B and C. Notice the elongation of contraction and one-half relaxation times of the
denervated EAS muscles compared with the innervated one.

56

Table 5. Contractile data for innervated and denervated cat external anal sphincter
muscles.

 

 

 

 

 

EAS EAS EAS
innervated 10 day denervated 60 day denervated
(n84) (n-4) (n-4)
CONTRACTION
TIME 36.5 :1: 1.0 50.5 :i: 28* 49.5 r 2.4*
(ms)
RELAXATION
TIME 25.3 :i: 1.2 46.5 :1: 3.3' 44.5 1 2.4’
(ms)
FAT'fofNDE" 53 :1: 12 10 1 as 0 1 0s
FORCE PER
secgggeem, 0.10 1: 0.01 0.12 r 0.02 0.11 :i: 0.02
(kc/cm”)

 

 

 

 

 

Values are mean :1: SE, n = number of muscles studied. Abbreviation: EAS, external anal
sphincter. *, p < 0.006 vs. innervated EAS; #, p < 0.002 vs. innervated EAS; S, p < 0.01
vs. innervated EAS. Fatigue index is the percent decrease of peak muscle tension
developed after 30 sec of tetanic stimulation at 20 Hz.

57
4. Manama

Susceptibility to fatigue of innervated and denervated EAS muscles was
determined by stimulating muscle strips at supramaximal intensity, 1.5 ms duration at 20
Hz to produce tetanus. Examples of tetanic stimulation of innervated (control) and
denervated EAS muscles are shown in Figure 21. The mean (:1: SE) fatigue index was
determined for each muscle group (Table 5). The fatigue index represents the percent
decrease of peak muscle tension after 30 sec of tetanic stimulation.

The fatigue indexes ranged from 39-68% for innervated (n = 4), 3-20% for 10 day
denervated (n = 4), and were always 0% for 60 day denervated (n = 4) EAS muscles.
Compared with the innervated EAS muscles, the denervated muscles became signiﬁcantly
less susceptible to fatigue (p < 0.003 among all three groups, ANOVA). The decreases in
fatigue susceptibility were signiﬁcant between innervated and 10 day denervated (p <
0.01) and between innervated and 60 day denervated (p < 0.01) EAS muscles, but not

between the 10 day and 60 day denervated EAS muscles (Bonferroni post tests).

58

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 21. Fatigue responses of innervated and denervated cat external anal sphincter
(EAS) muscles. Panel AIS from an innervated EAS, B Is from a 10 day denervated EAS,
and C rs from a 60 day denervated EAS. Muscles were stimulated at supramaximal
intensities (10-12 V), 1. 5 ms duration at 20 Hz for more than 30 sec. Pen recorder paper
speed was two min/sec. Vertical calibration bar in panel A equals 2 g and also applies to
panels B and C. Horizontal calibration bar in panel A equals 5 sec and also applies to
panels B and C.

59

5. WWW

Muscle force per cross-sectional area measurements were made with the muscle
strip stretched to its optimal length (L,). Peak twitch amplitudes were measured ﬁ'om the
single twitches used to determine contraction and one-half relaxation times. Cross-
sectional area was determined using the wet weights of the strips as described in methods.
Table 5 shows the mean (:1: SE) force per cross-sectional area (kg/cm’) for each of the
three EAS muscle groups. Values ranged from 008-011 kg/cm2 for innervated (n = 4),
0.10-0.15 kg/cm2 for 10 day denervated (n = 4), and 0.10-0. 13 kg/cm2 for 60 day
denervated (n = 4) EAS muscles. There were no statistically signiﬁcant differences among

all three groups (AN OVA), or between pairs of groups (Bonferroni post tests).

IV. DISCUSSION

The morphological, biochemical, and contractile properties of innervated and
denervated limb and trunk muscles have been studied over the last several decades. As
early as 1915, Langley showed that the contractions of a fast-twitch muscle became
slower following denervation. Since then, muscle masses, myoﬁbrillar protein contents,
isoform distributions and more contractile data have been reported for perturbed limb and
trunk muscles. These studies Show that muscles similar in ﬁber type react to comparable
perturbations in a predictable way (Matsuda et al., 1984). The aims of the present
research were to describe the properties of sphincteric muscles and to compare changes in
the denervated cat EAS with those of a denervated muscle of similar ﬁber type

composition.

A. Morphologic properties

Denervation of a limb or trunk muscle leads to a decrease in its mass and protein
content (Babij and Booth, 1988a; Babij and Booth, 1988b; Midrio et al., 1988). Babij and
Booth (1988a) showed that muscle mass decreases 26% and protein content decreases
31% after seven days denervation of three rat hindlimb muscles. The present study
reports that the denervated cat EDL and EAS muscles decrease in wet weight and protein

60

61

content similarly after 10 days of denervation.

The loss of muscle mass in denervated limb and trunk muscles is time dependent
(VVrcks and Hood, 1991). There is a rapid reduction in muscle mass in the ﬁrst 14 days
after denervation, followed by a more gradual reduction. The present study shows a
similar pattern over 60 days of denervation. The present study also shows a decrease in
muscle ﬁber cross-sectional areas (Figure 5). The mean cross-sectional muscle ﬁber area
shows a large initial decrease (10 day denervated EAS) and then a modest reduction over
the next 20 days (30 day denervated EAS). This is another indication that muscle mass
showed an initial rapid and then a gradual reduction. It also exempliﬁes the principle that
muscle size is intimately related to its mass (Babij and Booth, 1988b).

