HISTOCHEMICAL AND HISTOIGGICAL OBSERVATIONS
(IN RAT MYOCARDIUM FOLLOWING EXERCISE

A Dissevi‘aflon
Ior II“ Degree of DH. D.
MICHIGAN STATE UNIVERSITY
Kwok-Wai Ho

I975

THESIS

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Histochemical and Histological Observations
on Rat Myocardium Following Exercise

presented by

Kwok-wai Ho

has been accepted towards fulfillment
of the requirements for

Ph.D. Physical Education

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ABSTRACT

HISTOCHEMICAL AND HISTOLOGICAL OBSERVATIONS
ON RAT MYOCARDIUM FOLLOWING EXERCISE

By
Kwok-wai Ho

The purpose of this study was to determine the effects of two
programs of endurance running on selected morphologic, metabolic, and
histopathologic parameters in the hearts of adult male albino rats.

The training regimens were the Short and Long Controlled-Running Wheel
programs previously reported from this laboratory.

Absolute and relative heart weights, ventricular weights, and
atrial weights were determined. Histochemical procedures were used to
evaluate relative glycogen, fatty acids, succinate dehydrogenase, and
lactate dehydrogenase values of fibers in the apical portion of the
left ventricle. Histochemical stain intensities were measured objec-
tively using a Histochemical Photometer. Histological cross-sections
at five separate levels in each heart were examined by light microscope
for lesions.

I Eighty-four Sprague-Dawley rats were assigned randomly to three
treatment groups: a sedentary-control group (Control); a high-intensity,
short-duration running group (Short); and a low-intensity, long-

duration running group (Long).

Kwok-Wai Ho

The treatments were initiated when the animals were 84 days old.
Animals were sacrificed at the beginning of the study (0 weeks) and
after 8 and 16 weeks of treatments. At the 8-wk and l6-wk treatment
durations, sacrifices were conducted l5 minutes and 72 hours following
the last exercise periods of the Short and Long animals. Performance
criteria were used as the basis for animal selection in the Short and
Long groups. The final sample consisted of 72 animals with four animals
in each treatment-duration-sacrifice subgroup.

At both 8 and 16 weeks of training, the two exercise regimens
produced similar increases in the heart weight/body weight ratio.
These significant increases in relative heart size were caused by the
fact that the trained animals gained less body weight over time than
did the sedentary animals; they could not be attributed to a larger
than normal gain in the absolute heart weights of the trained animals.
The exercise-induced increase in relative heart weight appeared to be
age-dependent.

The changes in metabolic activity of cardiac muscle that were
produced by the Short and Long training programs could not be differ-
entiated by histochemical determinations 0f succinate dehydrogenase,
lactate dehydrogenase, glycogen, or fatty acids. This observation was
supported by the significant succinate dehydrogenase increases that
occurred in both the Short and Long groups. The lactate dehydrogenase

values of the 72-hr animals were significantly higher than those of the

Kwok-Nai Ho

15-min animals in both trained groups at 8 weeks; whereas, at 16 weeks,
the 15-min values were higher than the 72-hr values in both groups.
There were no significant Short versus Long contrasts at any time.

The values of succinate dehydrogenase, periodic acid-Schiff, and
lactate dehydrogenase all suggest that the hearts of the exercised
animals were subjected to a period of metabolic adjustment and that
they gradually adapted to the physical stresses imposed by the training
programs. Therefore, a 16-week period of training with the Short or
Long Controlled-Running Wheel programs is adequate for metabolic adapta-
tions to take place in the heart.

The results of the histopathological observations show that
neither the Short nor the Long training program, as presently con—
structed, produces lesions in the heart muscle of the rat when they are

applied for periods of up to 16 weeks.

HISTOCHEMICAL AND HISTOLOGICAL OBSERVATIONS
ON RAT MYOCARDIUM FOLLOWING EXERCISE

By

Kwok-Nai Ho

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Health, Physical Education, and Recreation

1975

DEDICATION

To My Familiy

1'1'

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr. William W.
Heusner and Dr. Wayne D. Van Huss for their invaluable advice and
guidance in the preparation of this study.

I wish also to thank Dr. Rexford E. Carrow and Dr. Glenn L.
Waxler for their assistance on the doctoral committee.

Acknowledgment is also made of the understanding, encouragement,
and assistance rendered by the staff of the Human Energy Research
Laboratory and the Neuromuscular Research Laboratory at Michigan State
University.

 

TABLE OF CONTENTS

 

CHAPTER Page
LIST OF TABLES ............................................ vi

LIST OF FIGURES ........................................... vii

I. THE PROBLEM ............................................... 1
Statement of the Problem ............................... 2
Rationale for the Study ................................ 2
Significance of the Problem ............................ 4
Limitations of the Study ............................... 4

II. REVIEW OF RELATED LITERATURE .............................. 6
Myocardial Metabolism .................................. 6
Myocardial Pathology ................................... 28

III. METHODS AND MATERIALS ..................................... 36
Sampling Procedures .................................... 36
Research Design ........................................ 37
Training Procedures .................................... 39
Training Programs ...................................... 41

Animal Care ............................................ 43
Sacrifice Procedures ................................... 43

Tissue Analyses ........................................ 45
Statistical Procedures ................................. 47

IV. RESULTS AND DISCUSSION .................................... 48
Training Results ....................................... 48

Body Weight and Heart Size Results ..................... 50
Histochemical Results .................................. 60
Histopathological Results .............................. 66
Discussion ............................................. 66

iv

TABLE OF CONTENTS--Continued

CHAPTER Page

V. CONCLUSIONS AND RECOMMENDATIONS..... ..................... 75

Conclusions ........................................... 77

Recommendations ....................................... 79

REFERENCES ...................................................... 80
APPENDICES

A. TRAINING PROGRAMS ........................................ 93

B. F-RATIOS AND P-VALUES FOR ALL EFFECTS .................... 96

C. MORPHOLOGICAL AND HISTOCHEMICAL RAW DATA ................. 101

LIST OF TABLES

TABLE Page

1. Experimental design with final cell frequencies ............ 38

2. Controlled running wheel programs for the Short and Long
groups on the 37th day of training ......................... 42

vi

LIST OF FIGURES

FIGURE

1.

10.

Schematic diagram of a myocardial cell illustrating the
major pathways of cardiac metabolism, the three phases of
the metabolic processes and their interrelationships ........

. Mean daily percent shock-free time (PSF) and percent

expected meters (PEM) for the CRW Short program .............

. Mean daily percent shock—free time (PSF) and percent

expected meters (PEM) for the CRW Long program ..............

. Means and standard errors of the mean for body weight (gm)

by treatment, duration, and treatment x duration with sigu
nificant (P<:.05) Student-NewmandKeuls (SNK) contrasts
shown below the figures .....................................

. Means and standard errors of the mean for total heart weight

(gm) by treatment, duration, and treatment x duration with
significant (P<:.05) SNK contrasts shown below the figures..

Means and standard errors of the mean for ventricular weight
(gm) by treatment, duration, and treatment x duration with
significant (P< .05) SNK contrasts shown below the figures..

. Means and standard errors of the mean for atrial weight (gm)

by treatment, duration, and treatment x duration with sig-
nificant (P<:.05) SNK contrasts shown below the figures .....

. Means and standard errgrs of the mean for total heart

weight/body weight (10 ) by treatment, duration, and treat-
ment x duration with significant (P< .05) SNK contrasts
shown below the figures .....................................

. Means and standard errgrs of the mean for ventrivular

weight/body weight (10 ) by treatment, duration, and
treatment x duration with significant (P<:.05) SNK
contrasts shown below the figures ..........................

Means and standard errors of the mean for atrial weight/
body weight (103) by treatment, duration, and treatment x
duration with significant (P<:.05) SNK contrasts shown
below the figures ..........................................

vii

Page

49

51

52

54

55

56

57

58

59

LIST OF FIGURES--Continued

FIGURE

ll.

12.

l3.

14.

Means and standard errors of the mean for succinate
dehydrogenase (% light absorbed) by treatment, duration,
and treatment x duration with significant (P<:.05) SNK
contrasts shown below the figures .............. . ...........

Means and standard errors of the mean for fatty acid
(% light absorbed) by treatment, duration, and treatment x
duration .......................... . ........................

Means and standard errors of the mean for periodic acid-
Schiff (% light absorbed) by treatment, duration, and
treatment x duration with significant (P<:.05) SNK con-
trasts shown below the figures .............................

Means and standard errors of the means for lactate dehydro-
genase (% light absorbed) by treatment, duration, sacrifice,
treatment x sacrifice, duration x sacrifice, treatment x
duration, treatment x duration x sacrifice with signifi-
cant (P<<.05) SNK contrasts shown below the figures ........

viii

Page

61

62

63

64

CHAPTER I

THE PROBLEM

Numerous investigators have demonstrated that prolonged regimens
of proper exercise can produce morphological, physiological, and
biochemical changes in the mammalian heart. These fundamental adapta-
tions are believed to be beneficial to cardiac function (35).

Recent evidence suggests that "exercise" consists of a variety of
specific activities each of which elicits a specific response within
the organism. For instance, both histochemical and biochemical observa-
tions have shown that mammalian skeletal muscle consists of hetero-
geneous fiber types. Although a few muscles are predominately composed
of one fiber population, most skeletal muscles show little fiber-type
homogeneity. Each type of muscle fiber has unique metabolic character-
istics and, presumably, its particular function in physical movements.
However, a recent experiment using a high-intensity (sprint) running
program in rats showed that the soleus, normally a slow-twitch oxidative
muscle, responded with an enzymatic shift toward glycolytic metabolism
(126).

Although there is some contradictory evidence in the literature
(42,60,79), it is believed that the ventricular myocardium possesses a

homogeneous fiber population with slow-twitch oxidative metabolic

characteristics (12). However, little is known about the specific
effects of different exercise regimens on the heart. Cureton (20) has
suggested that athletes who compete in high-resistance sport activities
and sprint or middle-distance running events may have an enlargement
of the right ventricle while the hearts of long-distance runners may be
more typically enlarged on the left side. Other studies have shown
that cardiac hypertrophy develops in animals subjected to hypoxia in a
decompression chamber (135,137) and in men living at high altitudes
(71,112). The hypertrophy involves mainly the right ventricle and is
produced presumably by the increased work load associated with anoxia-
induced pulmonary hypertension (135,l37). Comparable hypoxic condi-
tions might result during sprint and middle-distance running events and
in certain power-type activities such as weight lifting.

In light of these findings, this study was undertaken to deter-
mine the histochemical effects of selected programs of interval running

on the myocardial metabolism of rats.

Statement Qf_the Problem

 

The purpose of this study was to determine the effects of three
different levels of prolonged physical activity on the metabolic and
morphologic characteristics of the myocardium in the adult male albino

rat.

Rationale for the Study,
Exercise generally is assumed to be beneficial to the heart, yet

there are numerous reports of myocardial "accidents" occurring during

exercise. Since there is a recognizable continuum of physical activity,
it may be that the response of the heart is specific to different types
and intensities of exercise. This study was designed to provide pre-
liminary information regarding this possibility. It was hypothesized
that as the type of exercise progresses from the short-duration, high-
intensity (SHT) end of the physical activity continuum toward the long-
duration, low-intensity (LON) end of the physical activity continuum,
the metabolic responses of the myocardium should be altered from those
of aerobiosis with partial anaerobiosis to those of total aerobiosis.

Earlier investigators have lacked an apparatus which could pro-
duce a wide range of exercise intensities in small laboratory animals.
Therefore, while the duration of exercise has been controllable, the
intensity was not. Several years of work at Michigan State University
has resulted in the development of the Controlled-Running Wheel for
Small Animals (CRW) which can be used to simulate specific human train-
ing programs in the rat (142). Thus, both the need and the opportunity
now exist to obtain data on specific myocardial effects of defined
exercise regimens.

Myocardial damage following severe exercise stress has been
reported and has been referred to as resulting from physical "overstrain"
of the heart (74,78). This investigator hypothesized that if a specific
regimen of exercise is found to be detrimental to the heart, the sub-
endocardium would suffer more from hypoxia than would the rest of the
myocardium. This hypothesis was based on the supposition that the
endocardium probably has the most anaerobic type of metabolism with

the lowest oxygen tension during exercise.

Exercise-induced histochemical alterations may be dependent upon
the duration of the training program. Thus, zero-week, eight-week, and
sixteen-week periods of training were incorporated in the research
design. Because the responses to training may be reflected differently
immediately after an exercise bout and following a recovery period,
animals were sacrificed fifteen minutes and seventy-two hours after

their last exercise bout.

