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This is to certify that the

thesis entitled

The Effects of a Program of Vertical
Jumping on the Myosin ATPase Activities
of the Rat Soleus

presented'by

Michael Robert Ball

has been accepted towards fulfillment
of the requirements for

M. A. degree in The School of Health
Education, Counseling Psychology and
Human Performance

QIJM.HD

Major professor

Date November 14, 1986

O"! 639 MS U is an Affirmative Action/Equal Opportunity Institution

 

MSU

 

 

 

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THE EFFECTS OF A PROGRAM OF VERTICAL
JUMPING ON THE MYOSIN ATPASE ACTIVITIES

OF THE RAT SOLEUS

By

Michael Robert Ball

A THESIS

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

MASTER OF ARTS

Department of Health, Education
and Counseling Psychology

1986

ABSTRACT

THE EFFECTS OF HIGH POWER OUTPUT JUMPING
ON THE MYOSIN ATPASE ACTIVITIES OF THE RAT SOLEUS
By

Michael Robert Ball

The purpose of this investigation was to determine the
effects of a high power output jumping program on the myosin
ATPase activities of the surgically overloaded rat soleus.

Twenty animals were brought into the laboratory and
randomly assigned to three treatment groups: a sedentary-
control group; a sedentary-surgery group; and an exercise-
surgery group. Treatments were administered to the
exercise-surgery group five days per week under controlled
environmental conditions. All animals were provided with
food and water ad libitum.

All animals were sacrificed four months after they were
received by the laboratory. Body weight and soleus weight
were obtained for each animal at the beginning and end of
the four-month period. Due to unexpected complications the

final sample size consisted of seventeen animals}

ACKNOWLEDGMENTS

This study is dedicated, with special appreciation,
to my major advisor Dr. Kwok-Wai Ho. I would also like to
thank Dr. William Heusner and Dr. Wayne VanHuss for their

thoughtful assistance throughout my graduate career.

ii

TABLE OF CONTENTS

Chapter
I O mE PROBLEM O O O O O O I O O O 0 O O O 0 O 0

Statement of the Problem
Importance of the Problem
Research Plan . . . . . .
Limitations of the Study

II. REVIEW OF RELATED LITERATURE . . . . . . . . .
III. RESEARCH METHODS . . . . . . . . . . . . . . .

Treatment Groups
SCG . . . .
SSG . . . .
ESG . . . . .
Treatment Procedures
Surgical Procedures .
Sacrifice Procedures .
Histochemical Procedures
Methods of Tissue Analysis
Statistical Procedures . .

O O O O O O O O O O
o O o O o o 0 o o o
o o o o o 0 O 0 O
O 0 O O O O O O O 0
O O O O O O 0 0 0 O
o o O O o 0 o o o
o o o o o o o 0 0

IV. RESULTS AND DISCUSSION . . . . . . . . . . . .

Histochemical Results . . . . . . . . . .
DiSCUSSion O O O O O O O O O O O O O O O

V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS . . .

suma ry O O O I O O O O O O O O O O O O O

cone lus i ons O O O 0 O O O O O 0 I O O O 0

Recommendations . . . . . . . . . . . . .

L I ST OF REFERENCES 0 I O O O O O O O O O O O O O O O 0
APPENDIX

A-I. Body Weight and Soleus Weight Before and After
Four Month Treatment Period . . . . . . . . .

A-Il. Three Month Jumping Routine for Male Albino
Rats O O O O O O O O O O O O O O O O O O O O 0

iii

Page

hector-I

17

17
17
18
18
18
19
20
20
21
21

22

22
23

28
28
29
29

30

37

38

Table

II.

III.

LIST OF TABLES

Page
Muscle Fiber Composition Before and After
Four-Month Treatment Period . . . . . . . . . . 26
Summary of the Results of T-Test on the
Percentage of Fiber Types in Rat Soleus Before
and After the Four-Month Treatment Period . . . 27
Summary of the Results of Analysis of Variance
on the Changes in the Mean Percentages of Fiber
Types in the Rat Soleus Before and After Four-
Month Treatment Period . . . . . . . . . . . . 27
Body Weight and Soleus Weight Before and After
Four'Month Treatment PeriOd o o o o c c o c o o 37
Three-Month Jumping Routine for Male Albino
Rats O O O O O O O O O O I O O O O O O 0 O O O 38

iv

CHAPTER I

THE PROBLEM

A large number of studies have been conducted in recent
years to determine the mutability of skeletal muscle fiber
types. The usual procedure is to place the muscle under
various experimental conditions and subsequently to deter-
mine if any changes in energy metabolism and/or contractile
characteristics can be induced. These experimental pro-
cedures have included denervation (6, 26). cross-innervation
(8, 17), electrical stimulation (44, 61, 67). surgical over-
load (5, 37, 66) and various exercise routines (1, 28, 32”
47). As could be expected. the results of many of these
studies have been contradictory. Therefore, continued re-
search is needed to further understand the adaptability of
skeletal muscle to various stimuli, including selected

regimens of well-defined physical activity.