The decreases in muscle mass and muscle ﬁber cross-sectional areas are
proportional. Muscle mass does not decrease because of degeneration and subsequent
loss of muscle ﬁbers (Allen et al., 1995; Midrio et al., 1988; Schiafﬁno and Reggiani,
1994; Talmadge et al., 1993b). Figures 3 and 4 show that the number of muscle ﬁbers
does not decrease in the denervated EAS muscles. Midrio et a]. (1988) counted the
number of muscle ﬁbers in control and denervated rat EDL muscles and found there to be
no change. Thus, the decrease in mass of atrophic muscles is because of a decrease in the
size of their ﬁbers and not a decrease in ﬁber number.

, The loss of muscle mass and cross-sectional area following denervation is time
dependent and related to the loss of muscle proteins (Babij and Booth, 1988b; Morrison et
al., 1987). The present study shows a similar time dependent decrease in muscle protein
content, with the largest decrease occurring 10 days after denervation, followed by smaller

decreases. The decreases in muscle masses, ﬁber cross-sectional areas, and muscle protein

62

contents in the denervated cat EDL and EAS muscles are characteristic of muscle atrophy
(Goldspink, 1976; Midrio et al., 1988).

The relationship between the percent decreases in protein content and wet weight
for the denervated EDL and EAS muscles are not alike. Protein content of the denervated
cat EDL muscle decreases more than its wet weight for each denervated group (Table 2).
In contrast, the denervated EAS shows a greater wet weight loss for each group. I
propose that this discrepancy is attributable to an increase in the connective tissue protein
content, including elastin and collagen, of denervated EAS muscles.

Krier er a1. (1989) showed that the cat EAS has more connective tissue than do
limb and trunk muscles, which they suggest plays a signiﬁcant role in maintaining fecal
continence by giving the muscle a greater passive tension at its resting length. I propose
that the connective tissue proteins do not degenerate as much as do the other muscle
proteins such as the myoﬁbrillar proteins. They may even increase synthesis rate. This
selective maintenance of speciﬁc muscle proteins in denervated limb and trunk muscles is
known to occur for other muscle proteins (Covault et al., 1986; Goldman et al., 1985;
Matsuda et al., 1984).

The proposed increase in the synthesis of connective tissue muscle proteins in the
denervated EAS muscle groups would account for the smaller ratios between the percent
decrease in the muscle protein content and the percent decrease in muscle wet weight in
the denervated EAS muscles compared with the ratios in the denervated EDL muscles at
the same time points (Table 2). This conclusion is supported by the fact that the
denervated EAS muscle groups show a signiﬁcant increase in passive tensions at all

muscle lengths compared with the control EAS group (Figure 19). This indicates that the

63

denervated EAS muscles selectively maintain or increase synthesis of connective tissue
proteins. The eﬁ‘ect is to provide greater passive force to impede the passage of feces and

thereby preserve some degree of fecal continence.

B. Biochemical data of striated, sphincteric muscles

1. MLC Qmmsitiens

The MLC isoform contents of the cat striated, sphincteric muscles are more similar
to the fast-twitch EDL than the slow-twitch SOL (Figure 7). The EDL and striated,
sphincteric muscles have a predominance of MLle, 2f, and 3f. These are the MLC
isoforms that compose FG muscle ﬁbers. They also have a small content of MLC 1 sa and
lsb, indicating that there are some FOG muscle ﬁbers present. The SOL is the only
muscle that displayed the MLCZS isoform. MLC2s is only present in SO muscle ﬁbers
(Figure 2). This indicates that there are no SO muscle ﬁbers present in the EDL or
striated, sphincteric muscles.

Table 3 shows the relative concentrations of MLC3f expressed by the ratio of
MLC isoforms: 3009-30. This ratio is indicative of the predominant ﬁber type content of
the muscle (W ada et al., 1993). A larger ratio is associated with a fast-twitch muscle
composed of predominantly FG ﬁbers and a smaller ratio with a fast-twitch muscle
composed of predominantly FOG ﬁbers. The EUS and DU have the largest ratio,
indicating they are the fastest muscles. This is supported by data that show the cat and
rabbit EUS are fast-twitch muscles (Krier et al., 1990; Tokunaka et al., 1986) and in the

cat, the EUS has faster mechanical properties than the EAS (Bowen et al., 1976). The

64

E80 has the smallest ratio, indicating it is the slowest of these fast-twitch muscles. This is
consistent with data that Show that the cat ESO has contraction times that are slower than

the cat EDL (Floyd and Morrison, 1975).

2. MH m i ' ns

Figure 9 shows the percent distribution of each MHC isoform for cat striated,
sphincteric muscles. The cat soleus (not shown) has 100% MHC type I (Ariano et al. ,
1973). In contrast, the EDL has only 2.9% MHC type I, but has 68.5% MHC type IIB,
the MHC isoform found in FG muscle ﬁbers. This is consistent with histochemical data
that show the cat soleus is 100% SO and the cat EDL is predominantly FG (Ariano et al.,
1973). All striated, sphincteric muscles also show a predominance of MHC type IIB,
which is consistent with histochemical and mechanical data that deﬁne the cat EAS, EUS
and ESO as fast-twitch muscles (Bowen et al., 1976; Floyd and Morrison, 1975; Krier et
al., 1988). Consistent with the relative MLC3f concentrations shown in Table 3, the EUS
has the greatest content of MHC type IIB (80.6%), the MHC isoform found in F6 ﬁbers.
The ESO, with the lowest MLC3f concentration (ratio in Table 3), has the smallest
content of MHC type HB (45.6%), indicating that it is the muscle with the least amount of
FG ﬁbers.