Significance 9f.the Problem

 

Determination of the histochemical effects of various training
programs should contribute to an understanding of the functional nature
of the aerobic-anaerobic exercise continuum. The search for exercise-
induced myocardial alterations should provide insight into the mechan-
isms of metabolic adaptation and may supply information concerning the
type(s) of exercise which can be of most benefit to the heart. The
study of possible myocardial damage following specific CRW exercise
programs is crucial for future animal research which might employ

similar regimens of physical activity.

Limitations 9f_thg_$tudy

 

l. The results of this study cannot be applied directly to humans or
generally to other types of animals.

2. The exercise programs were designed to stimulate distinct aerobic
and anaerobic metabolic processes. However, at this time it is
impossible to rule out the possibility of overlapping metabolic

requirements.

The shock stimulus that was employed to motivate the animals to run
may have had unknown side effects causing morphological alterations
in the myocardium.

The limited number of histochemical methods that were employed
cannot be expected to provide a complete picture of metabolic
adaptations in the myocardium.

The histochemical methods that were employed are quantitatively
limited in that relative rather than actual concentrations are

measured.

CHAPTER II

REVIEW OF RELATED LITERATURE

Myocardial Metabolism

Under physiological conditions, substrates which can be used for
energy supply in myocardial metabolism are glucose, lactate, pyruvate,
free fatty acids, ketone bodies, and amino acids. However, the contri-
butions of the different substrates in the oxidative metabolism of the
normal heart may vary under resting conditions and during and after
different defined exercise regimens. The purpose of this section is to
review the patterns of substrate utilization and the metabolism of the

heart under various conditions.

General Principles gj_Myocardial Metabolism

The heart, as the result of its unique structure and function,
has evolved a complex metabolic system which is primarily aerobic (95,
98) and which provides a constant supply of high-energy phosphate bonds
for mechanical and chemical work. The relative amounts of different
substrates utilized by the heart differ markedly with different states
of nutrition, activity, and endocrine balance. The myocardial uptake
of most substrates is directly related to the arterial concentration
once the extraction threshold is exceeded. In general, the heart

utilizes free fatty acids as its predominant fuel. It also uses

significant quantities of glucose and lactate, lesser amounts of
pyruvate and ketone bodies, and only traces of amino acids (6,95,96).
The heart has an extraordinary concentration of mitochondria crowded
between the myofibrils (128). As in skeletal muscle, many important
enzymatic reactions take place in the mitochondria. This applies
particularly to the tricarboxylic acid cycle (TCA cycle) and oxidative
phosphorylation.

The metabolic processes in heart muscle may be divided into three
phases: (a) energy liberation, (b) energy conservation, and (c) energy
utilization (93). A schematic representation of these processes and
their interrelationships is depicted in Figure l. The first phase
involves TCA cycle liberation of nascent hydrogen and the oxidation of
hydride ions to water via the electron transport chain. Energy conserva-
tion is concerned with the capture of this energy as adenosine triphos-
phate (ATP) and the interaction of ATP with creatine to form creatine-
phOSphate (CP) where the energy is stored for later use. The energy-
utilization phase is the hydrolysis of ATP in the performance of cellu-
lar work. Ninety percent of the energy released is used to generate
mechanical work in the heart; the rest is used for other chemical
processes including the maintenance of the normal sodium and potassium
ion gradients responsible for transmembrane electrical potentials.

ATP utilization by cardiac muscle to perform mechanical work is ini-
tiated by depolarization of the muscle cell and the attached T-tubule
system which admits a pulse of calcium from the extracellular Space.

This in turn liberates large stores of calcium ions from the vesicles

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of the reticulum. The free calcium binds to a specific regulatory
protein (troponin in the troponin-tropomyosin-actin complex) which
results in conformational changes that permit cross-bridge formation
between the myosin heads on the thick filaments and the actin-combining
sites on the thin filaments. The formation of actomyosin results in
enhanced myosin adenosine triphosphatase (myosin ATPase) activity,
translocation of myofilaments, and shortening of the myofibril. The
adenosine diphosphate (ADP) formed from the hydrolysis of ATP is
resynthesized to ATP by combining with another phosphate group obtained
either from CP or during the processes of glycolysis and oxidative
phosphorylation in the electron transport chain. The ADP and Pi
(inorganic phosphate) formed from the hydrolysis of ATP also serve as
regulatory feed-back controls for intermediary metabolism. That is,
high levels of ADP and Pi stimulate oxidative phosphorylation, TCA
cycle activity, and glycolysis; whereas these processes are relatively
depressed by high levels of ATP and low levels of ADP and Pi (36,59,93,
106,119,140,l4l).

Free Fatty Acids (FFA)

Under resting conditions, the normal heart obtains 60+% of its
energy from the oxidation of plasma nonesterified FFA (6,95). The
myocardial uptake of individual FFA is directly related to their
arterial concentrations. However, there is no apparent explanation for
the existence of an arterial threshold below which no myocardial extrac-
tion of FFA takes place. The threshold for total FFA is about 0.350 mM

(15,16). FFA bind noncovalently to plasma albumin which serves to

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transport FFA. Infusions of catecholamines, which increase arterial
FFA levels, are associated with an increased extraction of FFA by the
myocardium (41). Insulin, on the other hand, decreases arterial levels
of FFA and thus indirectly causes a decreased myocardial uptake (40).
There are no known direct hormonal influences on FFA uptake by the heart
(95). Both animal and human studies have shown that there may be a
slightly preferential uptake of oleic acid (unsaturated) by the myo-
cardium. Uptake of FFA decreases as the chain length increases (15,16,
30,145,146). FFA not only provide fuel for oxidation but also signifi-
cantly influence intracellular carbohydrate metabolism. This occurs
both by a nonspecific competition between pyruvate and fatty-acid
derivatives for entry into the TCA cycle and by the production of
citrate which inhibits phosphofructokinase, one of the main rate-
1imiting enzymes of glycolysis (31,41,109,125).

The FFA undergoing oxidation in the myocardium come either from
endogenous intracellular lipid or extracellular fluid. The major endo-
genous source is stored fat droplets in the cytoplasm which consist
largely of triglycerides. It is the triglyceride fraction that under-
goes hydrolysis by intracellular lipases to yield FFA and glycerol, not
the phosphoglycerides which are chiefly structural (19,24,94). However,
there is little evidence to suggest that endogenous lipid is an impor-
tant energy source for the jn_§jtg_heart of the normal resting subject.
Triglycerides are used in the perfused working rat heart (19) and when
rats run to exhaustion in motor driven drums at a velocity of about

1 ft/sec (llO). Scheuer and co-workers reported that myocardial

ll

triglyceride levels fell both in conditioned and control rats after a
single 75-minute swim (120). This was confirmed by Frdberg in 1971 (33).
In a recent human study, Kaijser gt_al: (68) found that during pro-
longed exercise on a bicycle ergometer, at 50% of maximum load for
supine leg exercise, there was an efflux of fatty acids into coronary
sinus blood from endogenous lipid stores. It has been concluded that
the myocardium does derive energy from endogenous stores during pro-
longed exercise; but the heart is much less dependent on these stores
than is skeletal muscle which, under similar conditions derives about
70% of the FFA oxidized from endogenous sources (34,68).

During exercise, the liver is not a significant source of lipid
substrates for total energy expenditure (113). Therefore, the increased
entry of exogenous FFA into the blood is primarily from adipose tissue
(49,50). In fact, the composition of the FFA liberated by adipose
tissue approaches that of simultaneously obtained arterial FFA (6).

It is well-known that there is an elevated plasma level of catechol-
amines, especially norepinephrine, during exercise (3). Since catechol-
amines are potent lipolytic agents, it is believed that the adrenergic
neuro-humoral system is responsible for the mobilization of fat during
exercise. In exercised rats, an increased sensitivity of the lipolytic
response of isolated fat cells to norepinephrine has been observed (39).
In dogs, there are regional differences of physiologic importance in

the ability of the adrenergic neurorhumoral system to elevate the lipo-
lytic rate. Electrical stimulation of appropriate sympathetic nerves
causes marked lipolysis in subcutaneous adipose tissue and omentum but

not in mesenteric adipose tissue (lll).

12

Circulating catecholamines come from the adrenals and the adren-
ergic nerve terminals. The catecholamine levels in arterial blood dur-
ing exercise have been correlated to relative work load. The norepine-
phrine level increases slowly up to a work load corresponding to an
oxygen consumption of about 75% of maximum at which time that level
reaches 1.0 to 1.5 ug/l of plasma. Thereafter, the level increases
rapidly to about 10 ug/l of plasma at supramaximal work loads (43).

It has been concluded that the adrenals contribute very little to the
catecholamine levels during mild or moderate exercise and that activity
at sympathetic nerve endings in adipose tissue appears to be a more
critical factor in the mobilization of fat than is the level of circu-
lating catecholamines (43,111). Adrenergic stimulation of the adenyl
cyclase-cyclic AMP system in adipose tissue, leading to activation of
hormone-sensitive lipase, appears to be the main mechanism for the
release of FFA from stored triglycerides (48).

Increased secretion of growth hormone may contribute to fat
mobilization during the later stages of prolonged exercise, but the
concentration of growth hormone usually does not rise until 30 to 40
minutes after the onset of activity (45,46). The secretion of growth
hormone is believed to be regulated through effectors in the hypo-
thalamus which promote the release of a growth hormone-releasing factor
to the pituitary. Thus, secretion of growth hormone as well as nor-
epinephrine from sympathetic nerve endings during exercise may be
related to central adrenergic stimulation. Such adrenergic activation
also could promote fat mobilization through stimulation of alpha recep-

tors known to inhibit the secretion of insulin (105).

13

Some other hormones including adrenocorticotropic hormone (ACTH),
glucagon, and the adrenal glucocorticoids are known as adipokinetic
agents (89,130). However, the role of these hormones in activating
lipolysis during exercise is not fully understood.

It is known that reserpine depletes the norepinephrine stores of
sympathetically innervated organs. It blocks transmission from the
postganglionic sympathetic nerves which serves to inhibit the impulse-
induced release of norepinephrine into the blood. Mobilization of FFA
persisted in rats that were exercised for 16 weeks on a motor-driven
treadmill and then given reserpine in doses which were adequate to
severely deplete the tissue catecholamines (130). Thus, there may be
more than one hormonal system involved in activating lipolysis in the
rat during exercise.

Studies have shown that arterial FFA concentrations fall below
resting levels during the first 10 to 30 minutes of exercise and that
this is accompanied by a fall in the oxygen extraction ratio for FFA in
the heart (68,72). In general, the higher the rate of work, the greater
the early fall in FFA concentration and the higher the rise in lactate
concentration. When exercise is prolonged, the arterial FFA continue
to rise. The use of FFA in the production of energy may reach 68% of
the total energy production at the end of two hours of exercise. It has
been suggested that the mobilization and utilization of FFA by the heart
muscle is more pronounced in athletes than in untrained subjects (72).
Scheuer and co-workers (120) also found that rats forced to swim 75

minutes twice daily, 5 days a week for 6 weeks, had significantly

14

reduced levels of myocardial triglycerides. N6cker gt_gl, (92) used
coronary sinus catheterization to determine that the hearts of untrained
subjects derive 34%, 21%, and 23% of their energy from FFA during rest,
submaximal exercise (200 Watt, 12 minutes), and recovery 15 minutes
after exercise respectively. Values for athletes (members of the German

national team) were 49%, 30%, and 46% under similar conditions.

Ketone Bodies

The ketone bodies, B-hydroxybutyrate and acetoacetate, can be
taken up and oxidized by the heart (6,95). In many vertebrates, the
liver has the enzymatic capacity to divert some of the acetyl CoA
derived from fatty acid and pyruvate oxidation into free acetoacetate
and B-hydroxybutyrate. These ketone bodies are transported via the
blood to the peripheral tissues where the B-hydroxybutyrate is oxidized
by a nicotinamide adenine dinucleotide (MAD-dependent) dehydrogenase
to acetoacetate. Acetoacetate then is converted to acetoacetyl CoA
which is cleaved to acetyl CoA. The latter is oxidized via the TCA
cycle (6,95). The liver, therefore, is the main producer; and the
muscles, including the heart muscle, are the main utilizers of these
two ketone bodies.

The arterial concentration of ketone bodies is the factor which
decides their rate of uptake by the heart muscle. Normally, the con-
centration of ketone bodies in the blood is very low. The major ketone
body taken up by the human heart is B-hydroxybutyrate. Its arterial
concentration exceeds that of acetoacetate by three to eight times

(95).

15

There is no evidence that ketone bodies are major substrates for
myocardial metabolism in humans either at rest or during exercise.
Plasma ketone bodies account for only 2 to 9% of the heart's energy
supply at rest (6,68,92). Their arterial concentrations and their
relative contributions to myocardial oxidative metabolism both fall

during exercise (14,92).