Statement 2f the Problem

 

 

The purpose of this study was to determine the effects
of a three-month program of vertical jumping on myosin
ATPase in the soleus muscle of the male albino rat.

Vertical jumping is an activity which requires a high power
output. The soleus is a predominantly slow-twitch muscle in

the rat.

Importance gt the Problem

 

 

At least two major types of fibers, "fast-twitch" and
"slow-twitch", exist in the skeletal muscles of all classes
of vertebrates. The so-called "fast-twitch" muscle fibers
have a faster speed of contraction than do the "slow-twitch"
fibers. From a biochemical point of view, the contractile
speed of a muscle is determined by the ability of myosin
ATPase to catalyze the breakdown of ATP which allows the
cross-bridges formed between actin and myosin to shorten the
sarcomere (7, 11). The intrinsic contractile speed and
other metabolic factors, such as the glycolytic and/or
oxidative capacities of the various fiber types, determine
which fibers are selectively recruited under various exer-
cise conditions. In general, fast-twitch fibers are used
primarily when high degrees of muscle tension are generated;
slow-twitch fibers are recruited preferentially during lower
intensity muscle contractions.

Accumulating evidence has shown that low-intensity,
chronic types of stimulation, including endurance exercise,
can change the properties of myosin ATPase within fast-
twitch muscle fibers (35, 53, 66). This results in an
increase in the percentage of slow-twitch fibers within the
muscle. However, changes in fiber composition from slow-
twitch to fast-twitch, when either exercise or other experi-
mental conditions have been applied, have not been as

successful (15, 67, 69, 73).

3
Based upon the foregoing information, the present study
was conducted to determine if the myosin ATPase activities
in a slow muscle of adult rats could be changed by a program

of vertical jumping.

Research Plan

 

The rats used in this study were divided into three
groups. In the sedentary-control group surgery was
performed to remove the soleus muscle from either the right
or the left leg of each animal. No surgery was performed on
the contralateral leg. Consequently, any change in fiber
profile between the initially removed soleus and the soleus
in the other leg represents the effect of aging. In the
sedentary-surgery group the achilles tendon attachments of
the gastrocnemius and plantaris muscles were cut on either
the right or the left leg. In the opposite leg, the
achilles tendon attachment of the gastrocnemius again was
cut and the soleus was removed. Any change in fiber compo-
sition between the initially removed soleus and the soleus
in the other leg would represent the combined effects of
aging and surgical procedures. The animals in the exercise-
surgery group underwent the same surgery as the animals in
the sedentary-surgery group. In addition, each animal was
subjected to a progressive program of vertical jumping. Any
change in fiber composition in the group would represent the
combined effects of aging, surgery, and exercise.

Jumping was chosen as the form of exercise to be used

in this investigation because the resistance to movement

4

occurs primarily at the beginning of the jump and is not
continuous as is the case in activities such as running or
weightlifting. The nature of jumping, therefore, requires a
high velocity of muscular contraction which is conducive to
the selective recruitment of fast-twitch motor units.

Following the initial and final muscle removal, serial
cross sections were cut from the soleus and stained for
myosin ATPase (ph 4.3) and NADH. The stained sections then
were analyzed to determine the percentages of slow-and fast-

twitch muscle fibers.

Limitations of the Study

 

 

1. The results of this study can be applied only to adult
male rats and to the unique conditions to which they
were subjected.

2. The duration of the treatment period and the training
program were selected arbitrarily and, therefore, were
not necessarily optimal for obtaining significant
results.

3. A more complete understanding of the training effects
would have been obtained if contractile and metabolic
properties of the muscle had been studied in addition to
the histochemical method used.

4. The attempt to isolate the soleus by means of surgery
was unsuccessful. Consequently the training effects on
this muscle were greatly reduced.

5. A necessary and sufficient sample size was not obtained

and this limited the power of the statistical analysis.

CHAPTER II

REVIEW OF RELATED LITERATURE

Electrical stimulation of skeletal muscle has been a
successful means of producing fiber-type conversions. Both
external and implantable electrodes have been used, with the
former being more popular because of convenience. Many
studies have demonstrated an increase in contraction time as
well as decreased ATPase activity in fast-twitch fibers when
they are stimulated at a frequency characteristic of slow-
twitch fibers (44, 61, 62, 68). In a classic experiment,
Salmons and Vrbova (67) observed that chronic, low-frequency
stimulation of the lateral peroneal nerve causes a slowing
of contraction and half-relaxation speed in the fast-twitch
tibialis anterior and extensor digitorum longus muscles of
the rabbit. The nerve was stimulated at a frequency of 10
Hz, which is thought to be the typical impulse pattern of
slow motorneurons (22), for 24 hours a day during the entire
eight-week experimental period.