This study provides data for the ﬁrst biochemical classiﬁcation of the ﬁber type
distribution of the cat EAS. They Show that the innervated cat EAS is composed of 66%
FG, 23% FOG, and 11% SO ﬁbers. Fiber type distribution had been measured
histochemically before the technique of separating MHC isoforms in a SDS-
polyacrylanride gel was developed. The ﬁber type composition of the cat EAS classiﬁed

by histochemical staining is 73% FG, 23% FOG, and 4% SO (Krier et al., 1988). The

65

results of both methods are similar, as are comparisons between histochemical and
biochemical ﬁber type distribution studies of limb and trunk muscles. For example,
Talmadge et al. (1995b) deﬁned the cat plantaris as containing 56% FG, 21% FOG, and
23% SO ﬁbers using SDS-polyacrylamide gel electrophoresis. Histochernical ﬁber typing
showed it to contain 46% FG, 28% FOG, and 26% SO ﬁbers (Ariano et al., 1973).
Talmadge et al. (1995a) ascribe the differences in content values to the many variables
inherent in the histochemical methods employed.

There is a discrepancy between the MLC and MHC data for the EDL and striated,
sphincteric muscles. Present MLC data for the fast-twitch muscles (EDL, EAS, EUS,
DU, and ESO) show the absence of MLC2S (Figure 7). This MLC isoform is only present
in S0 muscle ﬁbers (Figure 2). The present MHC isoform data, however, show that these
muscles contain 80 ﬁbers (Figure 9). It is unknown why the MLC2s isoform is not
detected in these fast-twitch muscles. Rubenstein and Kelly (1978) and Tsika et al. (1987)
have also shown that the MLCZS isoform band is absent in their SDS polyacrylamide gels
with rat EDL muscle samples (which have a SO ﬁber content ranging ﬁom 2.2% (Tsika et
al., 1987) to 4.1% (Midrio et al., 1988)). Furthermore, Carraro et al. (1983) have shown
the absence of the MLCZS band in the rat EDL using the two-dimensional SDS
polyacrylamide gel electrophoretic technique.

A possible explanation for these apparent discrepancies may be because of the
abundances of the MLC subunits and their ability to be detected on a gel. The present
study shows the cat EDL contains 2.9% 80 ﬁbers and 68.5% F G ﬁbers. Tsika et a1.
(1987) showed the rat EDL contains 2.2% SO ﬁbers and 84.8% FG ﬁbers. FG ﬁbers have

three different MLC combinations (fm,_,) (Figure 2). The ﬁnl isomyosin contains two

66

MLC3f subunits and the ﬁn2 contains one. Tsika er a1. (1987), using nondissociating
conditions to electrophoretically separate the native myosin molecules, showed that the
breakdown of the PG ﬁbers of the rat EDL is 18.2% ﬁnl and 30.4% ﬁnz. Therefore, in a
sample of 100 muscle ﬁbers (400 MLC subunits), there are 67 MLC3f subunits ([18.2 x 2]
+ [30.4 x 1]) and only four MLC28 subunits (2.2 x 2). As shown in Figure 10, where the
cat EDL lane has twice as much protein loaded to intensify the MLC3f band, the MLC3f
band intensity is weak compared to the other MLC isoform bands. Although this band
represents the MLC3f of the cat EDL, that of the rat EDL is equally as weak (Rubenstein
and Kelly (1978); Tsika er al., 1987). Because there are 17 times more MLC3f than 25
subunits (67/4) in the rat EDL, the MLC2s isoform band would appear 1/17 as intense as

the MLC3f band and would not be detectable.

C. Biochemical data of denervated external anal sphincter muscles

The present data deﬁne the transition of the MHC isoform proﬁles of the cat EDL
and EAS muscles caused by denervation. Denervated muscles were compared with
control muscles using their MHC and MLC isoform distributions. MHC and MLC
isoform distributions are commonly used to detect changes in muscle ﬁber type
composition alter perturbations of limb and trunk muscles (Adams et al., 1994; Diﬁ‘ee et
al., 1991; Termin et al., 1989). Previous limb and trunk muscle perturbation studies have
shown that the MHC isoform transitions induced by equal perturbations of muscles of the
same ﬁber type composition are generically similar (Matsuda et al., 1984). Therefore,

changes in the cat EAS and EDL muscle MHC isoform distributions after denervation

67

were compared because they are both fast-twitch muscles that contain predominantly FG

ﬁbers.

1. MHC iseferm ttapeitions

Denervation of the cat EDL was performed to demonstrate the effects on the
MHC isoform distribution of a denervated limb muscle that is similar in MHC isoform
content to the cat EAS. Data for the denervated cat EDL can also be compared with
those for denervation of the rat EDL. The 35 day denervated rat EDL showed a 98%
increase in MHC type I content, a 57% increase in type IIA content and a 46% decrease in
type IIB content (Midrio et al., 1988). This is consistent with other studies that show
denervation of fast-twitch limb and trunk muscles causes a “fast-to-slow” ﬁber type
transition (Eisenberg and Hood, 1994; Tyc and Vrbova, 1995; Wicks and Hood, 1991).