Amino Acids

Under ordinary conditions, insignificant amounts of amino acids
are used by the heart for myocardial energy metabolism both at rest
(6,15,95,96) and during exercise (15,92). However, the intermolecular
transfer of amino groups in the myocardium is active because of large
amounts of two transaminases, glutamic-oxaloacetic transaminase and
glutamic-pyruvate transaminase; and the uptake and release of individual
amino acids have been reported (82,95). The relative roles in the heart

of uptake from circulation and transamination are not known.

Glucose and Glycogen.

Under physiological resting conditions, two carbohydrate sub-
strates, glucose and lactate, account for about 30 to 35% of the total
oxygen extracted by the heart (6,95). Pyruvate is used by the human
heart, but the blood concentration is low and so is its myocardial
utilization (6). It has been estimated that the human heart oxidizes
about 11 grams of glucose and 10 grams of lactate per day (6).

In both the perfused rat heart and the human or dog heart ig_situ,

 

there is a threshold for extraction of glucose; furthermore, the glucose

16

uptake approaches maximum when the plasma glucose concentration rises
to a certain level (6,85,95). Thus, glucose uptake by the heart muscle
appears to be limited by the rate of transport of glucose across the
cell membrane (85). This process is believed to occur at steroscopic
sites on the cell membrane under the influence of insulin (32), and it
is known to be facilitated by hypoxia (32,85). When the rate of mem-
brane transport exceeds the rate of phosphorylation of intracellular
glucose, phosphorylation becomes the limiting factor for glucose uptake
(85).

It has been shown that, despite a major reduction of hepatic
blood flow, glucose production by the liver can contribute substantially
to the total pool of metabolic substrates during moderate to heavy exer-
cise. Rowell (113) reported that there is a 6-fold increase in glucose
production above resting values, while Hultmen and Nilsson (58) found a
9 to 11—fold increase at the end of heavy exercise. More recently,
Wahren gt_al, (139) reported that splanchnic glucose production rises
2 to 3-fold during exercise but returns rapidly to basal levels after
exercise. The differences between these reported values for increased
hepatic output of glucose apparently are related to the duration and
intensity of the exercise bouts imposed by the various investigators.
The increased hepatic production of glucose, primarily by means of
augmented glycogenolysis, contributes to blood glucose homeostatsis in
exercise. Using a needle biopsy technique, Hultman and Nilsson (58)
have shown that the liver glycogen content depends on the composition

of the food ingested. After a carbohydrate-free diet, the glycogen

17

stores in the liver decrease rapidly as does the blood glucose level
during exercise.

It is generally agreed that blood insulin decreases during differ-
ent durations and intensities of exercise and then rises rapidly during
recovery to a level above the basal concentration (44,45,107,139).
Thus, the normal relationship between blood glucose and insulin is
restored shortly after the cessation of exercise (107,139). Pruett
(107) reported that the age of the individual, the subject's state of
training, and the subject's diet appear to play only minor roles in the
behavior of the plasma insulin concentration during exercise. However,
Hartley gt-al, (44,45) consistently found increased insulin levels dur-
ing prolonged bicycle exercise, at various work loads, after a seven-
week physical training program. They believe that following training
lowered plasma norepinephrine levels during work may permit higher con-
centrations of insulin. It is known that infusion of catecholamines
results in a reduction of circulating insulin even in cases of glucose-
induced hyperglycemia. Porte and Baum (5,105) suggest the existence of
an alpha-receptor mechanism for the catecholamine suppression of insulin
release in pancreatic tissue. Thus, following systematic training, it
is possible that decreased catecholamine levels during exercise might
be responsible for the higher arterial insulin level. In comparison
with the diaphragm and gastrocnemius, the rat heart appears to be rela-
tively insensitive to the glycogenic action of insulin (116). The role
of insulin in glucose transport across the myocardial cell membrane

needs further elucidation. Certainly, it cannot be stated categorically

18

from the observation of decreased plasma insulin levels during exercise
that the activity of insulin also is reduced.

In a study of the regulation of glucose uptake in the rat heart,
Morgan gt_al, (85) indicated that the extracellular transfer of glucose
from the capillary to the cell membrane is very rapid since glucose
moves by simple diffusion and there is an increase in capillary perme-
ability with anoxia. Also, the transfer in heart muscle is much faster
than in skeletal muscle because the diffusion distance is less (85).
Therefore, extracellular transfer does not appear to be an important
limiting step for glucose uptake.

As mentioned previously, the transport of glucose across the cell
membrane is believed to occur at specific membrane sites (32). This
transport process is a translocation rather than a transformation of the
substrate. In the absence of insulin, glucose transport through the
cell membrane is the major limiting step for uptake within the normal
range of blood glucose concentrations. At higher concentrations,
phosphorylation becomes the predominantly limiting factor (85). With
anoxia, glucose uptake is increased. This is due to an acceleration of
transport, an increase in the phosphorylation capacity, and a decrease
in the apparent phosphorylation Michaelis-Menten constant (Km). With
anoxia plus insulin, glucose uptake is further accelerated because
transport is facilitated by insulin.

After glucose is transported across the cell membrane, it is
rapidly phosphorylated to glucose—6—phosphate (G-6-P) by hexokinase.

G-6-P may then be metabolized in three ways: (a) anaerobic glycolysis

19

in the cytoplasm, (b) oxidation via the pentose shunt, or (c) conversion
to glycogen. The pentose shunt is relatively inactive in normal heart
tissue (6). In the several steps of metabolism of G-6-P to pyruvate,
one of the most important rate-limiting steps is the phosphorylation of
fructose-6-phosphate to fructose-l,6-diphosphate, a reaction catalyzed
by the enzyme phosphofructokinase (PFK). PFK is highly sensitive to
inhibition by GP, ATP, and citrate. This inhibition is released by
cyclic 3',5'-AMP; AMP; Pi; fructose-6-phosphate; ADP, and fructose-1,6-
diphosphate. A rise in pH also appears to release ATP inhibition of
PFK activity (90.95.98).

Transported glucose is the only effective exogenous glycogen
precursor in the heart because of the absence of the enzyme fructose-
l,6-diphosphatase. Thus, gluconeogenesis from pyruvate cannot take
place in heart muscle (95). An increase in the circulating glucose
concentration promotes glycogen synthesis. Glycogen synthesis occurs
by transfer of the glucose moiety of uridine diphosphate glucose (UDP-
glucose), the most active glucosyl donor, to an acceptor polyglucose
chain under the influence of glycogen synthetase. Glycogen synthetase
exists in phosphorylated and dephosohorylated forms. The phosphorylated
(D or dependent) form is dependent on the glycogen precursor, glucose-6-
phosphate, for its maximal activity. The dephosphorylated (I or inde-
pendent) form is active in the absence of glucose-6-phosphate. In the
working rat heart which is isolated and perfused, over 90% of the
synthetase is in the D form (95). The D form is dephosphorylated to
the I form by the action of the enzyme glycogen synthetase phosphatase;

20

whereas, the I form can be rephosphorylated to D form by glycogen syn-
thetase kinase with the expenditure of ATP. This kinase, a regulatory
enzyme, is activated by cyclic 3',5'-AMP. Cyclic 3',5'-AMP is formed
from ATP by adenyl cyclase which is present in the cell membrane. The
formation of cyclic 3',5'-AMP is greatly stimulated by epinephrine
secreted from the adrenal medulla during physiological stress. Cyclic
3',5'-AMP appears to function as a positive modulator for glycogen
synthetase kinase which, in turn, generates the D form of glycogen
synthetase. The overall effect of epinephrine then is to inhibit the
synthesis of glycogen (83). On the other hand, by some unknown mechan-
ism, growth hormone causes a significantly increased glycogen deposition
in rat hearts (116). Russell and Bloom (116) have stated that insulin
is somewhat more effective than growth hormone in promoting glycogen
deposition in the gastrocnemius muscle although it has less effect on
the heart in rats. Recently, Das (21) concluded that the insulin-
induced acceleration of glycogen synthesis in the normal perfused rat
heart is due mainly to an increase in substrate concentration because
no change in the I/D ratio of synthetase forms has been found.

The degradation of glycogen starts by the action of glchgen phos-
phorylase which removes the terminal glucose residue to yield glucose-l-
phosphate. The glycogen phosphorylase in heart muscle occurs in two
forms: the active form (phosphorylase a) and a much less active form
phosphorylase b). Phosphorylase b can undergo dimerization and phos-
phorylation to yield the active a form under the catalysis of phosphoryl-

ase b kinase. This kinase in turn is activated by cyclic 3',5'-AMP.

 

21

It has been demonstrated that catecholamines stimulate the enzyme adenyl
cyclase which catalyzes the formation of cyclic 3' ,5'-AMP both 11mg
and jg_yitrg_(83). It is clear that epinephrine has a dual action: it
promotes the breakdown of glycogen and inhibits glycogen syntheSis
through its effect on glycogen synthetase. However, in comparison with
the situation found in skeletal muscle, cardiac glycogenolysis usually
responds only to a large dose of epinephrine which probably is in

excess of that required to produce a maximal positive inotropic response
of the heart. Thus, the significance of the adrenergic mechanism which
directly regulates cardiac glycogen levels is questionable (61,83).

It is well-known that anoxia rapidly depletes myocardial glycogen,
but the role of catecholamines in glycogen degradation during anoxia
has been questioned by Mayer and co-workers (83). They showed that the
degradation of cardiac glycogen subsequent to anoxia appears to be
delayed even after 75% of the phosphorylase has been converted to the
a form. During hypoxia or muscular contraction, decreased ATP,
increased Pi’ and decreased G-6-P all contribute to enhanced glycogenoly-
sis. Increased AMP levels, arising from the action of myokinase on the
increased ADP levels, also increase the affinity of phosphorylase b for
its substrates, glycogen and Pi (95).

In rats, thyroxin and glucagon both have been shown to influence
cardiac phosphorylase activity (57,147). Also reported is the fact that
the concentration of glycogen in the rat heart is decreased following
adrenalectomy. Phosphorylase and phosphorylase a activities are un—

changed, but glycogen synthetase activity is lowered. Administration of

22

glucocorticoids prevents this decrease (22,104). Therefore, it seems
likely that there is a dual mechanism, hormonal and cellular, which
controls glycogen metabolism.

In a study of the circadian rhythm of cardiac glycogen synthesis
in rats, Bockman gt_al, (8) reported that there is a diurnal variation
of glycogen levels in the heart with a peak and a nadir occurring two
hours after lights-on and two hours after lights-off respectively.

They found no significant rhythm in either total glycogen synthetase
activity or glycogen synthetase I'activity. Therefore, the reciprocal
relationship between the glycogen level and the percentage of synthetase
l'may not be an important control system in the normal rat heart since
glycogen is always above the levels at which this relationship holds.
Poland and Trauner (104) confirmed the existence of a diurnal glycogen
variation and found that adrenalectomy appears to eliminate the peak
value.

Cardiac glycogen in rats decreases during exercise, and the extent
of the glycogenolysis depends on the severity of exercise (67,101,103,
120). Recovery following exercise involves a period during which the
cardiac glycogen rises to values significantly above control levels
(67,103,104). This exercise-related supercompensation of cardiac gly-
cogen reaches a peak about 4 hours after exhaustive exercise and then
lasts for at least 16 hours. Thus, approximately 24 hours are required
for cardiac glycogen to return to its pre-exercise level (104). It has
been shown that adrenalectomy essentially eliminates cardiac glycogen

supercompensation following exercise; but daily treatment of

23

adrenalectomized rats with dexamethasone, a potent synthetic gluco-
corticoid having very little mineralocorticoid activity, completely
restores the capacity for supercompensation to take place (104).

Other studies have indicated that the extent of glycogenolysis
during exercise as well as the pattern of glycogen recovery following
exercise are similar in trained and untrained rats. However, the
maximum glycogen level reached during recovery is significantly higher
in trained animals (67,103). Increased cardiac glycogen storage in
conditioned animals has been observed by several investigators (26,102,
103,124). Scheuer et_al, (120) also reported an elevated cardiac
glycogen level in rats forced to swim 75 minutes twice daily, 5 days a
week, for 6 weeks. Using intubation of the coronary sinus, Keul (72)
studied six well-trained professional cyclists and 27 healthy untrained
subjects of the same age. All subjects were tested at rest, during
light and hard ergometric exercise, and during recovery. He found that
both the arteriovenous glucose difference and the contribution of
exogenous glucose to the energy production of the heart were similar in
the untrained subjects and in the athletes at rest. During exercise,
the glucose uptake of the heart and the contribution of exogenous glu-
cose to oxidative metabolism were diminished in both groups. The
mechanism of the lowered utilization of glucose is to be sought in the
large rise of arterial lactate during exercise. The lactate is ex-
tracted and oxidized by the heart muscle as an energy-supplying sub-

strate.