There is evidence to indicate that alterations in
contractile properties occur in several stages and are due
to entirely different circumstances. The increase in time-
to-peak tension and half-relaxation time during the first

few weeks of low-frequency stimulation possibly is due to

6
alterations in the sarcoplasmic reticulum affecting calcium
release (38, 39, 55). The delayed changes are believed to
be the result of changes in myosin composition. Additional
evidence for a change in myosin isoform can be seen in the
transcription process following chronic stimulation. Co-
expression of fast- and slow-type myosin light chains has
been reported following long-term, low-frequency stimulation
of fast muscles. This change in myosin light chain compo-
sition is believed to be due to the presence of mRNA
normally found only in slow-twitch muscle (63).

In studies where predominantly slow-twitch muscle is
stimulated by frequencies found in fast motorneurons the
results are not so obvious. Jones (50) observed a short-
ening of contraction time in the soleus muscle of rats less
than two-weeks-old when this muscle was stimulated at
frequencies of 25 or 40 Hz. The increased speed of con-
traction was not compared with ATPase activity because the
stained fibers are not easily differentiated at this young
stage.

Lomo and Westgaard (53) used histochemistry and
isometric twitch contraction time to show that fiber types
are interchangeable if a slow muscle is stimulated
"phasically" and fast muscle is stimulated "tonically".
They divided rats into two groups both of which had their
sciatic nerves cut. In the first group, the soleus muscle
was stimulated tetanically at 100 Hz for 0.5 seconds every

25 seconds. In the second group, the denervated soleus

7

received stimuli of 10 Hz lasting 10 seconds and repeated
every 50 seconds. After five weeks the animals stimulated
at 100 Hz had a mean contraction time of one-half that of a
normal (innervated) soleus while those stimulated at 10 Hz
had mean contraction times almost identical to a normal
soleus. The results were supported histochemically by an
increase in ATPase staining intensity among the fibers
(preincubated at ph 10.4) in the soleus muscle stimulated at
100 Hz.

Other studies seem to contradict these results.
Salmons and Vrbova (67) and Brown et al (15) found no effect
on contraction time if slow muscle was stimulated inter-
mittently at 40 Hz. Clearly, more work is needed regarding
the effects of intermittent, high-frequency stimulation of
slow muscle to determine if it can be made "faster" and, if
it can, what conditions must be met with regards to age,
stimulation frequency, etc.

Cross-innervation is known to change contraction time
(8, 16, 17, 21). A slow muscle innervated by a nerve from a
fast muscle becomes faster, and a fast muscle innervated by
a nerve to a slow muscle becomes slower. These changes are
supported by alterations in myosin ATPase activity following
cross-innervation (8, 17).

Chronic spinal cord transection is known to cause a
regression toward more fast-twitch fibers in the soleus
muscle of adult and immature animals (6, 26). Increases in

fast-myosin isozyme also have been observed in fast-twitch

8
muscles under similar conditions (27, 49). Comparable
results are seen following immobilization of slow muscle
(13, 54).

Roy et al (66) showed that both fast and slow plantar
flexors in rats have an increased percentage of slow-twitch
fibers after functional overload. The soleus or medial
gastrocnemius were overloaded by surgical removal of their
major synergists bilaterally. After 12-14 weeks, histo-
chemical analysis of myosin ATPase following alkaline
preincubation indicated an increased percentage of slow-
twitch fibers. Other fast-to-slow fiber transitions are
well documented following surgical overload (5, 37, 46, 65).

Much research has been conducted with humans regarding
histochemical changes in muscle following training.
Oxidative enzyme capacities are enhanced in both fast- and
slow-twitch fibers after endurance training (1, 32, 43, 45).

On the other hand, enhanced glycolytic capacity is known to
occur following high-intensity, anaerobic training (28, 32).
However, no significant changes in the percentage of slow
and fast muscle fibers as identified histochemically have
been found in response to endurance training (28, 32),
weightlifting (73) or short-term anaerobic work (69). Thus,
most investigators agree that while individual human muscle
fibers can adapt metabolically to the load placed upon them,
it is not possible, at least given the relatively short time
span in which most studies are conducted, to change the

contractile characteristics of a given fiber.

9

In contrast, one study showed a small amount of muscle
fiber conversion. Jansson et al (47) reported a significant
decrease in the percentage of slow-twitch fibers in long-
distance runners following anaerobic training. Initially,
the subjects were trained aerobically at about their
anaerobic threshold level (70-80% V02 max) by running
approximately 110 km a week. The subjects remained on this
training schedule for an average of 18 weeks. Subsequently,
the subjects were trained with an interval program at
90-100% of their V02 max for 7-13 weeks. The cumulative
distance per week, which also included some distance
running, was reduced to 71 km. Muscle biopsies were taken
from the vastus lateralis after each training period.
Muscle sections were stained for myosin ATPase activity.
The percentage of slow-twitch fibers decreased from 69%
following aerobic training to 52% following anaerobic
training. The increase in fast-twitch fibers occurred
primarily in an intermediate staining type. However, the
results of this study must be considered carefully not only
because of the small number of subjects (n=4) but also
because of the interpretation of stained serial muscle
sections used for classification of fiber types. Photo-
graphs of the sections printed in the article indicate that
fibers from the vastus lateralis which appeared dark after
staining for myosin ATPase (ph 4.3) are labeled as both
slow-twitch and intermediate types but the latter were

included with fast-twitch fibers when percentages were

10

determined. Also, fast-twitch fibers were further categor-
ized based on their oxidative capacity at this low pre-
incubation ph. Experience in our laboratory has indicated
that no determination of oxidative enzyme activities is
possible after staining for myosin ATPase in an acid ph.