The transitions among MHC isoforms following denervation are greater in the cat
EDL (present study) than they are in the rat EDL (Midrio er al., 1988). The rat EDL did
not show an increase in any MHC isoform of more than 98% after 35 days denervation.
By contrast, MHC type I in the cat EDL increased 300% and MHC type IIA increased
189% after only 10 days of denervation. The diﬁ‘erence in the magnitude of the MHC
isoform transitions exhibited by the cat and rat EDL muscles may be a species-speciﬁc
phenomenon or may be a ﬁmction of the methods of denervation.

The rat EDL was denervated by cutting the sciatic nerve, whereas in the cat, only
the nerve branches to the EDL were sectioned. By sectioning the sciatic nerve, all
muscles in that leg are denervated and it cannot bear weight. In experiments of rat hind-

limb muscle “unweighting” by tail suspension, the ability to express slower myosins (MHC

68

types I and 11A) is repressed (Tsika et al., 1987). In fact, the fast-twitch rat medial
gastrocnernius muscle assumed faster contractile properties after four weeks of tail
suspension (Winiarski et al. , 1987). The “unweighting” produced by sciatic nerve section
may contribute to the fact that the rat EDL did not Show as large a transition toward the
slower isoforms as did the cat EDL.

Like the denervated rat and cat EDL muscles, denervation of the cat EAS
produced a transition toward a slower MHC isoform type distribution. In contrast to the
denervated rat and cat EDL muscles, however, the denervated cat EAS did not increase
MHC type I isoform content. Why MHC type I content increases in one denervated fast-
twitch muscle and not in another is unknown.

Developmental, neuronal, mechanical and hormonal factors all determine a muscle
ﬁber’s MHC isoform composition (Crow and Stockdale, 1986; Gunning and Hardeman,
1991; Schiafﬁno and Reggiani, 1994). The absence of an increase in the MHC type I
isoform content after denervation of the EAS may relate to two of these factors:
developmental and mechanical. The embryological origin and anatomy of the EAS differ
from those of limb and trunk muscles and may therefore be responsible for its unique
MHC isoform transition.

The embryologic origin of the EAS is controversial (Levi et al., 1991). It
originates either from the splanchnic mesoderrn (Hamilton and Mossman, 1972) or ﬁom
the cloacal sphincter (Levi et al. , 1991). The cloacal sphincter is a derivative of the
endodermal intestine and the ectodermal proctodeum (Torrey and Feduccia, 1979).
Unlike limb and trunk muscles that develop ﬁom somatic and somitic mesoderrnal tissue,

respectively (Oppenheimer and Lefevre, 1989), the EAS derives either ﬁom splanchnic

69

mesoderrn or from fusion of endodennal and ectoderrnal tissues. Because myoblasts
originating from diﬁ'erent embryologic tissues may diﬁ‘er in transcriptional control of their
MHC genes, the discrepancy in the SO ﬁber content transitions induced by denervation of
the EDL and EAS muscles in this study may reﬂect the fundamental difference between
the embryologic origins of each muscle’s myoblasts (Gunning and Hardeman, 1991).

The unique anatomy of the cat EAS may be another factor that is responsible for
the unconventional MIIC isoform transition. The EAS has a greater amount of connective
tissue in parallel with its muscle ﬁbers than do limb and trunk muscles (Krier et al., 1989).
This large amount of connective tissue increases passive “stiffness” of the EAS as shown
by its passive length-tension curve (Krier et al., 1989). At optimal length (L,), the passive
tension of the EAS is much greater than zero, whereas for limb and trunk muscles, it is
nearly equal to zero (Krier et al., 1989). Because the EAS has no bony insertions as do
limb and trunk muscles, it maintains a higher passive tension (“stiffness”) to prevent over-
stretching when a fecal bolus passes through its lumen during defecation. When the EAS
is denervated, the passive tensions at all muscle lengths show a signiﬁcant increase (Figure
19). The difference in passive tensions between the EAS and limb and trunk muscles may
contribute to the variation in MHC isoform transitions exhibited by the muscles when
denervated because the mechanical environment of muscle ﬁbers inﬂuences their MHC
isoform content (Crow and Stockdale, 1986; Gunning and Hardeman, 1991; Schiafﬁno

and Reggiani, 1994).

2. MLC i§oferm transitiens

Figure 11 compares MLC isoform contents of control and denervated EAS muscle

70

groups. There are no differences in the MLC isoform distributions among the four
groups. Failure to show changes in the MLC isoform proﬁle may be related to the allotted
time period after denervation. For example, denervation of a fast-twitch vertebrate
skeletal muscle leads to a decrease in the MLC317MLC1f ratio, but this isoform proﬁle
change is not detectable until several months post-denervation (Carraro, et al., 1979;
Matsuda et al., 1984).

The lag for changes in MLC isoform content to develop also occurs in other
perturbed limb and trunk muscles. Pette et al. (1976) have shown that there is no change
in the MLC isoform proﬁle in the chronically stimulated fast-twitch rat EDL muscle until
60 days after denervation, which they attributed to the slow turnover rates of MLC
subunits. Carraro et al. (1979) proposed that the post-surgical delay in detecting a change
in the content of MLC isoforms is because of their long half-lives. Swynghedauw (1986)
concludes that whereas the half-life of myosin is only one week, detection of MLC isoform
proﬁle changes requires perturbations that persist three to four times the half-life of
myosin.

A factor that determines the turnover rates for proteins is the rate at which they are
degraded (Wicks and Hood, 1991). Muscles contain serine protease and cathepsins that
ﬁmction to degrade myoﬁbrillar proteins (Obinata et al. , 1981). The cathepsins split the
actomyosin complexes and the serine protease then digests the MHC subunits. The MLC
subunits are spared. There are no muscle speciﬁc proteases that speciﬁcally degrade MLC
subunits (Obinata et al. , 1981). This sparing slows their turnover rates, thereby
prolonging their half-lives. This may be why there is no change in MLC isoform contents

among control and experimental EAS muscle groups in the present study.