24

Opie (95) has observed that there are very complex control mechan-
isms for the synthesis and degradation of cardiac glycogen. A high
cardiac glycogen level can be associated with a low turnover rate and

vice versa. However, the meaning of these facts is obscure at present.

Pyruvate

There are two main pathways for the metabolism of pyruvate in the
heart: (a) the pyruvate dehydrogenase system that converts pyruvate to
acetyl CoA which then is oxidized via the TCA cycle, and (b) the pyru-
vate carboxylase system which, in the presence of excess acetyl CoA,
converts pyruvate to oxaloacetate thus enabling the TCA cycle to oxidize
more acetyl CoA (77, p. 464). Under anaerobic conditions, pyruvate is
reduced to lactate rather than to acetyl CoA, but the endogenous produc-
tion of lactate is of minimal importance in normal cardiac muscle.

Under resting conditions, the oxidation of exogenous pyruvate
accounts for a very small proportion of the total energy production in
the human heart. This proportion may be even smaller during
exercise (92). Molé gt_al, (84) found the pyruvate concentration to be
lower in the blood of trained subjects than in that of untrained sub-
jects during submaximal exercise. Krasnow gt_al, (75) reported that
pyruvate extraction is a function of arterial concentration both in
dog and human hearts. Mild exercise did not alter the mean extraction
ratio of pyruvate in their human subjects. The rate of pyruvate entry
into the TCA cycle is decreased by an increased availability of circu-
lating FFA because pyruvate dehydrogenase activity is inhibited by an
accumulation of acetyl CoA and NADH (95).

25

Lactate

Under normal aerobic conditions, the heart extracts and metabo-
lizes lactate more or less in proportion to its arterial concentration
above the extraction threshold (6,72,75). During the early stages of
exercise, the relative contribution of FFA to myocardial metabolism
falls while that of lactate increases to as much as 30 to 60% of the
heart's oxygen uptake. The exact value depends largely on the work
load with higher loads being more dependent on lactate (68,72). After
two hours of exercise, the arterial lactate concentration falls to
about twice the resting level. There is an accompanying drop in glu-
cose concentration and an approximate doubling of FFA levels. Lactate
extraction at this time, although greater than at rest, is considerably
less than it is early in exercise (68). Keul (72) and Ndcker _t_al,
(92) have reported that, during maximal exercise, athletes have lower
arterial levels of lactate but higher extraction rates than do non-
athletes. Therefore, the arterial concentration may not be the sole
factor determining lactate extraction by the heart muscle.

Before lactate can be utilized for metabolic energy, it must
first be oxidized by lactate dehydrogenase (LDH); but the equilibrium
constant (1.2x10'12) for this enzyme greatly favors lactate formation.
Therefore, lactate oxidation can occur only if the hydrogen ion is
removed or pyruvate and/or NADH can be oxidized by a system having a
very low Km for these substrates. The oxidation of pyruvate to acetyl
CoA, which is catalyzed by the pyruvate dehydrogenase system, essen-

tially is irreversible under aerobic conditions because of the large

26

decrease in standard free energy (-8.0 Kcal). A number of mechanisms
have been proposed for the "shuttling" of NADH into the mitochondria
for oxidation via the respiratory chain. In contrast to skeletal
muscle, it is reported that the malate-aspartate shuttle appears to be
the primary mechanism for the transfer of reducing equivalents from

the cytosol to the mitochondrial electron transport chain both in rat
and rabbit hearts (117,144). Kaplan and Goodfriend (70) and Wilson
gt_al, (148) have suggested that the M-isoenzyme of LDH is present
mainly in muscle that can function anaerobically (such as skeletal
muscle), while the H-isoenzyme of LDH is present in muscle that func-
tions only aerobically (such as the heart). In studying the effects of
exercise on LDH activity in cardiac muscle, Gollnick gt_al, (37,38)
swam rats 60 minutes daily for 35 consecutive days. They found that
this endurance training program produced a significant increase in heart
LDH activity. However, a single exercise bout just before sacrifice
did not significantly alter the LDH activity in either trained or un-
trained animals. Increased LDH activity gives the heart a greater
capacity for lactate utilization and complements the oxidative pathways

by supplying additional substrate in the forms of pyruvate and NADH.

Summary gf_Myocardial Metabolism

The main substrates for the energy metabolism of cardiac muscle
are FFA, glucose, and lactate. Pyruvate and ketone bodies are used to
a lesser degree. Amino acids apparently are not important substrates
for the energetic reactions of cardiac muscle. Under physiological

resting conditions, the normal heart obtains approximately 60% of its

27

energy from the oxidation of plasma FFA, 30 to 35% from glucose and
lactate, and about 5% from pyruvate and ketone bodies.

For various substrates, particularly FFA, lactate, pyruvate, and
ketone bodies, arterial concentration is the primary factor which deter-
mines the rate of uptake by the heart. The extraction and utilization
of glucose is influenced by the insulin level of the blood rather than
by the arterial glucose level.

Exercise is accompanied both by increased myocardial energy re-
quirements and by marked modifications in the metabolic environment of
the heart. Changes occur in the arterial concentrations of a number of
important substrates, and alterations take place both in local sympa-
thetic activity and in the levels of circulating hormones. For these
reasons, exercise might be expected to modify the pattern of substrate
utilization by the heart. Early in exercise, the relative contribution
of FFA to myocardial oxidative metabolism falls and that of lactate
increases to as high as 60% of the total energy requirement. In general,
the higher the rate of work, the greater the initial fall in FFA concen-
tration and the higher the rise in lactate concentration. When exercise
is prolonged, the arterial lactate and glucose concentrations fall
while that of the FFA undergoes a marked rise. There is evidence that,
during prolonged exercise, some energy for myocardial metabolism is
derived from endogenous glycogen and triglycerides although the heart

is much less dependent on these sources than is skeletal muscle.

28

Myocardial Pathology,

 

Despite many recent medical advances, cardiovascular disease con-
tinues to be the nation's number one killer and claims many thousands
of lives in this country each year. The Heart and Lung Institute's
national task force on arteriosclerosis concluded that, "the totality
of existing knowledge, especially as it relates to the basic causes of
the disease, does not permit efficient or comprehensive control of
either atherosclerosis or its complications such as heart disease,
stroke, kidney failure, or vascular disease" (1). A major problem is
the lack of definitive information concerning the basic mechanisms that
underlie the development of atherosclerotic lesions. Various risk
factors have been identified, but the roles of these factors in the
clinical progress of the disease are not well-documented.

An understanding of the pathogenesis of atherosclerotic lesions
will require further study of the factor of hemodynamic forces, the
relation of fatty streaks to intimal plaque formation, the role of
altered arterial wall metabolism, the cellular reactions during lesion
formation, the transport of lipids across the arterial wall, and the
Inechanisms of the growth of fibrous plaques (122).

In the heart, atherosclerosis can produce various degrees of
narrowing and even complete occlusion of the arteries. The usual result
”is ischemic heart disease. Epidemiologic studies have demonstrated a
Correlation between clinical manifestations of atherosclerosis and a

number of predisposing factors. These include advancing age, male sex,

hypertension, impaired glucose tolerance, and obesity. Other factors

29

which may play a role are a family history of the disease, certain
personality traits, and emotional stress. Although exercise alone can-
not prevent atherosclerosis, lack of physical activity is considered

to be one of the important risk factors and often is associated with
serious cardiovascular events (lO,47,69,86,87,99,13l).

Myocardial ischemia develops whenever the arterial blood supply
is reduced to a level below that required by the myocardium for adequate
function. The point of necrosis undoubtedly is controlled by the com-
bined effects of metabolic breakdown, autolysis and protein denatura-
tion, and increased permeability of the sarcolemma that permits exces-
sive loss of electrolytes and coenzymes. Eventually, the impaired
biochemical processes of energy production can no longer maintain

cellular integrity (80).

Metabolic Changes

 

Under the aerobic conditions that normally exist in the myocardium,
most of the high-energy phosphate compounds are formed within mito-
chondria via the TCA cycle and the electron transport system. When mild
ischemia develops, however, oxygen depletion causes an excess of
hydrogen ions to accumulate in the respiratory chain, and oxidative
phosphorylation via mitochondrial metabolism is inhibited. The alterna-
tive for cellular production of high-energy phosphate compounds is the
process of anaerobic glycolysis. Since sufficient glucose cannot be
SUpplied to the ischemic cells via the impaired coronary circulation,
stored glycogen is used. Furthermore, because ATP levels cannot be

maintained even partially for more than a few seconds by conversion of

30

CP stores, ATP is rapidly broken down to ADP, AMP, and Pi‘ These
changes, in turn, affect the rate of anaerobic glycolysis. For example,
PFK, the rate limiting enzyme of glycolysis which converts fructose-6-
phosphate to fructose-l,6-diphosphate, is inhibited allosterically by
ATP; but this inhibition is released by ADP, AMP, and Pi' Glycogen
breakdown also is facilitated during mild ischemia by conversion of the
enzyme phosphorylase b to phosphorylase a which is the active form for
degradation of glycogen. Ischemia apparently causes the release of
endogenous catecholamines to activate adenyl cyclase which catalyzes

the formation of cyclic 3',5'-AMP. Cyclic 3',5'-AMP enables activation
of phosphorylase b kinase which catalyzes the formation of phosphorylase
a (150).

During severe ischemia, areas of irreversibly injured myocardium
are incapable of maintaining adequate levels of ATP by glycolytic means.
Neill §t_gl, (91) have shown under controlled conditions that anaerobic
glycolysis can account for only about 15% of the total myocardial energy
production. Eventually, even anaerobic glycolysis ceases within the
affected cells. Cessation of anaerobic glycolysis may occur for a
variety of reasons. Stored glycogen may be depleted, or glycolysis may
be inhibited even though there still is glycogen present within the cell.
It has been shown, for example, that the activity of PFK in the acutely
infarcting myocardium can be limited by an accumulation of hydrogen
ions; i.e., by an intracellular acidosis resulting principally from
increased lactate (9,76,97,150). The heart is extremely susceptible to

an accumulation of hydrogen ions because the myocardium has a poor

31

intrinsic buffering capacity which enhances changes in pH. A lowered

pH might denature some of the enzymes of anaerobic glycolysis, or it

might facilitate the degradation of critical cofactors such as nicotine

adenine dinucleotide which becomes unstable in an acid environment (65).
Many studies have demonstrated that a marked reduction in high-

energy phosphate levels is accompanied by impaired cardiac function.

In other words, anaerobic glycolytic energy production eventually fails

to maintain the functional viability of the cell.

Cell Membrane Defects and Electrolyte Changes

It is well-known that cardiac ischemia results in pronounced and
stereotyped alterations of the myocardial electrolyte content.

Magnesium (Mg++) loss is the earliest and most persistent derangement.
This is followed by a reduction of inorganic phosphate (Pi)’ an accumu-
lation of sodium (Na+), and finally a loss of myocardial potassium (K+)
(108). The impairment of the electrolyte balance is believed to be an
acute effect of ATP depletion which results in a suppression of active
transmembrane transport and a generalized increase in membrane perme-
ability during ischemia (65).

Mitochondrial function and integrity are dependent upon Mg++.
Decreased oxidative activity and/or uncoupling oxidative phosphorylation
have been shown in the cardiac mitochondria of animals deficient in Mg++
(138). Welt and Tosteson (143) reported that Na+-KI-ATPase (membrane
adenosine triphosphatase) is Mg++-dependent and is involved in the
transport of Na+ and KT. They concluded that Mg++ is necessary for the

action of "pump" ATPase. Without optimal activity of this ATPase,

32

there is an apparent loss of the capacity to maintain an appropriate
concentration gradient across the cell membrane. This results in shifts
of K+ out of the cell and Na+ into the cell.