A number of studies have demonstrated the presence of
intermediate type fibers in humans following endurance
training (1, 34, 45). It has been hypothesized that fibers
of an intermediate type are in transition from either fast-
to-slow or vice versa. Studies in humans indicate that
intermediate fibers contain both fast and slow myosin (12)
and that their cross-sectional areas fall in between the
size of fast-twitch and slow-twitch fibers (70). Also,
ATPase intermediate fibers in the rat soleus have contrac-
tion times in between those of fast and slow fibers (51,
52). The results of these studies are encouraging but more
research is needed to determine if a true exercise-induced
fiber conversion is possible in humans or if the inter-
mediate fibers are simply a by-product of nerve damage or
other factors.

Animals subjected to endurance training show concomi-
tant increases in the ability to oxidize carbohydrates and
fatty acids (3, 9, 42, 56). Recent evidence suggests that
contractile parameters also may be subject to change. Green
et al (35) found a 17% increase in the percentage of histo-
chemically defined slow-twitch fibers in the deep portion of

the vastus lateralis of endurance-trained rats. During the

11
first seven weeks of training the animals ran once per day
on a motor driven treadmill at a constant speed of 27 meters
per minute. Grade and run duration were progressively
increased. During the final eight weeks of training, the
animals ran in the morning and the evening. The animals
began the twice-per-day exercise program by running for 15
minutes in the morning and 90 minutes in the evening. The
running time for the morning session was increased by 3
minutes per day while the duration of the evening session
was held constant. Grade continued to be progressively
increased as it was during the initial seven weeks of
training. By the end of the 15 weeks of training the
animals were running for 210 minutes per day at 27 meters
per minute and at a l5-degree grade.

Staudte et all (72) observed a decrease in isometric
twitch contraction time in the soleus muscle of sprint-
trained rats. The animals, 46 days of age at the initiation
of the experiment, were trained seven days a week for three
weeks. Each animal ran four times daily, twice in the
morning and twice in the afternoon, with each run lasting 45
minutes. At least 60 seconds rest was allowed between runs.
Speed and grade of the motor driven treadmill were increased
gradually throughout the three-week training period. At the
cessation of training the isometric twitch contraction time
of the trained soleus muscles decreased 15% when compared to

that of the control muscles.

12

Jaweed et al (48) demonstrated alterations in myosin
ATPase activity in rats following weightlifting exercises of
various intensities. The trained animals were divided into
two groups. The "high-intensity, prolonged-duration" group
climbed a 50° inclined ladder (90 cm in length) a total of
50 times each day. The "high-intensity, short-duration"
group climbed a 50° inclined ladder (45 cm in length) a
total of 25 times a day. Additional weight was added to
each animal as the training proceeded to increase the work
load. Each group trained four days a week for six weeks.
The results of the study showed a 3.7% decrease in the
percentage of slow-twitch fibers in the soleus of the "short
duration" group but an 8.2% increase in the percentage of
slow-twitch fibers in the "prolonged-duration" group.

Exner et al (30) reported a 20% decrease in contraction
time of fast muscle in female rats trained isometrically for
25 consecutive days. The animals exercised by holding a
static body position in a 600 inclined tube with weights
attached to their tails. The training consisted of three
sessions, performed twice daily, 12 hours apart. Between
each session the animals were allowed to rest for 30
minutes. No session lasted longer than five minutes. If an
animal could hold itself in the tube for five minutes the
amount of weight attached to its tail was increased in the
next session.

Surprisingly, when slow muscle from these rats was

analyzed, a 20% increase in contraction time was observed.

13
In addition, no change was observed in contraction time in
slow or fast muscle in male rats trained identically for 35
consecutive days (31).

Bagby et al (2) found no significant effect on myosin
ATPase activities in the rat gastrocnemius following sprint
of endurance training. All animals in the exercise groups
were trained in motor-driven wheels by progressively in-
creasing the distance run during daily sessions (5
days/week). The endurance group improved until they were
running continuously for one hour at 28.4 meters per minute
during each session. The sprint group alternated 30-second
sprints with 30-second rest periods. At the completion of
training this group was sprinting 18 times per day at a
speed of 80.4 meters per minute. The duration of training
for both groups was 11 weeks.