71

In contrast, changes in MHC isoform composition have been detected eight days
after a perturbation. Termin et al. (1989) showed that chronic, low-frequency electrical
stimulation of rat fast-twitch tibialis anterior muscles resulted in decreased MHC type IIB
and increased MHC type IIA isoform contents only eight days after stimulation began.
Likewise, the denervated cat EDL and EAS muscles in the present study had MHC
isoform changes after only 10 days of denervation (Figures 13 and 15).

It is surprising that MHC isoform contents change before the contents of the MLC
isoforms do because there is a preferential association between MHC and MLC isoforms
(Figure 2) (Bottinelli et al., 1994; Tsika et al., 1987). In the present study, with the
signiﬁcant increases in MHC type HA and decreases in MHC type IIB in the denervated
EAS muscle groups, it would be expected that there would be decreases in the MLC3f
contents and increases in the MLC 1 sa and lsb contents. However, there are no signiﬁcant
differences in the MLC isoform contents (Figure 11). This inconsistency is present in
other muscle perturbation studies (Carraro et al., 1979; Matsuda et al., 1984; Pette et a1. ,
1976). The half-lives of MLC subunits being longer than those of MHC subunits may
explain why there are no changes detected in MLC isoform contents.

It does not explain why the MHC isoform composition changes without a
concomitant change in the MLC isoform content, as would be expected from Figure 2. It
may be that, because the MLC subunits are not degraded as fast as the MHC subunits are,
the MLC3f isoform remains in the myoﬁber longer. Therefore, the MLC3f isoform would
still appear in the SDS polyacrylamide gels. These excess MLC3f subunits would prevent
a detection of a decrease in MLC3f content and would also limit the detection of an

increase in the MLCls isoform contents. The fact that the MLCls isoform exists as two

72

distinct isoforms (1 sa and lsb) could also limit the detection of an increase in its content
because the increase could be spread between the two isoforms.

The unassociated, obsolete MLC subunits may remain in the myoﬁber longer than
the obsolete MHC subunit because the conversion of ﬁber types occurs by a change in the
transcription of MHC and MLC isoform genes and not by degeneration of selected ﬁbers
(Allen et al., 1995; Schiaﬁino and Reggiani, 1994; Talmadge et al., 1993b). The shift in
MHC isoform transcription occurs in the following sequence of types: IHIIAHIIB (Betto
et al., 1986; Schiamno and Reggiani, 1994). This transition commonly occurs when
myoﬁbers undergo transformations as they do when they are denervated (Talmadge et al. ,
1993b). Thus, a myoﬁber undergoing a transformation from PC to FOG will co-express
MHC types IIB and HA However, that myoﬁber contains a serine protease that will
degrade the MHC subunits, thereby causing it to contain eventually only the recently
transcribed MHC type IIA isoform subunit. Since the MLC isoforms have longer half-

lives, they will be detected in the myoﬁber longer than the obsolete MHC isoforms.
D. Contractile data

EAS muscle strips were stimulated with square wave pulses at 1.5 ms duration.
This duration has been shown to produce a transmural electrical activation of the muscle
ﬁber whose response is not decreased by the addition of a neuromuscular blocking agent
(Parlani et al., 1992). This is consistent with present data that show no decrease in the
amplitude of twitch contractile responses after d-tubocurarine (200 pM) was added to the

organ bath (Figure 16). This shows that at 1.5 ms duration pulses, the twitch contractile

73

responses represent direct activation of the muscle membrane and are not due to activation

of cholinergic motor axons within the muscle.

1. Twiteh contregile date

Single twitches of the EAS muscle strips were used to calculate contraction and
one-half relaxation (Tm) times. The innervated (control) EAS muscle strips had
contraction times and T,,2 times characteristic of fast-twitch muscles composed of
predominantly FG ﬁbers (Burke et al., 1971; Burke and Tsairis, 1974). The mean
contraction and Tl,2 times for the innervated EAS in the present study were 36.5 and 25.3
ms, respectively. These are consistent with those shown by Krier et al. (1989) (34 and 22
ms contraction and T1,2 times, respectively).

The denervated EAS muscle groups had prolonged contraction and T1,2 times
(Table 5). This is consistent with past studies of other mammalian denervated limb and
trunk muscles (Finol et al., 1981; Gauthier et al., 1982; Gutmann et al., 1972; Langley,
1915; Lewis, 1972; Tyc and Vrbova, 1995; Wicks and Hood, 1991). The prolongation in
contraction times is compatible with the MHC isoform transitions that occurred in the
denervated EAS muscles. The contraction time of a ﬁber is controlled principally by its
MHC isoform content (I-Ioh, 1992). A switch in isoform composition ﬁom MHC type IIB
to MHC type IIA has been shown to increase muscle ﬁber contraction times (Bottinelli et
al., 1991; Reiser et al., 1985; Sweeney et al., 1986). This is because MHC type IIB has a
greater myosin ATPase activity than MHC type IIA (I-Ioh and Hughes, 1988; Reiser et al.,
1988; Schiaﬁino and Reggiani, 1994). The myosin ATPase activity of a muscle ﬁber

correlates with the contraction time, so that the greater the activity, the shorter the

74

contraction time (Swynghedauw, 1986; Thomason et al., 1986). Because the
predominant ﬁber type of the denervated muscles becomes FOG (ﬁbers with MHC type
IIA), there is less myosin ATPase activity and thus a longer contraction time.