In a study of experimental myocardial ischemia in dogs, Jennings
and Shen (66) occluded the circumflex branch of the left coronary
artery for 40 min with a Goldblatt clamp. After blood flow had been
re-established fOr 20 min, they found that the damaged tissue exhibited

a 10-fold increase in calcium (Ca++) as well as increases in H 0 and

2
Na+. Tissue Mg++ and KI were decreased. In particular, the accumulated
Ca++ was located intramitochondrially. They suggested that two mechan-
isms may have been operative. The first possibility is that the irre-
versible ischemic injury may have caused the cell membranes of the
affected myocardial cells to become more permeable to Ca++. The second
is that ischemia may have induced a defect in the mitochondria of the
injured cells which lead to the excessive Ca++ accumulation. Either
mechanism could have been involved with or without the other, but the
results indicate both were operative. The effect of Ca++ accumulation
on mitochondrial function 1n_yjyg_remains to be established. However,
Chance (18) has shown that respiration is deficient in mitochondria
containing high Ca++ in.yitrg,

In another experimental study of enzymatic changes in acute myo-
cardial ischemia, Jennings gt_gl, (64) ligated the circumflex artery to
produce irreversible infarcts in the left posterior papillary muscles

of dogs. They found that glutamic oxaloacetate transaminase (GOT), a

small cytoplasmic enzyme, had totally diffused from the cell by one

33

hour after ligation. By two hours, even the large enzyme LDH was lost.
They also reported that there is good evidence to indicate that GOT and
LDH are not structurally altered by ischemia. That is, after their
release to the general circulation from the dead or dying fibers,
appreciable amounts of active enzymes were demonstrated in the serum.
The levels of enzyme activity in the serum were directly related to the
size of the myocardial infarcts produced. Thus, there must have been
defects of sufficient size in the semipermeable fiber membranes to
allow these enzymes to leak out.

de la Iglesia and Lumb (23) reduced the coronary flow in pigs by
20%. Their technique was to constrict the anterior descending branch
of the left coronary artery. Samples of myocardium taken from the left
ventricles of the animals showed important ultrastructure alterations
in the cell membranes. In addition, small vacuoles formed within one
hour, and large vacuoles appeared five hours after the reduction of
coronary flow. Although no descriptions of comparable vacuoles have
been found in the literature and no definite significance has been
attached to them, the authors claimed that these vacuoles appeared to

be intracellular in nature.

Mitochondrial Defects

Mitochondria in cardiac muscle are especially sensitive to ischemic
injury. Mitochondrial swelling has been observed following as little as
20 to 30 min of experimentally induced mild ischemia in both dogs and
rats (11,52). Although this early swelling may be reversible, later

changes observed in mitochondria probably are not reversible.

34

Studies have shown that severely ischemic cells develop striking
changes in mitochondrial structure and function at about the same time
that necrotic foci become manifest. These changes include severe swell-
ing associated with uncoupling oxidative phosphorylation (l7) and
increased fragility (63). Usually there is deposition of an intra-
matrical density having either (a) an amorphous or smudged appearance
or (b) a discrete microcrystalline appearance. The amorphous type of
dense body is believed to consist of lipid plus coagulated or denatured
mitochondrial protein (52,62). The microcrystalline granules represent
the accumulation of an as yet unidentified form of calcium phosphate
(66). The exact relationship between these two types of aberrations is
not clear. However, Jennings and Shen (66) found intramitochondrial
dense bodies in reperfused ischemic myocardium to be structurally dif-
ferent from those in myocardial cells irreversibly injured by permanent
ischemia. In the former case, the dense bodies were primarily micro-
crystalline; in the latter, they appeared to be mostly amorphous.

As mentioned previously, increased Ca++ accumulation may result
in depressed mitochondrial function. For example, respiration in yitrg_
is deficient in mitochondria containing high Ca++ (18). The mitochron-
drial production of high-energy phosphate compounds also is impaired if
the rate of removal of phosphate, as calcium phosphate granules, suffi-
ciently decreases the inorganic phosphate concentration (66).

The ischemia-induced defect in mitochondrial respiration generally
is believed to be structural since little protection is obtained by

adding various mitochondrial cofactors to the medium. In a study of

35

structural and functional abnormalities of mitochondria isolated from
ischemic dog myocardium, 60 min of ischemic conditions caused increased
mitochondrial fragility and inability to consume oxygen with pyruvate
as the oxidizable substrate (63). Use of C14-labelled pyruvate indi-
cated that although the pyruvate dehydrogenase complex appeared to be
intact, TCA cycle activity was disrupted (63). The uncoupling oxidative

phosphorylation may or may not be due to structural disorganization (65).

Endgplasmic Reticulum Alterations

Early membrane alterations during ischemic injury do not occur
only in the plasma membranes but also in the membranes of organelles.
In the dog and rat, dilation and vesiculation of the endoplasmic reticu-
lum take place within about 30 min of myocardial ischemia (11,52).
It is not known if these early alterations in the endoplasmic reticulum
are associated with any defect in the protein synthesizing capacity of

the myocardial cell.

CHAPTER III

METHODS AND MATERIALS

In classical exercise physiology, gross measurements of total-body
oxygen debt and oxygen uptake have been used to reflect metabolic re-
sponses to physical activity. Repeated bouts of exhaustive exercise
lead to an increased tolerance of oxygen debt which presumatfly reflects
a greater capacity for the generation of metabolic energy via anaerobic
metabolism. Training regimens based on this type of exercise commonly
are described as being "anaerobic" endurance programs and are character-
ized by maximal workloads and relatively short periods of activity.

In contrast, training of an "aerobic" endurance nature is thought to be
dependent chiefly upon oxidative muscle metabolism and tends to increase
total-body oxygen uptake capacity. Moderate or light workloads and
relatively long periods of activity are typical of "aerobic" endurance
programs. This study was designed to investigate cellular-level altera-
tions in the left ventricular myocardium of the white rat following

various durations of "aerobic“ and ''anaerobic" endurance training.

Sampling,Procedures
Eighty-four normal, male, albino rats of the Sprague-Dawley strain
were obtained from Hormone Assay, Inc., Chicago, Illinois. They were

received in two shipments of 72 and 12 animals respectively.

36

37

The animals were assigned randomly to three treatment groups and then
were given a period of 12 days to adjust to laboratory conditions
before the treatments were begun. The 72 animals in the first shipment
were treated for either eight or sixteen weeks; those in the second
shipment served as zero-week controls. Initiation of treatments for all

animals began at 84 days of age.

Research Design

 

The study was organized as a three-way (3x3x2) factorial design.
The first factor, Treatment, consisted of three levels of physical
activity. The following groups of animals were used to represent these
levels: (a) Short--an exercised group that was subjected to an
"anaerobic" endurance training program composed of high-intensity work-
loads maintained for short periods of time, (b) Long--an exercised
group that was subjected to an uaerobic" endurance training program
composed of low—intensity workloads maintained for long periods of time,
and (c) Control—«a nonexercised group that remained relatively inactive
throughout the experiment. The second factor, Treatment Duration, also
was represented by three groups of animals: (a) O-wk--a group that was
sacrificed after the 12-day adjustment period and thus received the
treatments for zero weeks, (b) 8-wk--a group that was sacrificed after
receiving the treatments for eight weeks, and (c) 16-wk--a group that
was sacrificed after receiving the treatments for sixteen weeks. The
third factor, Sacrifice Time, divided the animals assigned to each treat-

ment duration into two subgroups in order to distinguish immediate and

38

transitory reSponses to exercise from persistent training adaptations.
The two subgroups were: (a) lS-min--a subgroup that was sacrificed

15 min after the last exercise bouts of the two training programs were
completed, and (b) 72-hr--a subgroup that was sacrificed 72 hr after
the training programs were completed.

Anticipated limitations of time and personnel at sacrifice re-
stricted the research design to a final sample of n = 4 animals in each
of the 18 Treatment-Duration-Sacrifice Time cells (see Table 1). Since
one of the purposes of the study was to compare various parameters in
two groups of highly trained animals and a group of untrained animals,
three extra rats originally were assigned to each 8-wk and 16-wk cell
of the two exercised groups. Those animals with the best training per-
formances over each treatment duration then were selected for sacrifice.

The criteria used for this selection procedure will be described later.

Table l.--Experimental design with final cell frequencies.

 

 

Treatment Duration

 

O-weeks 8vweeks l6-weeks

 

 

 

Treatment Sacrifice Time SacrifiCe Time Sacrifice Time

 

 

 

15 min 72 hr 15 min 72 hr 15 min 72 hr

 

Control 4 4 4 4 4 4
Short 4 4 4 4 4 4
Long 4 4 4 4 4 4

 

39

Although six sacrifices of 12 animals are suggested by Table 1,
only five were necessary. Two sacrifices of 8-wk animals and two
sacrifices of 16-wk animals were conducted, but only one sacrifice of
O-wk animals followed the adjustment period. Because the O-wk animals
were to receive no training, it was determined that the same animals
could be used as both l5-min and 72-hr subjects. Therefore, to facili-
tate a balanced statistical analysis, each O-wk animal was assigned
randomly to one of the three treatment groups and then reassigned to a
second but different group. This technique reduced the number of ani-
mals needed while preserving the expected error variance in the six

O-wk cells.

Training Procedures

 

The exercise treatments were performed in a battery of individual
Controlled-Running Wheels (CRW) developed at the Human Energy Research
Laboratory, Michigan State University. The CRW apparatus can be
described as, ". . . a unique animal-powered wheel which is capable of
inducing small laboratory animals to participate in highly specific
programs of controlled, reproducible exercise“ (142). The animals learn
to run by avoidance-response operant conditioning. A low-intensity con-
trolled shock current provides motivation for the animals to run.

A typical training period consists of alternated work and rest
intervals. The wheel is braked automatically during all rest intervals
to prevent spontaneous activity. A specified number of work and rest

intervals constitutes one bout of exercise. A single training period

4o

usually includes several such bouts separated by a relatively long rest
time between bouts.

A light above the wheel signals the start of each work interval.
The animal is given a specified period of time (acceleration period) to
reach a preset running speed. If the animal does not reach the preset
speed by the end of the acceleration period, the light remains on and a
controlled shock current is applied through the running surface. As
soon as the animal attains the prescribed speed, the light is ex-
tinguished and the shock is discontinued. If the animal responds to the
light by running at or faster than the preset speed during the accelera-
tion period, the light is extinguished immediately and shock is avoided.
If the animal fails to maintain the prescribed speed while running, the
light-shock sequence is repeated.

The performance data for each animal are displayed in terms of the
total meters run (TMR) and the cumulative duration of shock (CDS). The
TMR and the total expected meters (TEM) are used to calculate the per-

centage of expected meters (PEM):
PEM = 100(TMR/TEM)

PEM values are the chief criterion used to evaluate and compare train-
ing performances. A secondary criterion is provided by the percentage
of shock-free time (PSF) which is calculated from the C05 and the total
work time (TWT):

PSF = 100 - 100(CDS/TWT)

41

TraininggPrggrams
In this study, all exercise treatments were administered five days
per week between 12:00 noon and 9:00 p.m. Body weights of the exercised
animals were recorded before and after each training period. The intens-
ity of the exercise was increased gradually during the first eight

weeks and then was held constant through sixteen weeks.

312::

The Short group was subjected to a high-intensity, short-duration
endurance program which was progressive in nature. That is, the intens-
ity of the program was increased gradually until, on the 37th day of
training and thereafter, the animals were expected to complete eight
bouts of exercise with 2.5 min of inactivity between bouts. Each bout
included six lO-sec work intervals alternated with 40-sec rest intervals.
During the work intervals, the animals were required to run at the rela-
tively fast speed of 99 m/min (see Table 2). A description of the

complete training program is given in Appendix A.

Long

The Long group was subjected to a low-intensity, long-duration
endurance program which also was progressive in nature. By the 37th
day of training, and thereafter, these animals were expected to complete
four bouts of exercise with 2.5 min of inactivity between bouts. Each
bout consisted of one 12.5-min continuous run at a speed of only 36
m/min (see Table 2). A description of the complete training program is

given in Appendix A.

42

 

 

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43

Animal Care

 

All of the animals were housed in individual sedentary cages
(24 cm x 18 cm x 18 cm) throughout the investigation. Since rats normal-
ly are more active at dusk and during the first few hours of darkness
than they are during daylight hours, the light sequence in the animal
quarters was automatically set to alter the rats' active period by
having the lights off between 1:00 p.m. and 1:00 a.m.

A relatively constant environment was maintained for the animals
by automatic temperature control. All animals were handled daily. The
cages were cleaned regularly. The animals had access to food (Wayne

Laboratory Blox) and water ag_libitum.

Sacrifice Procedures

Final body weights were recorded prior to sacrifice. Each animal
then was anesthetized by an intraperitoneal injection (4 mg/100 g body
weight) of a 6.48% sodium pentabarbital (Halatal) solution. The heart
was removed and washed free of blood with Ringer's solution. The great
vessels of the heart were trimmed. A dissection along the artrioventric-
ular groove was performed to separate the atria from the ventricles
which then were transversely sectioned approximately one-third of the
ventricular length from the apex. The three heart sections were blotted
dry and weighed. The apical portion of the ventricles was mounted, apex
up, on an Ames Lab-Tek Cryostat Chuck using 5% gum tragacanth. The pre-
pared chuck was held with forceps and lowered, for approximately 20

seconds, into 2-methylbutane (isopentane) precooled to a viscous fluid

44

(-l4O to -l85°C) by liquid nitrogen. This apical portion of the heart
was sectioned approximately 300p from the apex. Serial sections, lOu
thick, were cut on a microtome in a cryostat at a temperature of -20°C.
These sections were used to study the morphology and the relative con-
centrations of substrates and enzymes in the apical portion of the left
ventricle.