Baldwin et a1 (4)-trained rats by means of a program of
treadmill running for durations of between 18 and 24 weeks.
All animals were trained progressively for the first 12
weeks and then kept on a routine that consisted of two hours
of continuous running at 1.2 miles per hour up a 15% grade,
five days per week. No changes in myosin ATPase activity or
in the total amount of actomyosin recovered from the vastus
lateralis muscle occurred in response to training.

Barnard et al (10) exercised male guinea pigs on a
program of treadmill running. The animals ran five days per
week for a total of nine or eighteen weeks. The training

was progressively increased each week through the first nine

14
weeks then kept at this workload for the remainder of the
experiment.

On Tuesday and Thursday of the first week the animals
ran for ten minutes at 20.8 meters per minute, 2% grade. On
Monday, Wednesday, and Friday of the first week they ran ten
minutes of wind sprints with 30 seconds of running at 40
meters per minute alternated with 30-second rest periods.
Each session was preceded by a five minute warm-up at 20.8
meters per minute, 0% grade, and concluded by a 15-minute
run at 33.3 meters per minute, 0% grade.

During the 9th week the program consisted of a five-
minute warm-up at 27.5 meters per minute, 0% grade. On
Tuesday and Thursday the animals ran for 20 minutes at 40
meters per minute, 2% grade. On Monday, Wednesday and
Friday the animals alternated 30 seconds of walking at 20.8
meters per minute, 0% grade, with 30 seconds of sprints at
49.3 meters per minute, 0% grade, for a total of 20 minutes.
All sessions were concluded with a 15-minute run at 36.8
meters per minute, 2% grade.

There were no significant differences between control
and trained gastrocnemius or plantaris muscles in the
proportion of myosin ATPase dark- and light-staining fibers
after nine or eighteen weeks of running.

Edgerton (24) found no changes in the percentages of
slow and fast muscle fibers as identified histochemically in
the soleus muscle of rats subjected to endurance exercise.

The trained rats were divided into two groups: one group

15
was forced to swim for one 30-minute period per day with a
weight equal to 3% of body weight attached to the tip of the
tail; the second group was forced to swim for two 30-minute
periods per day with a weight equal to 4% of the body weight
attached to the tail. In addition, the rats in this group
were permitted to exercise at will in a revolving drum
adjacent to their cages. A third group of rats served as
sedentary controls. The trained rats exercised six days per
week for a total of fifty-two days.

In a study involving non-human primates, no changes
were found in the percentage of muscle fiber types in the
soleus, plantaris or tibialis anterior muscles following a
six-month training program (25). The adult galago
senegalenius (lesser bushbaby) was trained to run upright on
a treadmill by using a horizontal bar to support its fore-
limbs. The animals learned to run in this position within
five training sessions. After this the training load was
progressively increased. At the end of the first month the
animals ran for an average of 25 minutes at 30 meters per
minute, 0% grade. During the last two months all animals
were able to run 60 minutes at 43 meters per minute, 4%
grade.

Roy et al (64) found no change in the percentage of
fast- and slow-twitch fibers in the rectus femoris of rats
subjected to a weightlifting program. The rats were trained
to stand upright on their hind legs by the use of light

stimulus followed by electic shock. Each rat performed

16
sixteen lifts daily, five days per week, for eight weeks.
The duration of each lift was approximately two seconds and
a rest interval of 30 seconds was provided between lifts.
The training load was increased gradually, and by the end of
the study each exercised animal was capable of lifting 130%
of its body weight.

Goneya et a1 (33) trained cats to move a bar with
their right fore-limbs to receive a food reward. The
weights attached to the bar were gradually increased during
19-46 weeks of training. At the conclusion of the experi-
ment, serial sections from the non-exercised left and
exercised right flexor carpi radialis muscle were processed
for alkali- and acid-stable myosin ATPase activity.

The question of whether the intrinsic contractile
characteristics of skeletal muscle can be altered by train-
ing alone has yet to be answered. At the present time it
would appear most beneficial in terms of economy,
efficiency, and accuracy to use animal models for this kind
of research. Apparently, specific species-related muscle
adaptations do result from exercise, so any attempt to
completely correlate the results in animals with humans
would be unwise. Rigorous variable control and extreme
exercise conditions are necessities in this field of re-
search and until these conditions are consistently met any

"answers" should remain assumptions.

CHAPTER III

RESEARCH METHODS

TWenty male, albino rats (Sprague-Dawley strain) were
received by the laboratory at 140 days of age. The rats
were assigned randomly to one of the three treatment groups.
Surgery was performed on all animals two days after their
arrival. Following surgery the rats were housed in seden-
tary cages for thirty days to allow recovery prior to the
initiation of training.

One of the rats died during surgery and myosin ATPase
activity in the stained serial sections of two other rats
could not be accurately determined. Consequently, data was

obtained from 17 animals in this study.

Treatment Groups

The three treatment groups used in the investigation
were as follows:

Sedentary-Control Group (SCG) --- These rats were
housed in individual sedentary cages (24 cm long by 18 cm
wide by 18 cm tall) for the duration of the experiment.