The prolonged T1,2 times of the denervated EAS muscle groups are also
attributable to differential protein expression of different muscle ﬁber types, even though it
is not a myoﬁbrillar protein. The calcium binding protein, parvalbumin, shows a ﬁber type
speciﬁc distribution. The relative amounts of parvalbumin among ﬁber types is: FG >
FOG > SO (Schmitt and Pette, 1991). Parvalburnin is involved in the relaxation rate of
the muscle ﬁber because it binds the free cytoplasmic calcium, making it unavailable for
further tension generation (Talmadge et a1. , 1993). The greater the amount of
parvalbumin in the muscle ﬁber, the faster the calcium is sequestered and the shorter the
T1,2 time. In the denervated EAS muscles, the predominant ﬁber type becomes FOG, so
that there is less parvalbumin than was previously available in the PG ﬁber. The increased
T1,2 times of the denervated EAS muscles is consistent with the decreased parvalbumin
content of FOG ﬁbers (Schnritt and Pette, 1991; Talmadge et al., 1993).

The contraction and T1,2 times of the denervated EAS muscle groups were greater
than those of the innervated groups, but there was no difference between the 10 day and
60 day denervated groups for either time. By 10 days denervation, the EAS muscles had
already switched ﬁ'om a predominance of PG ﬁbers to a predominance of FOG ﬁbers
(Figure 15). The MHC type IIA content had increased 35% within 10 days after
denervation and had become the predominant MHC isoform. This large increase in MHC
type IIA content and the switch in ﬁber type predominance accounts for the signiﬁcant
increase in contraction times between the innervated and 10 day denervated groups.

Although there was a signiﬁcant increase in MHC type HA content between the 10

75

day and 60 day denervated groups (12%), the muscles were still predominantly FOG. The
S0 ﬁber contents were not diﬁ'erent between the two groups. There would have to be an
increase in the 80 ﬁber content or a greater increase than 12% in the MHC type IIA
content for there to be an increase in contraction times between the 10 day and 60 day
denervated groups.

The T1,2 times did not increase between the 10 day and 60 day denervated groups
also because there was only a 12% increase in MHC type IIA content and no increase in
S0 ﬁber content. The number of ﬁbers with a lower parvalbumin concentration would
have to increase greater than 12% or there would have to be an increase in the content of
SO ﬁbers (ﬁbers with the lowest parvalbumin concentration) for there to be a detectable
increase in Tl,2 times between the two groups.

A 12% increase in the MHC type IIA content could lead to a 12% increase in the
contraction and T1,2 times. Ifso, a 12% increase in a 50.5 ms contraction time or a 46.5
ms T1,2 time would be only about 6 ms. The standard error of these measurements was A:
3 ms. To show a 12% increase in times, there would be an increase in the pen trace twitch
time span of only 0.5 mm, a span too small to be consistently revealed. The increases in
times between the innervated and 10 day denervated groups, however, were 38 and 84%
for contraction and T1,2 times, respectively. These resulted in increases in pen trace twitch
time spans that were large enough to be consistently detected (2.8 and 4.2 mm for

contraction and Tl,2 times, respectively).

2.311%

The denervated EAS muscle groups showed decreases in their fatigue indexes,

76

demonstrating that the muscles became more resistant to fatigue (Table 5). The
denervated fast-twitch rat EDL also shows a decrease in its fatigue index (T yc and
Vrbova, 1995). The increased fatigue resistance is primarily due to the inherent oxidative
capacities exhibited by ﬁbers with different MHC isoform compositions.

Oxidative capacities are highest in ﬁbers with MHC type 1, intermediate in type
IIA ﬁbers and lowest in type IIB ﬁbers (Burke and Tsairis, 1974; Hamalainen and Pette,
1993). The increased MHC type IIA contents in the denervated EAS muscle groups
thereby give the muscles a higher oxidative capacity. The higher oxidative capacity allows
the muscle to withstand tetanic stimulation for longer periods (Baldwin et al., 1973). The
increased fatigue resistance is also a result, in part, of the MHC type IIA subunit exhibiting
a slower cross-bridge cycling than the MHC type IIB subunit (Goldspink et al., 1970).
The decreases in fatigue indexes of the denervated muscle groups are consistent with the
higher oxidative capacity of MHC type IIA ﬁbers, the predominant ﬁber type of the
denervated EAS muscles, compared with the capacity of type IIB ﬁbers, the predominant

ﬁber type of the innervated EAS muscles.

1W

The innervated EAS muscle twitch force per cross-sectional area in the present
study was 0.10 :i: 0.01 kg/cm’. This is consistent with the value of 0.25 i 0.20 kg/cm2
determined for the cat EAS by Krier et al. (1989). The muscle force per cross-sectional
area for the denervated EAS muscle groups was the same as for controls (Table 5). This
is consistent with data shown by Finol et al. (1981), in which the denervated rat EDL

showed a small, transient increase in force per cross—sectional area at 10 days denervation,

77

but returned to control levels and showed no signiﬁcant diﬁ‘erence ﬁom controls at 40
days. The present data showed a tendency for a transient increase at 10 days, although it
was not signiﬁcant. The absence of change in force per cross-sectional area with
denervation indicates that, although the whole muscle size decreased, the force produced

by control and denervated muscle strips of equal cross-sectional areas remained constant.