The upper half of the ventricles was fixed in a 10% formaldehyde
solution and dehydrated in graded concentrations of alcohol. Following
dehydration, it was embedded in paraffin and sectioned on a microtome.
Sections 8p thick were taken at four separate levels from this portion

of the heart for morphological study of the myocardium.

Histochemical Methods

The frozen sections from the apical portion of the heart were
used for histochemical evaluations. The presence of glycogen was
investigated with the Periodic Acid-Schiff (PAS) reaction (81, p. 132);
Fatty acids (FA) were demonstrated by Holczinger's technique (56); LDH
activity was studied using nitro blue tetrazolium (NBT) as the electron
acceptor and diphosphopyridine nucleotide (oxidized) as the cofactor
(lOO); SDH activity was examined by the method of Barka and Anderson
(4) with NBT as the electron acceptor. Incubation times were varied
according to the procedure. A fresh frozen hematoxylin-eosin (H & E)
technique (81, p. 29) also was used to examine the morphology of the
apical portion of the heart. Sections stained with H & E and PAS were

mounted in permount while other sections were mounted in glycerin jelly.

45

PAS-stained sections have been used to evaluate the glycogen con-
tent in both skeletal and cardiac muscle by several previous investiga-
tors (127,149,151). Holczinger (56) has reported that fatty acids may
be seen histochemically in fresh frozen tissue by the use of a copper
acetate incubating medium. The reaction was found to be quite sensitive
in that it demonstrates both saturated and unsaturated fatty acids in
the same manner. In a recent study of histochemical fiber typing in
cat and rat skeletal muscles, Schmalbruch and Kamieniecka (121) con-
cluded that LDH- and SDH-stained sections can be used to identify dif-
ferent fiber populations in the gastrocnemius and soleus muscles. The
methods employed to determine fiber types were: (a) subjective ratings
of projected color micrographs, and (b) measurements of the transmission

of light through cross-sections of individual fibers.

Histological Methods

 

The paraffin embedded sections from the upper half of the ven-_.
tricular portion of the heart were stained with H & E for the evaluation
of general cellular structure, modified Gomori's trichrome for collagen
(29), and Von Kossa's stain for the demonstration of calcification (81,

p. 152).

Tissue Analyses

 

An area of the left ventricle that could be located consistently
in each tissue section was needed to study the metabolic characteristics
of the left ventricular myocardium. Therefore, the constrictor band of

muscle (115) which corresponds closely to the cylindrical layer of muscle

46

in the left ventricle (132) was used in this investigation.

Histochemical Evaluations

Each histochemical stain was evaluated objectively with the use
of a Histochemical Photometer (HCP) at a magnification of 80X. The
operator of the HCP is able to center the photometric beam within the
cross-section of a single muscle fiber. The meter registers the percent-
age of light that is transmitted through the fiber on a scale from 0 to
100. Repeated measures of the same fibers on different days have shown
the average percent error for the HCP to be 10.3%. Thirty fibers1 were
selected at random from the constrictor band of muscle in the apical
portion of the heart for light transmission readings. That is, thirty
readings were taken of each stain for each animal. The percentage of
light transmitted was converted to percentage of light absorbed so that

higher HCP values reflect higher values of substrates and enzymes.

Pathology Evaluations
The heart sections stained with H & E, modified Gomori's trichrome,

and Von Kossa's stain were evaluated subjectively under a light

 

1In a previous study, a sample of 30 fibers was calculated to be
more than necessary and sufficient for a fourvway (7x4x8x10) mixedvmodel
nested analysis of variance that was run on HCP data when: (a) the
probability of making a type I statistical error was limited to the .01
level, (b) the probability of making a type II statistical error was
limited to the .05 level, (c) the minimal mean difference to be detected
as significant was set at 0.5 standard deviations, and (d) a moderate
variability between subgroup means was assumed. Consequently. standard
laboratory protocol now is to take readings on 30 fibers in each muscle
area of interest. This procedure is applied routinely to both skeletal
and cardiac muscle.

47

microscope. They were rated on a 0 to 4 scale (54) according to the
lesions exhibited. Both the severity and the number of lesions were
considered. A low rating (0 to l) was assigned to those sections with
no damage or only a few small foci of damage. A higher rating was used

to indicate more extensive pathological involvement.

Statistical Procedures

The body weights, the absolute and relative heart weights, and the
histochemical data were analyzed using a three-way (3x3x2) fixed-effects
analysis of variance routine on the Michigan State University Control
Data 6500 Computer (CDC 6500). Student-Newman-Kuels tests were used to
evaluate differences between pairs of means whenever a significant
F-ratio was obtained. Chi-square contingency tests were planned for
the pathology data, but an overall lack of lesions made the conduct of
these tests unnecessary. The 0.05 level of significance was established

for all statistical analyses.

CHAPTER IV

RESULTS AND DISCUSSION

The results of this study are presented in the following order:
training data from the controlled running wheel (CRW) programs, data on
body weight and morphological changes of the heart, data on histochem-
ical observations of the heart, and finally data on histopathological
observations of the heart. The LDH results provided the only signifi-
cant F-ratios for sacrifice time. That is, similar mean values were
obtained for the 15-min and 72-hr sacrifices in all other dependent
variables. Therefore, with the exception of the LDH values, the data
were pooled across sacrifice time and the final results are presented
only by treatment, duration, and treatment x duration. The F-ratios
and P values for all effects, including sacrifice time, are summarized
in Appendix B. The raw data may be found in Appendix C. An overall
discussion of the more important findings is presented at the end of

this chapter.

Training Results
For both CRW training groups, a criterion of 75 percent of expect-
ed meters (PEM) was set as the minimum acceptable performance level.
The training results for the Short group are shown in Figure 2. The

data indicate that these animals maintained a PEM of approximately 80

48

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50

during the later stages of training when running velocities of 90 and
99 m/min were required. A search of the literature has revealed no
other programs for the training of laboratory rats which have incorpor-
ated such high running velocities. Percent shock-free time (PSF) values
were approximately 85 to 90 during the more strenuous portions of the
training program. These results show that the animals generally re-
sponded to the conditioned light stimuli rather than to the uncondi-
tioned shock stimuli.

The training data for the Long program are presented in Figure 3.
PEM values were approximately 85 for the last fourteen weeks of training.
A running velocity of 36 m/min was required throughout this period. The
PSF values were in the range of 85 to 95 during the last eleven weeks of
training. These results again provide evidence that the animals re-

sponded well to the training program.

Body Weight and Heart Size Results
The final results for body weight, total heart weight, ventricular
weight, atrial weight, total heart weight/body weight, ventricular
weight/body weight, and atrial weight/body weight are shown in Figures 4
through 10. These results are summarized here in terms of treatment,

duration, and treatment x duration effects.

Treatment Effects

 

Overall, the Control animals weighed significantly more than did
either the Short or the Long animals (Figure 4). There were no important

differences in the three measures of absolute heart size (Figures 5

51

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53

through 7); but the two exercised groups had larger than normal ratios
of total heart weight/body weight (Figure 8), ventricular weight/body
weight (Figure 9), and atrial weight/body weight (Figure 10). These
significant contrasts in relative heart size may reflect differences in

cardiac function which favored the trained animals.

Duration Effects

The data on body weight (Figure 4), total heart weight (Figure 5),
and ventricular weight (Figure 6) show that there was continuing growth
across all groups of animals between the O-wk and the l6-wk sacrifices.
It is of interest to note that the atrial weight at 8 weeks was signifi-
cantly less than it was at either 0 weeks or 16 weeks (Figure 7). Since
no explanation can be offered for this decrease in absolute atrial
weight at 8 weeks, the possibility of a measurement artifact cannot be
ruled out.

The data on relative heart size show that the O-wk animals had
significantly greater values than did either the 8-wk or the l6-wk
animals (Figures 8 through 10). This age-related effect was anticipated
(7,13,136).

Treatment 5.0uration Effects

As expected, there were no significant differences in body weight,
absolute heart size, or relative heart size among the treatment groups
at 0 weeks (Figures 4 through 10). However, the Control animals
weighed significantly more than did the exercised animals at both 8 and

16 weeks (Figure 4).

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Significant increases in the relative heart and ventricular sizes
of the Short and Long groups were found after 8 and l6 weeks of treat-
ment (Figures 8 and 9). The atrial weight/body weight ratio of the
Short group was significantly higher than that of the Control group at
l6 weeks (Figure l0). The implications of these findings will be dis-

cussed later.

Histochemical Results
The final histochemical photometer results for SDH, FA, PAS, and
LDH are shown in Figures ll through 14. These results are summarized
here in terms of main and interaction effects. They will be discussed

in the final section of this chapter.

Treatment Effects

The exercised animals, in both the Short and Long groups, had
significantly higher overall SDH values (percent light absorbed) than
did the CON animals (Figure ll). There were no significant treatment

effects for FA, PAS, or LDH (Figures l2 through 14).

Duration Effects

There were significant declines in the SDH, PAS, and LDH values
between the O-wk and 8-wk sacrifices. These initial decreases were
followed by marked increases at 16 weeks (Figures ll, l3, and T4). The
FA values had a different pattern over time with a temporary nonsignifi-

cant rise at 8 weeks (Figure 12).

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64

Lactate Dehydragenase (“lo L19“ Absorbed)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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W Mia! Mb) Tatum : Nation
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70
.l.
J- I-‘fl-h ‘- i-Iun-b noun-- baton- 1 Iain- Cullen-u nun-u
an art Lara a-n a-wu 1e-w11
Sum“ Tmi I Sacrifice Mien x Saaritice
rhyme-0111 04111 72-1:>e-w11 15-00

M IQ-min > IB-Wl 72-111

Fa .0 m
of.

ion-1 73-. um- n-o 10.00 71- 0-11— n-~ 1301111 n-ur 15-00 n-u 10011- 11.. 13010 n-u 0-1-1 71-1.
M SIT can SHT LON can S"? La!
O-H O-Ul IO-W
Tum-11 3 Duration 11 Sacrifice

e-aau ‘12-" >8-OON 15-00 16-5141' Ifmmn >t6-SH1’ TZ-tv
O-Q't‘l’ 724': > O-SHT 1.5-111111 IG-W t5-min>t6-Lm 7247
CW 72-111 > 84.01 15-0111

00M IS-min > B-SHT 113-00 > 04.01! tS-min

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

fig t4 Means md standad errors at the mean for lactate dehydrogenase (‘7. tight
(Method) by treatment. duratton, socr1t1ce. treatment 11 sacrifice. duration 11
sacrifice, treatment 11 durahon, treatment 1: duration 11 sacntice wtth
Stqntftcont (p < .05) SNK contrasts Show be'ow the figures.

65

Treatment §_Duratian Effects

The SDH value of the Short group was significantly higher than
that of the Control group at l6 weeks (Figure ll). The LDH value of
the Control group was significantly higher than those of the Short and
Lang groups at 8 weeks (Figure l4).

Sacrifice Effects

A comparison of the data for the lS-min and 72-hr sacrifices
shows that the 72-hr animals had significantly higher LDH values than
did the lS-min animals (Figure 14).

Duration §_Sacrifice Effects
At 8 weeks, the 72-hr animals had significantly higher LDH values
than did the l5-min animals, whereas the situation was reversed at 16

weeks (Figure.l4).

Treatment §_Duratian §_Sacrifice Effects

The 8-wk LDH values for all three treatment groups were signifi-
cantly higher in the 72-hr animals than they were in the lS-min animals
(Figure 14). There were no significant differences between the three
groups of 72-hr animals at 8 weeks; however, the lS-min Control value
was higher than the lS-min Short value which, in turn, was higher than
the lS-min Lang value.

By l5 weeks. the LDH pattern had reversed in the Short and Lang
groups. The l5-min values were higher than the 72-hr values in these

two groups of animals. There were no significant differences within

66

the Control group, between the three groups of lS-min animals, or be-

tween the three 72-hr groups.

Histgpathological Results

Cross-sections from five separate levels of each heart were evalu-
ated by microscopic examination. Heart damage was rated according to
the subjective scale previously described (54). Only three of the
seventy—two animals had focal areas of inflammatory reaction. Lympho-
cytes and other mononuclear cells were observed in the heart sections of
those three animals. The results of the histopathological observations
are presented in Appendix C. The coronary arteries and the intramyo-
cardial vessels that were observed appeared to be free aflesions.
There was no abnormal thickening of the vessel walls. Thus. the histo-
pathological observations suggested that a condition of normalcy existed

in the hearts of the animals in all comparison groups.