From either the right or the left leg, the soleus was sur-
gically removed two days after the animal arrived at the
laboratory. No surgery was performed on the contralateral
leg. Therefore, any change in fiber composition between the

17

18
initially removed soleus and the soleus in the other leg
should represent the effects of aging. Data were collected
on four animals in this group.

Sedentary-Surgery Group (SSG) --- This group of rats

 

also was housed in individual sedentary cages. During the
surgery, the achilles tendon attachments of the gastroc-
nemius and plantaris were severed in either the right or
left hindlimb. In the opposite leg the achilles tendon
attachment of the gastrocnemius again was cut and the soleus
was removed surgically. Therefore, any change in fiber
composition between the initially removed soleus and the
isolated soleus in the other leg should represent a combined
effect of aging and surgical procedures. Data were col-
lected on four animals in this group.

Exercise-Surgery Group (ESG) --- These animals were

 

housed in identical sedentary cages and were subjected to
the same surgical operations as the animals in the SSG
group. In addition, each animal was trained to jump to its
maximum height twenty times a day, five days a week, for
three months. Therefore, any change in fiber composition
between the initially removed soleus and the isolated soleus
in the other leg should represent a combined effect of
aging, surgical procedure, and training effects. Data were

collected on nine animals in this group.

Treatment Procedures

 

All animals were given commercial block food and water

ad libitum during the entire four-month experimental period.

19
Lighting (light-dark cycles of 12 hours light and 12 hours
dark) and room temperature (210-2300) were controlled auto-
matically.

A jumping chamber with an electric grid and landing
platform was used for training (figure 1). Each exercise
session was conducted in the evening during the animals dark
cycle. The rats were allowed approximately three minutes
rest between "good" jumps. A good jump was classified sub-
jectively based on the rat's effort. Often two or three
unsuccessful jumps preceded what was classified as a good
jump. Only good jumps were counted. Under all circum-
stances the rat remained in the chamber until a good jump
was made.

When a given rat could reach the landing platform on 15
out of 20 good jumps, the height was increased by one inch
in the next training session. Verbal encouragement as well
as electric shock was used to motivate the rats to jump.

The training program is shown in detail in Table I.

Surgical Procedures

 

Prior to the initial surgery, each animal was anesthe-
tized intra-peritoneally with approximately 1 cc. of a 1
percent pentobarbitol solution. A skin incision then was
made to expose the lateral portion of the lower hindlimb.
Following tendon detachment and/or soleus muscle removal as
previously described, the limbs were sutured and the animals
were returned to their cages. Sham operations were not

performed since it has been determined that no difference

20
between sham and normal muscles occurs with regards to the

physiological properties being observed (5).

Sacrifice Procedures

 

Seventy-two hours after the final training session,
each animal was weighed and then anesthetized with approxi-
mately 3 cc. of 1 percent pentobarbitol. Each animal's
right or left soleus muscle was excised and weighed. If
necessary, an additional injection of pentobarbitol was used

to kill the animal.

Histochemical Procedures

Following both the initial and final muscle removal, a
muscle block was obtained from the belly of the muscle and
placed vertically on a metal block with 5 percent gum
tragacanth. The metal block was placed in fluid 150 pentane
that had been cooled with liquid nitrogen. Serial cross
sections, 10 mm thick, then were cut from each muscle block
using an Ames lab-tek cryostat maintained at -20°C. The
serial cross sections were stained for myosin ATPase using
an acid (ph 4.3) preincubation (36). Other sections were
stained for nicotinamide dinucleotide diaphorase (NADH)
(58). Permount was used as the mounting medium for myosin
ATPase and glycerin jelly was used to mount those sections
stained for NADH. Thirty-minute incubation times were used

for all histochemical procedures.

21

Methods pf Tissue Analysis

 

The histochemically stained serial cross sections from
each muscle were projected with a microprojector in a dark-
room. All clearly differentiable fibers were counted and
classified. Initially, the myosin ATPase sections were
analyzed to differentiate between fast- and slow-twitch
fibers. Following this, the NADH-stained slides were used
to determine the general oxidative enzyme capacities of the
fast-twitch fibers in order to further sub-classify these
fibers.

A number of fibers of intermediate staining were ob-
served in some of the myosin ATPase stained sections. These
fibers were classified as intermediate type.

Muscle fiber splitting was exhibited in some of the
muscle sections. When this occurred, an estimation of the
number of "normal-sized" fibers that would have existed in
this area was made and added to the total fiber count of the
muscle section.

The numbers of fast-twitch, slow-twitch, and inter-
mediate fibers were obtained for each section. Following

this, the percentage of each fiber type was calculated.

Statistical Procedures

A dependent sample t-test was used for statistical com-
parisons within each group. A one-way analysis of variance
was used to compare the mean changes between groups. The

0.05 level was established for statistical significance.

CHAPTER IV

RESULTS AND DISCUSSION

The histochemical data and statistical results are
presented in the first part of this chapter. The last part
of the chapter consists of a discussion attempting to relate
the histochemical data with physiological and morphological

observations.