4. Length-teneiep reletienship

The length-tension relationship of the innervated control EAS muscle group
(Figure 18A) is similar to that obtained by Krier et al. (1989). They showed that the
passive tension curve is steeper in the EAS than it is in limb and truck muscles. Passive
tension at L, for the EAS is much greater than zero, whereas that of limb and trunk
muscles is nearly equal to zero (Podolsky and Schoenberg, 1983). The present study also
shows the cat EAS to have a passive tension at L, much greater than zero.

The total tension curves of limb and trunk muscles have a non-linear characteristic
beginning immediately after their peak in active tension (Podolsky and Schoenberg, 1983).
It occurs because the passive tension at L, for limb and trunk muscles is nearly equal to
zero, thus the subsequent decrease in active tension is not offset by the effects of passive
tension. The total tension curves of the EAS muscles ﬁom the present study and ﬁom
Krier et al. (1989) do not have this characteristic because the passive tension at L, in the
EAS muscle is much greater than zero, thereby offsetting the decrease in active tension.

The absence of non-linearity is also characteristic of the total tension curves of
cardiac and smooth muscles (Ekstrom and Uvelius, 1981; Grimm and Whitehorn, 1966),

both of which have passive tension curves that are steeper than those of limb and trunk

78
muscles (Ekstrom and Uvelius, 1981; Grimm and Whitehorrr, 1966). The passive tension
curve for the EAS is similar to those for smooth and cardiac muscles (Krier et al. , 1989),
both of which also surround hollow cavities and control ﬂow of matter through them.

The length of the muscle at which passive tension is ﬁrst observed (L,) is another
characteristic of cardiac and smooth muscles that is similar to the EAS. The ratio of L,./L,
is a constant for diﬁerent muscles. It is 0.67 for smooth muscle, 0.70 for cardiac muscle,
and approximately 1.00 for limb and trunk muscles (Podolsky and Schoenberg, 1983).
EAS muscles in the present study began to show passive tension at a length 65% of L,
(L/L, = 0.65). The L, of the EAS is therefore similar to those of cardiac and smooth
muscles.

Denervated EAS muscle groups in the present study had about a ﬁve fold increase
in passive tensions at L, compared with controls (Figure 19). Passive tension originates
from both the sarcolemmal membranes and from the connective tissue components of the
muscle which are arranged in parallel to the muscle ﬁbers and are thereby called the
“parallel elastic components” (PEC) (Arnold et al., 1987). These include collagen, elastin,
proteoglycans and other glucoproteins (Glavind et a1. , 1993). The differences in passive
tensions of smooth muscles from the lower esophagus, rectum, internal anal sphincter, and
uterine cervix have been correlated with their collagen contents and extent of collagen
cross-linking by hydroxyproline (Glavind et al. , 1993); the greater the contents of collagen
and hydroxyproline, the greater the passive tension. Glavind et al. (1993) also showed
smooth muscle tissues with lower collagen and hydroxyproline contents to be more
compliant.

I propose that the higher passive tensions at L, for the denervated EAS muscle

groups are a result of an increased content of PEC proteins, compared with that of the

79

control EAS muscles. It also originates from the increased ratio of sarcolemmal
membrane surface area to cross-sectional area of the denervated muscle ﬁbers (Arnold et
al., 1987). The greater sarcolemmal membrane to cross-sectional area ratio and increased
PEC content make the denervated EAS muscles less compliant and therefore more resis-
tant to passage of fecal material in the absence of active contractions. This resistance
must be able to counter the forces of pressure exerted by smooth muscle contractions of
the distal rectum and anal canal. Were there no such increases in passive tensions of
denervated EAS muscles, then even small increases in pressure would cause fecal leakage.

Because the EAS muscle has no bony attachments to protect it from being
overstretched, it relies on its relatively high passive tension to do so (Krier et al., 1989).
When a fecal bolus passes through the lumen of the sphincter, active contractions also
limit the amount of stretch endured. Because denervated EAS muscles cannot actively
contract, their increases in passive tensions serve to limit stretch to a greater degree by
making the muscle even less compliant.

Because the human rectum is a reservoir for fecal material, it is usually stretched
(Krier, 1989). This activates the smooth muscle cells of the rectum to contract,
generating pressure to push the fecal material anally. Because the denervated EAS cannot
generate active tension to counter the increase in pressure, its increased passive tension is
all that remains to maintain fecal continence. Fecal leakage will occur when the pressure
generated by the rectal smooth muscle contraction is greater than the passive tension of
the denervated EAS. Thus, the increase in passive tensions shown by denervated EAS
muscles functions to maintain some degree of fecal continence, the degree being
proportional to the difference in passive tensions between the innervated and denervated

EAS muscles at each muscle length.

V. SUMMARY AND CONCLUSIONS

The present study characterizes the biochemical proﬁle of the cat EAS muscle and
presents a model to investigate the changes in its morphological, biochemical, and
contractile properties after chronic denervation. These properties are compared with

those of the denervated cat EDL muscle. It is concluded from these studies that:

l. Denervation of cat EAS muscles results in decreased muscle wet weight and ﬁber
cross-sectional area as a result of decreased protein content. Denervated cat EDL

muscles also have decreased wet weight and protein content.

2. Denervated cat EDL muscles have a greater decline in protein content than wet
weight, whereas denervated cat EAS muscles Show a greater decline in wet weight

than protein content.

3. The MLC and MHC proﬁles of innervated cat EAS and other striated, sphincteric
muscles are representative of fast-twitch muscles containing predominantly FG

ﬁbers.