Discussion

 

In accord with the results of other studies (7,38,l29,134,l36,137),
both the Short and Long groups of exercised animals gained significantly
less body weight than did the sedentary controls (Figure 4). Although
there was continuing growth in total heart weight throughout the experi«
mental period, there were no significant differences between treatment
groups at different durations (Figure 5). Thus, the significant in.
creases in the heart weight/body weight ratios of the Short and Long
groups (Figure 8) can be attributed to the lower body weights of the

67

exercised animals. In spite of this simplistic explanation, it is

clear that, as compared to the Control animals, the trained animals had
relatively larger hearts to support relatively less body mass (Figures

8, 9, 10). If these anatomical data reflect differences in physiological
measures (e.g., stroke volume, oxygen pulse, and maximum oxygen uptake),
the capacity for increased cardiac function must have been much greater
in the trained animals than it was in the sedentary animals. Unfortu-
nately, direct evidence on small laboratory animals has not been found
in the literature to either corroborate or refute this hypothesis.

The data of Bloor and Leon (7) show that cardiac enlargement
results from exercise in young rats, whereas exercise in the old rat
causes a decline in heart weight due to a loss of myocardial fibers and
a decrease in fiber mass. The age-dependent response of the rat heart
to exercise also was demonstrated by Tomanek and his associates (134).
In a discussion of the regulation of organ and tissue growth, Burton
(13) stated that there are periods of exponential growth and regulated
growth in the rat heart. The early growth period, named for its char-
acteristic exponential nature, begins immediately following birth and
continues for approximately 45 days. At this time, the rate of growth
slows as the regulated phase begins and continues up to approximately
480 days of age.

The animals used in this study were 84 days old (postpubertal) at
the beginning of the treatments. According to the information supplied
by other investigators, the lack of a significant increase in absolute

heart weight with the exercise treatments should not be surprising (136).

68

Furthermore, the cellular mechanism of exercise-induced cardiac enlarge-
ment has not been determined. Myocardial fiber hyperplasia, fiber
hypertrophy, or both, may occur in the cardiac muscle of the exercised
animal. Evidence indicates that myocardial fiber hyperplasia does occur
for at least a short time beyond the immediate postnatal period in both
the rat and the human (118,123). Maintenance of a normal fiber diameter
is important to insure that the diffusion distance from surrounding
capillaries to central myofibrils is not increased. Therefore, hyper-
plasia and fiber lengthening have been postulated to be more advantageous
than fiber thickening (35). Using different ages of male Sprague-Dawley
rats, Tomanek (133) reported that the hearts of trained animals had no
greater myocardial fiber diameters than did those of the controls, but
the capillary/muscle fiber ratios were significantly increased in the
trained groups at all ages. The data from this study do not provide
sufficient information to speculate as to the mechanisms of the in-
creased heart weight/body weight ratios that were observed. Neverthe-
less, the results would suggest the existence of a great reserve in
cardiac work load per unit of total body mass in the exercised animals.
There are no apparent explanations for the significant decrease
in atrial weight across all treatment groups at 8 weeks (Figure 10).
However, it is of interest to note that the Short animals had signifi-
cantly greater overall atrial weight/body weight ratios than did either
the Control~or the Long animals. Valdivia (135) exposed guinea pigs to
simulated altitudes of 5500 m for various time periods up to 28 weeks.

He reported that the exposed animals developed marked hypertrophy and

69

dilatation of the right atrium. Under the hypoxic conditions described,
pulmonary artery hypertension appeared to be the etiologic factor.

It has been demonstrated in both animal subjects (27,28) and
human subjects (25,88) that acute hypoxia induces considerable pul-
monary hypertension. Although the mechanism of the hypoxic effect on
pulmonary circulation has not been identified, several authors agree
that the increase in pulmonary artery pressure is due to direct action
of hypoxia on the walls of the pulmonary vessels without intervention
of the autonomic nervous system (25,27,28,88). In normal human sub-
jects, this pulmonary vasoconstriction probably is due to low oxygen
tension in the blood acting directly on the walls of the pulmonary
arterioles (25). However, a similar vasoconstriction can be induced by
changes of oxygen tension in the alveoli (27).

Comparable hypoxic conditions might exist during some very strenu-
ous sport events. It has been reported that there is a marked reduction
of oxygen tension in the systemic venous blood during maximal exercise
despite a normal or even elevated partial oxygen pressure in the lung
alveoli (2). Of course, for this lowered oxygen tension in the blood
to cause pulmonary vasoconstriction, one would have to assume inadequate
compensation via the usual exercise-induced vasodilator activity of the
autonomic nervous system. At present, there is no evidence to evaluate
the relative effects of these two opposing factors during strenuous
work.

In the maximal effort of sprint running, such as that performed

by the Short animals in this study, deep breathing is impaired.

70

Rats may even hold their breath with the glottis closed as do human
sprinters. If so, the increased intrathoracic pressure would have
acted in conjunction with any hypoxic pulmonary hypertension to greatly
increase the work load of the right heart in the Short animals.
In fact, recent data obtained by the corrosion0cast technique show that,
in the same age and strain of rats, animals subjected to a repetitive
program of high-intensity weight lifting have significantly higher
right/left ratios for coronary cast weights than do either Control or
Long animals (55).

In contrast to the results obtained by other investigators (51,
114), the histochemical data of this study show that SDH values were
significantly increased in the hearts of both the Short and the Long
animals (Figure 11). It should be noted that the training programs
employed in the current study were either higher in intensity or longer
in duration than those used in the previous investigations. It is pos-
sible that the normally high oxidative capacity of the heart was not
challenged by the exercise stresses imposed in earlier studies and/or
that the duration of training must be extended for an adequate adjust-
ment to take place in the heart. The duration and treatment x duration
contrasts shown in Figure 11 appear to support the latter hypothesis.
That is, the data suggest that there may have been an initial age-
related decline in SDH values which was not counteracted by training
until after the 8-wk sacrifice had been completed.

The fact that LDH did not show a training effect was surprising
(Figure 14). Both SDH and LDH are well-recognized aerobic enzymes in

71

the heart, and increased values were expected. Using a biochemical
assay, Gollnick and associates (37,38) found significantly increased
LDH activity in the hearts of Sprague-Dawley rats after 60 minutes of
swimming for 35 consecutive days. Their animals were sacrificed on
the same day as the final exercise period. However, the effect was
attributed to training since a 30-min bout of swimming immediately
prior to sacrifice was shown not to alter enzymatic activity in the
hearts of either trained or untrained animals.

Several investigators have claimed that lactate is the preferen-
tial energy-supplying substrate for the heart muscle when work intensity
is high (68,72,92). In spite of a lower arterial level in trained sub-
jects, more lactate is extracted by the trained heart than by the un-
trained heart (72,92). Certainly there is little doubt concerning the
availability of arterial lactate to the heart during and immediately
following exercise. The highest arterial lactate concentrations are
found after exercise. It is difficult to state exactly when peak
values are reached since this is dependent on the duration and intensity
of the preceding work. However, the lactate level in the 10th minute
~after exercise already is reduced, and resting values are reached in
trained athletes 30 to 60 minutes after exercise at maximal work loads
(73). Considering the general availability of lactate and its rapid
return to resting levels, there does not appear to be a ready explana-
tion for the disparity between the LDH results of previous investiga-

tions (37,38) and those of the current study.

72

An interesting finding was that at 8 weeks the 72-hr animals in
all treatment groups had significantly higher LDH values than did the
corresponding 15-min animals (Figure 14). Among the lS-min animals,
the Control group had a significantly higher mean value than did either
the Short or the Long group. Except for the slightly depressed Control
group value at 15 min, these results might be an indication of temporary
enzymatic exhaustion or depletion of LDH. Furthermore, SDH, PAS, and
LDH each showed a significant decline at 8 weeks and then an increase at
16 weeks (Figures 11, 13, 14). Both trained groups had higher terminal
SDH values than did the Control group (Figure 11), and the 15-min values
of LDH in the trained groups were significantly higher than the 72-hr
values at 16 weeks (Figure 14). Taken together, these data suggest that
the hearts of the trained animals were subjected to a period of metabolic
adjustment and that they gradually adapted to the physical stresses
imposed by the exercise programs. The net result was a marked increase
in metabolic oxidative capacity.

Further evidence of an aerobic adaptation may be found in the
data on fatty acids (FA). There was a trend for FA to increase in the
exercised animals at both 8 and 16 weeks (Figure 12). This is in agree-
ment with the results of previous investigations (33,72,92,120) in
which pronounced utilization of lipids was found to occur in the hearts
of well-conditioned subjects. However, the PAS results failed to show
the increase in glycogen storage that has been reported by others (26,
103,120,124). Opie (95) has stated that there are very complex control

mechanisms for the synthesis and degradation of cardiac glycogen.

73

A high glycogen level can be associated with a low turnover rate and
vice versa. Therefore, the meaning of these observations is obscure.

At the beginning of this study, it was hypothesized that as the
type of exercise progresses from the short-duration, high-intensity end
of the physical activity continuum toward the long-duration, low-
intensity end, the metabolic responses of the myocardium might be a1-
tered from those of aerobiosis with partial anaerobiosis to those of
total aerobiosis. The histochemical data of this study do not support
that hypothesis since there is no evidence to indicate that the Short
and Long animals had different myocardial metabolic characteristics.

Recently, Lehninger (77, pp. 838-839) has taken the position that
the heart muscle has a completely aerobic metabolism under resting or
moderately active conditions and uses glycolysis as a source of extra
energy only in an emergency. This conclusion is based upon several sets
of observations:

1. Very little lactate is formed from pyruvate in the hearts of resting
animals. Fatty acids contribute 70% of the fuel source and glucose
the remainder.

2. Twelve seconds after the onset of a heavy work load, the contribu-
tion of glucose from the blood and glycogen rises to 82%, the rate
of breakdown of glucose 6-phosphate is increased over thirtyfold,
and 80% of the pyruvate formed from glycogen is reduced to lactate
which appears in the blood.

3. The supply of glycogen in the heart is limited and can suffice for

only a short period of transition from the resting to the working

74

state. Within 2 min after the imposition of a heavy work load, the
heart readjusts its metabolism. There is a large increase in the
amount of FA oxidized and a decrease in the rate of glucose utiliza-
tion.

The cells of heart muscle are extremely rich in mitochondria which
make up 40% or more of the cytoplasmic space (128). This abundance of
mitochondria, plus a sufficient coronary blood supply under even extreme
physiological conditions, tend to insure cardiac aerobiosis. With all
of these facts in mind, it is clear that unless a very specific
“anaerobic“ training regimen is employed, it will be difficult to alter
the basic aerobic metabolism of the heart. It is likely that the Short
and Long training programs used in this study were not sufficiently
divergent in nature to produce differentiated metabolic responses in
the resting heart. Certainly the Short program cannot be c1as$ified as
strictly “anaerobic" exercise. In fact, biochemical data on skeletal
muscles from the same animals failed to reflect differences in metabolic

activities between the Short and Long groups (53).

CHAPTER V

CONCLUSIONS AND RECOMMENDATIONS

The purpose of this study was to determine the effects of two
programs of endurance running on selected morphologic, metabolic, and
histopathologic parameters in the hearts of adult male albino rats.

The training regimens were the Short and Long Controlled-Running Wheel
programs previously reported from this laboratory (142).

Animals were assigned randomly to Control, Short, and Long treat-
ment groups. The treatments were initiated when the animals were 84
days of age. Animals were sacrificed at the beginning of the study
(0 weeks) and after 8 and 16 weeks of treatments. At the 8-wk and 16-wk
treatment durations, sacrifices were conducted 15 minutes and 72 hours
following the last exercise periods of the Short and Long animals. The
final sample consisted of 72 animals with four animals in each treatment-
duration-sacrifice subgroup.

Absolute and relative heart weights, ventricular weights, and
atrial weights were determined for gross morphological observations of
heart size. Four histochemical procedures were used to evaluate the
relative glycogen, fatty acids, succinate dehydrogenase, and lactate de-
hydrogenase values of cardiac fibers in the apical portion of the left

ventricle. Histochemical stain intensities were measured objectively

75

76

using a Histochemical Photometer. Histological cross-sections at five
separate levels in each heart were examined by light microscope for
indications of lesions.

The results showed that there were significant increases in the
mean heart weight/body weight ratios of the Short and Long groups after
8 and 16 weeks of treatments. However, the increases were due primarily
to relatively low body weights in the trained animals rather than to
dramatic increases in absolute heart weights.

The results also suggest that increased relative heart size is an
age-dependent response of the rat heart to exercise. This observation
is supported by the fact that at the beginning of the treatments the
animals were 84 days old (postpubertal), an age at which the growth
rate of the heart has slowed and has entered a regulated phase.
Furthermore, although there was continuing growth in total heart weight
throughout the experimental period, there were no significant differ-
ences between treatment groups at different durations.