Histochemical Results

The total fiber counts and fiber type percentages
before and after the four-month treatment periods are
presented in Table I. The t-test and analysis of variance
results are presented in Table II and Table III, respec-
tively. As can be seen in Table II, no significant changes
occurred in the percentages of muscle fiber types in any of
the three treatment groups. The results of the analysis of
variance indicate that the changes in the percentages of in-
termediate staining fibers in the three treatment groups are
significantly different from each other. In an effort to
determine the source of this significance, a number of
planned comparison and multiple range tests were run. These
tests did not reveal any further information regarding the

data.

22

23

Despite the lack of statistical significance in the
results, a number of interesting observations can be made.
In all three treatment groups the percentage of slow-twitch
fibers increased and the percentage of fast-twitch fibers
decreased following the experimental period. In addition,
though no fibers of an intermediate staining type were
present in any of the animals at the beginning of the ex-
periment, at least some animals in each treatment group had
fibers of this type at the conclusion of the experimental
period. Finally, fiber splitting, while not observed in any
of the animals at the initiation of the study, was present
in animals from each treatment group in the post-

experimental histochemical analysis.

Discussion

 

As previously described, the surgical procedure used in
this study in an attempt to isolate the soleus proved to be
unsuccessful. At some unknown time following the initial
surgery the severed tendons of the gastrocnemius and
plantaris reattached to the posterior surface of the
calcaneus. The observed appearance and size of these
muscles at the conclusion of the experiment indicated that
they were functional for a good portion of the treatment
period.

An argument could be raised that if the entire ankle
plantar flexor group was functional then little, if any,
additional stress was placed on the soleus in response to

training. However, electro-myographic data have shown that

24
motor unit activity in the soleus, while probably generating
only small degrees of tension, is nevertheless enhanced
during vertical jumps of increasing height. It is possible
that this greater muscle activity occurs in response to the
animal needing additional postural support prior to takeoff.

The changes in the percentages of the fiber types in
the soleus, though not significant, suggest that the muscle
was chronically overloaded to some degree since similar
shifts in fiber populations are seen following surgical
overload (5, 37, 46, 65, 66). However, it must be noted
that in treatment group three, where no surgery was
performed to isolate the soleus, changes indicative of
overload also were observed. These changes contradict
previous work where the effects of aging on the muscle were
reported to be a decreased percentage of slow-twitch fibers
(23, 29). One possible explanation for this discrepancy may
be that the animals in this study used the leg with the
soleus intact to a greater degree than the leg with the
soleus removed. Consequently, the increased activity of the
non-operated leg could have provided sufficient stimulus to
retard a slow-to-fast fiber conversion.

The presence of intermediate-staining fibers in the
post-treatment muscles seems to indicate that some fibers
were in the process of conversion. Although there was a
significant between-group difference in the percentage of
these fibers, no determination of exactly which groups

were different from each other could be made. Despite the

25

fact that the power of the statistical tests used allowed
the significance to be detected, simple inspection of the
final results indicates that no drastic changes occurred.

Fiber splitting has been known to occur in the muscles
of some animals following physical stress (20, 24, 33, 40).
The exact cause of this phenomenon is not known and further
research is needed to determine the functional ability of
the fibers following splitting (if indeed a complete
longitudinal division is occurring). In this study fiber
splitting was present in animals from each treatment group,
though it was more evident in the rats subjected to
intensive surgery. It is interesting to note that all of
the splitting occurred in slow-twitch fibers. Previous data
suggests that this is not always the case (21, 33, 40).
Also, one study found evidence of both slow- and fast-twitch
fibers splitting from one parent fiber (40). However,
regardless of the type of fiber undergoing splitting, most
activity of this kind seems to be limited to predominantly
slow-twitch muscle. The degree of overload required to
induce splitting, the pattern of innervation, the position
of nuclei, and other questions related to the status of

these fibers remains subject to speculation.

26

 

 

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27

TABLE II. Summary of the results of student's t-test on the per-
centage of fiber types in the rat soleus before and after
the 4 month treatment period.

 

 

Slow-twitch Fast-twitch Intermediate
Comparisons Fibers Fibers Fibers

Treatment Group I (before)

Treatment 6:28p I (after) N N N
Treatment Group II (before)

Treatment 6::up II (after) N N N
Treatment Group III (before)

Treatment G¥2up III (after) N N N

 

N= Not significant
S= Significant difference at .05 level.

TABLE III. Summary of the results of analysis of variance on the
changes in the mean percentages of fiber types in the rat
soleus before and after the 4 month treatment period.

 

Slow-twitch gngt-twitch Intermediate
Comparisons Fibers Fibers Fibers

Treatment Group (I)
vs. Treatment Group (II) N N S
vs. Treatment Group (III)

 

N= Not significant
8: Significant difference at .05 level.