4. Denervation of cat EAS muscles results in a switch in the predominance of PG

80

81
ﬁbers to FOG ﬁbers, just as occurs in denervated cat EDL muscles. Denervated
EDL muscles, however, have a progressive increase in MHC type I isoform

content, whereas denervated EAS muscles do not.

5. Denervated cat EAS muscles have increased twitch contraction and one-half

relaxation times, as well as an increased resistance to fatigue.

6. The passive length-tension curves of denervated cat EAS muscles become steeper,

with increased passive tension at all muscle lengths greater than L,.

The present study establishes that the cat EAS, and other striated, sphincteric
muscles, are composed of predominantly FG muscle ﬁbers. Fast contracting muscle ﬁbers
are important for continence because they rapidly close the anal canal.

Denervation of the cat EAS muscle leads to atrophic changes similar to those that
occur in the denervated EDL. The smaller ratios between the percent decrease in the
muscle protein content and the percent decrease in muscle wet weight for the denervated
EAS muscle groups, however, indicate a selective maintenance of speciﬁc muscle proteins,
presumably the PEC proteins. This presumption is supported further by the increases in
passive tensions exhibited by the denervated EAS muscles.

The PEC proteins are partially responsible for passive tension, and a presumed
increase in their content would increase the passive tension of the denervated EAS
muscles. This would increase resistance to the passage of fecal material, thereby
providing some degree of continence. Because the lack of active tension generation may

allow a fecal bolus to stretch the denervated EAS beyond its physiological limits, the

82
increased passive tension makes the muscle less compliant, thereby protecting it from
being over stretched.

Denervation of the cat EAS muscles increased the incidence of ﬁnding feces
outside the litter box. It was difﬁcult to distinguish if the feces were there because they
were knocked out of the box by a restless cat or because the cat could not make it to the
litter box in time. The cages of cats with denervated EAS muscles, however, did have
feces outside the litter box more often, suggesting that the denervation caused a degree of
incontinence. The majority of the time, however, cats with denervated EAS muscles were
able to make it to their litter boxes, presumably because the increased passive tensions
exhibited by these muscles gave them the time to do so.

Denervation of the cat EAS muscle results in transformation of muscle ﬁber types
ﬁom predominantly FG to predominantly FOG, similar to what occurs in the denervated
fast-twitch EDL. Denervated EAS muscles, however, do not increase SO ﬁber content,
such as occurs in the denervated EDL. This diﬁ‘erence may be because of differences in
their embryological origins and mechanical environments.

As expected with a switch in ﬁber type composition ﬁom PO to FOG, the
denervated EAS muscle groups have increases in their contraction and one-half relaxation
times, as well as an increase in their resistances to fatigue. The increased contraction
times are a result of the decreased myosin ATPase activity of MHC type IIA subunits
compared with that of the MHC type IIB subunits. The increased one-half relaxation
times are consistent with decreased parvalbumin concentrations in FOG ﬁbers compared
with FG ﬁbers. Increased resistance to fatigue shown by the denervated EAS muscles
occurs because FOG ﬁbers have a greater oxidative capacity than do FG ﬁbers, thereby

increasing muscle endurance.

APPENDIX

APPENDIX

Facsimile permission for use of ﬁgure
Page 1 of 2

15 1° 9.9 7.1-: :50: FL! :17 133 1131 sous In. parsrotocr 2201

FAX COVER SHEET

UNIVERSITY OF ILLINOIS
DEPARTMENT OF MOLECULAR ti INTEGRATIVE PHYSIOL 061’
524 BURRILL ML / 407 S. GOODMNAVE.
URBANA. ILLINOIS 61801 / USA.

c
FAX: (217)3334133 DAIZ: 5 /7/ “’
VOICE: (217)333-1733 TIME: 3T4

TO.- K tclxauL last: :7.

 

“tum 1(5'7) 35"- 5.”?

more lied-ark ‘73- I“-

REGARDING:

 

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83

84

 

 

APPENDIX
Facsimile permission for use of ﬁgure
Page 2 of 2
rmmzm It's 13:09 Fax 217 :13; 1133 mawrgr. Parsrorocr Persimmottsriiriiiu
pm, May 6, 1996 11...:4345 pm EST Iﬁgme-
To= Dr. Richard Tsika FNH'Richard Kustasz
: - _ _ F ' -

53“; 217 333 1133 v3.4.5” 355-5125

 

Dear Dr. Tsika:

I am a graduate student in the Department of Physiology at

Michigan State University in the late Dr. Jacob Krier's lab. i am

writing you to ask for your permission to use a ﬁgure from one

of your published papers. it is a schematic representation of
m: skeletal muscle isomyosins that I am interested in using for my
Doctoral Dissertation entitled "The Morphological, Biochemical and Physio-
logical Properties of Innervated and Denervated Cat Striated External Anal
Sphincter Muscle”. it is Figure 6 in your paper entitlled "Subunit composition
of rodent isomyosins and their distribution in hindlimb skeletal muscles” that
appears in the Journal of Applied Physiology, 63(5): 2101-2110. 1987. I will
clearly and properly acknowledge your figure and cite its origination.

Could you please fax this back to me with your signature on the desired line?

Yes. Richard Kustasz may use the figure cited abovesz

No, Richard Kustasz may not use the figure:

 

Thank you very much for your time.

Ricriard Kustasz FAX: 1-517-355-5125

BIBLIOGRAPHY

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Can‘aro, U., C. Catani, and D. Biral. Selective maintenance of neurotrophically regulated
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