CHAPTER V

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

The purpose of the present investigation was to deter-
mine the effect of jumping on the activity of myosin ATPase
in the soleus muscle of male albino rats.

Twenty animals were brought into the laboratory and
assigned randomly to one of three treatment groups. Due to
complications final data were collected on only seventeen
animals. In each animal one soleus muscle was removed,
weighed and stained for myosin ATPase and NADH at the be-
ginning and at the end of the four-month treatment period.
Changes in fiber composition between the initially and
finally removed soleus muscles represented the effects of
aging, aging and surgery, and exercise with aging and
surgery, respectively, in the three treatment groups. The
animals in the exercise group were trained to jump to their
maximum height twenty times a day, five days a week, for the
final three months of the treatment period.

Visual images of the entire transverse section of the
muscle were projected onto drawing paper with a micro-
projector. All discernable fibers in each section were

counted and classified based on their staining intensity.

28

29
No significant changes occurred in the percentages of
the three fiber types in any of the treatment groups. A
significant difference in the change in the percentage of
intermediate fibers between the three groups was present
but the source of this significance could not be found. In
addition, fiber splitting occurred in the slow-twitch fibers

of animals from each of the three treatment groups.

Conclusions

 

The results of the study have led to the following con-

clusions.

1. The myosin ATPase activities in the soleus muscle of
adult male albino rats are not significantly altered by
the type of high- intensity exercise used in this study.

2. The number of intermediate (transitionary) fibers in the
adult muscle seems to be related to the severity of the

load to which the muscle is subjected.

Recommendations

1. In order to completely isolate the soleus muscle its
major synergists should be removed.

2. To obtain a thorough understanding of the cellular a1-
terations taking place, additional enzyme activities
and isometric twitch contraction times should be de-
termined.

3. Animals should be sacrificed periodically during the
treatment period to ascertain the time course of any

involved changes.

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Salmons, S. and F. A. Sreter. Significance of
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APPENDIX

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38

TABLE A-II. THREE MONTH JUMPING ROUTINE FOR MALE ALBINO RATS.

 

 

 

 

 

 

TOTAL JUMPS/ AVERAGE HEIGHT
TOTAL AVERAGE NO. (inches)
WEEK DAY TRAINING TIME SUCCESSFUL JUMPS ATTAINED
M 123 minutes 20/20 5.0
TU 147 20/20 6.0
1 W 142 20/20 7.0
TH 138 20/20 7.0
F 119 20/20 7.0
M 137 25/25 8.0
TU 141 20/20 9.0
W 140 20/20 10.0
2 TH 98 15/15 10.0
F 111 20/20 10.0
SA 153 20/20 10.0
SU 90 20/20 10.0
M 114 20/20 11.0
TU 132 20/20 12.0
3 TH 155 35/35 13.0
F 119 20/20 13.0
SA 127 20l20 13.6
SD 120 20/20 13.6
M 116 20/19.5 14.6
TU 132 20/19.2 14.6
4 W 108 20/18.6 14.6
TH 82 10/7.2 15.6
F 113 20/17.6 15.6
SA 124 20l18.0 15.6
M 157 20/19.5 16.6
TU 116 20/18.6 17.6
5 W 113 20/17.4 17.6
TH 106 20/18.0 17.6
F 134 20/17.5 17.6
M 112 20l16.2 18.0
TU 107 20/15.4 18.2
6 W 94 20/14.0 19.7
TH 138 20/13.7 19.7
F 100 20/14.2 19.7
SA 94 20/14.6 19.7

 

39

TABLE A-II (cont'd.)

 

 

 

 

 

 

 

TOTAL JUMPS/ AVERAGE HEIGHT
TOTAL AVERAGE NO. (inches)
WEEK DAY TRAINING TIME SUCCESSFUL JUMPS ATTAINED

M 116 20/11.6 20.0

TU 121 15/7.2 20.0

7 W 111 15/8.4 20.0
TH 192 30/14.8 20.0

SA 123 20/16.1 19.5

M 132 20/14.7 19.01

W 114 20/14.7 19.0

8 TH 72 10/5.2 19.4
F 139 20/14.2 19.6

SA 201 20/16.6 18.7

M 117 20/11.0 20.0

9 TU 130 20/9.3 20.4
F 147 20/8.7 21.3

SA 110 20/9.0 19.8

M 109 20/12.9 21.1

TU 124 20/13.6 21.0

10 W 133 20/14.0 21.0
TH 119 20/12.0 20.6

F 117 20/12.7 20.6

SA 127 20/12.5 19.8

M 109 20/13.3 19.4

TU 121 20/11.7 20.3

11 W 126 20/14.6 20.7
TH 140 20/13.0 21.0

F 107 15/13.1 21.0

M 119 20/8.4 22.1

TU 91 20/7.6 22.3

12 W 137 20/9.7 21.4
TH 42 20/10.2 21.2

SU 107 20/15.8 19.3

M 93 15/4.7 20.0

13 W 115 10/9.2 19.6
F 38 5/5.0 14.3