The histochemical data suggest that, over time, the two training
programs tend to produce similar metabolic adaptations in cardiac
muscle. Evidence for this observation is provided by the significant
succinate dehydrogenase increases that occurred in both the Short and
Long groups throughout the experimental period. The lactate dehydro-
genase values of the 72-hr animals were significantly higher than those
of the lS-min animals in both trained groups at 8 weeks; whereas, at
16 weeks, the situation was reversed and the 15-min values were higher

than the 72-hr values in both groups. Further evidence is found in the

77

corresponding nonsignificant fatty acid increases that were seen in the
two exercised groups. There were no significant Short versus Long con-
trasts at any time. Therefore, the results of this study strongly
indicate that increased aerobic metabolic capacity was developed in the
hearts of both the Short and Long animals.

In addition to the increased aerobic capacity of the trained
animals, the values of succinate dehydrogenase, periodic acid—Schiff,
and lactate dehydrogenase each showed a significant decline at 8 weeks
and then an increase at 16 weeks. Both trained groups had higher
terminal succinate dehydrogenase values than did the Control group, and
the lS-min values of lactate dehydrogenase in the trained groups were
significantly higher than the 72-hr values at 16 weeks. Together,
these data suggest that the trained hearts were subjected to a period
of metabolic adjustment and that they gradually adapted to the physical
stresses imposed by the exercise programs. Therefore, the duration of
training must be quite extended for adequate adjustment to take place
in the heart.

The results of the histopathological observations showed the
hearts of the animals in all three treatment groups to be essentially
normal. Little evidence of lesions was seen, and none of that could be

attributed to the training regimens.

Conclusions

 

The data from this study can be generalized only to adult male

rats of the Sprague-Dawley strain. Furthermore, the training effects

78

undoubtedly are specific to the Short and Long Controlled-Running Wheel

programs that were used in the investigation. With these limitations

in mind, the following conclusions appear to be warranted:

1.

At both 8 and 16 weeks of training, the two exercise regimens pro-
duce similar increases in the heart weight/body weight ratio.

These significant increases in proportional heart size are caused
by the fact that the trained animal gains less body weight over
time than does the sedentary animal; they cannot be attributed to a
larger than normal gain in the absolute heart weight of the trained
animal. Regardless of the cause, the net effect is that the
trained animal has a relatively large heart mass to support a rela-
tively small body mass.

The exercise-induced increase in relative heart size appears to be
dependent upon age.

The changes in metabolic activity of cardiac muscle that are pro-
duced by the Short and Long training programs cannot be differen-
tiated by histochemical determinations of succinate dehydrogenase,
lactate dehydrogenase, glycogen, or fatty acids.

Both training programs produce an increase in the aerobic metabolic
capacity of the myocardium.

Although the Short training program originally was conceived as
being largely dependent upon anaerobic metabolic processes, its
primary effect in the heart is to stimulate aerobic metabolism.

A l6-week period of training with the Short or Long Controlled-

Running Wheel programs is adequate for metabolic adjustments of the

79

heart to be detected by histochemical techniques.
Neither the Short nor the Long training program, as presently con-
structed, produce significant lesions in the heart muscle of the

rat when they are applied for periods of up to 16 weeks.

Recommendations

 

High-intensity exercise regimens for animals should be developed to
facilitate study of the adaptations to anaerobic training.
Power-type events, such as high-jumping and weight—lifting, are
needed for animals so that resultant adaptations can be compared
with those resulting from activities across the endurance continuum.
A quantitative biochemical analysis of the myocardium should accom-
pany any further histochemical research of this nature.

A study is needed to determine the effects of different exercise
regimens on coronary circulation at both the arterial and capillary
levels.

A study is needed to determine the effects of different exercise

regimens on the metabolic responses of the heart to hypoxia.

10.

11.

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APPENDICES

APPENDIX A
TRAINING PROGRAMS

93

94

APPENDIX A

TRAINING PROGRAMS

Standard Bight-Week. Short-Duration, High-Intensity
Endurance Training Program for Postpubertal and
Adult Hale Rats in Controlled-Running wheels

 

Total
Acc- Time Time Total
eler— Wbrk Repeti- Bet— of Total Work
Day Day ation Time Rest tions No. ween Run Prog. Ex . Time
of of Time (min: Time pct of Bouts Shock Speed (min: Hegers (sec)
Hk.‘ Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) (m/min) sec)- TEH TNT
0 4dr -2 3.0 40:00 10 l 1 5.0 0.0 27 40:00 --- --
5-2 -1 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- --—
1 l-H 1 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200
2.? 2 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200
3'" 3 3.0 00:10 10 4O 3 5.0 1.2 27 49:30 540 1200
4-T 4 2.5 00:10 10 40 3 5.0 1.2 36 49:30 720 1200
s-r 5 2.0 00:10 10 40 3 5.0 1.2 36 49:30 720 1200
2 1-M 6 1.5 00:10 10 28 4 5.0 1.2 45 51:40 840 1120
2-T 7 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
3." 8 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
4-T 9 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
S-P 10 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
3 1-H 11 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
2'? 12 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
3-w 13 1.5 00:10 20 23 4 5.0 1. 2 63 59140 966 920
4-T 14 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
S-P 15 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
4 1-H )6 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
2'? 17 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
3-w 18 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
4-T 19 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
SI? 20 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
S l-M 21 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
2-T 22 1.5 00:10 30 ,16 4 5.0 1.0 81 55:40 864 640
3-H 23 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
4-T 24 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
S-P 25 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
6 l-M 26 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
2-T 27 2.0 00:10 35 10 5 5.0 1.0 90 54:35 750 500
3t" 28 2.0 00:10 ' 35 10 5 5.0 1.0 90 54:35 750 500
4-T 29 2.0 00:10 35 10 5 5.0 1,0 90 54:35 750 500
Set 30 2.0 00:10 35 10 S 5.0 1.0 90 54:35 750 500
7 l-H 31 2.0 00:10 35 10 S 5.0 1.0 90 54:35 750 500
2'? 32 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
3d" 33 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
4d? 34 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
S-P 35 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
8 1-H 36 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
247 37 2.0 00:10 40 6 8 2.5 1.0 99 52.10 792 480
3dfl 38 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480
4-T 39 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480
Sn? 40 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480

 

This standard program was designed using male rats of the Sprague-Dawley strain. All
animals were between 70 and 170 days-of-age at the beginning of the program. The duration and
intensity of the program were established so that 75 per cent of all such animals should have P3P
and PER scores of 75 or higher during the final two weeks. Alterations in the work time, number
of bouts, or time between bouts can be used to affect changes in these values. Other strains or
ages of animals could be expected to respond differently to the program.

All animals should be exposed to a minimum of one week of voluntary running in a wheel
prior to the start of the program. Failure to provide this adjustment period will impose a double
learning situation on the animals and will seriously impair the effectiveness of the training
programs.

95

Standard Eight-week. Long-Duration, Low—Intensity
Endurance Training Program for Postpubertal and
Adult Hale Rats in Controlled-Running Wheels

 

 

Total

Acc- Time Time Total

eler- work Repeti- Bet- of Total Work

Day Day ation Time Rest tions No. ween Run Prog. Exp. Time
of of Time (min: Time per of 80uts Shock Speed (min: Meters (sec)

Wk. "k- Tr. (see) sec) (sec) Bout Bouts (min) (ma) (m/min) sec) TEN Twr

0 4-T -2 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- ---

s-r -1 3.0 40:00 10 1 1 5.0 0.0 27 40:00 --- ---

1 1-H 1 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540- 1200
2dT 2 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200

3-fl 3 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200

4'? 4_ 2.5 00:20 10 30 2 5.0 1.2 27 34:40 540 1200

S-r 5 2.5 00:30 15 20 2 5.0 1.2 27 34:30 540 1200

2 1'" 6 2.0 00:40 20 15 2 5.0 1.2 36 34:20 720 1200
2~T 7 2.0 00:50 25 12 2 5.0 1.2 36 34:10 720 1200

3d" 8 1.5 01:00 30 10 2 5.0 1.2 36 34:00 720 1200

4-T 9 1.5 02:30 60 4 2 5.0 1.2 36 31:00 720 1200

SI? 10 1.0 02:30 60 4 2 5.0 1.2 36 31:00 720 1200

3 1'! 11 1.0 02:30 60 4 2 5.0 1.2 36 31:00 720 1200
2~T 12 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500

3-H 13 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500
4iT 14 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500

s-r 15 1.0 05:00 0 1 s 2.5 1.2 36 35:00 900 1500

4 l-M 16 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500
2dT 17 1.0 07:30 0 l 4 2.5 1.0 36 37:30 1080 1800

3-H 18 1.0 07:30 0 1 4 2.5 1.0 36 7:30 1080 1800
4dr 19 1.0 07:30 0 1 4 2.5 1.0 36 37:30 1080 1800
S-r 20 1.0 07:30 0 1 4 2.5 1.0 36 37:30 1080 1800

5 1-1: 21 1.0 07:30 0 1 4 2. s 1.0 36 37:30 1080 1800
2-T 22 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1350 2250
3-W 23 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1350 2250
4-T 24 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1350 2250
S-F 25 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1350 2250

6 1-H 26 1.0 07:30 0 1 S 2.5 1.0 36 47:30 1350 2250
2-T 27 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1440 2400
3-H 28 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1440 2400
4-T 29 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1440 2400
5-2 30 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1440 2400

7 1-H 31 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1440 2400
2uT 32 1.0 10:00 0 l S 2.5 1.0. 36 60:00 1800 3000
3‘“ 33 1.0 10:00 0 1 5 2.5 1.0 36 60:00 1800 3000
48T 34 1.0 10:00 0 1 S 2.5 1.0 36 60:00 1800 3000
S-P 35 1.0 10:00 0 1 5 2.5 1.0 36 60:00 1800 3000

8 1!“ 36 1.0 10:00 0 l 5 2.5 1.0 36 60:00 1800 3000
2-T 37 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000
3dfl 38 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000
4dr 39 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000
s-r 40 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000

 

This standard program was designed using male rats of the Sprague-Dawley strain. All
animals were between 70 and 170 days-of-age at the beginning of the program. The duration and
intensity of the program were established so that 75 per cent of all such animals should have PSF
and PER scores of 75 or higher during the final two weeks. Alterations in the rest time,
repetitions per bout. number of bouts, or time between bouts can be used to affect changes in
these values. Other strains or ages of animals could be expected to respond differently to the
program.

All animals should be exposed to a minimum of one week of voluntary running in a wheel
prior to the start of the program. Failure to provide this adjustment period will impose a
double learning situation on the animals and will seriously impair the effectiveness of the
training program.

APPENDIX B
F-RATIOS AND P-VALUES FOR ALL EFFECTS

96

97

APPENDIX B--F-Ratios and P-Values for Body Weight.

 

 

 

 

 

 

 

 

 

 

 

Effect F-Ratio P
Treatment 48.7815 <0.0005
Duration 147.8279 <0.0005
Sacrifice Time .2475 .621
Treatment x Duration 8.2605 <0.0005
Treatment x Sacrifice Time .3511 .706
Duration x Sacrifice Time 1.2111 .306
Treatment x Duration x Sacrifice Time 2.8689 .062
APPENDIX B--F-Ratios and P-Values for Total Heart Weight

Effect F-Ratio P
Treatment .7597 .473
Duration 56.6863 <0.0005
Sacrifice Time .3882 .536
Treatment x Duration .5445 .697
Treatment x Sacrifice Time .4914 .614
Duration x Sacrifice Time .5578 .576
Treatment x Duration x Sacrifice Time .9082 .466
APPENDIX B--F-Ratios and P-Values for Ventricular Weight

Effect F-Ratio P
Treatment 1.0293 .364
Duration 64.0966 <0.0005
Sacrifice Time .3140 .578
Treatment x Duration .4647 .761
Treatment x Sacrifice Time .8053 .452
Duration x Sacrifice Time .8955 .414
Treatment x Duration x Sacrifice Time 1.2619 .296

 

98

APPENDIX B--F-Ratios and P-Values for Atrial Weight.

 

 

 

Effect F-Ratio P
Treatment 1.6066 .201
Duration 19.8623 <0.0005
Sacrifice Time .3805 .540
Treatment x Duration 1.6194 .183
Treatment x Sacrifice Time 1.0682 .351
Duration x Sacrifice Time .5255 .594
Treatment x Duration x Sacrifice Time 2.3316 .067

 

APPENDIX B--F-Ratios and P-Values for Total Heart Weight/Body Weight.

 

 

 

Effect F-Ratio P

Treatment 22.8623 <0.0005
Duration 15.4894 <0.0005
Sacrifice Time .0210 .885
Treatment x Duration 5.9701 <0.0005
Treatment x Sacrifice Time .8895 .417
Duration x Sacrifice Time .0055 .994
Treatment x Duration x Sacrifice Time .6648 .619

 

APPENDIX B--F-Ratios and P-Values for Ventricular Weight/Body Weight.

 

 

 

Effect F-Ratio P
Treatment 22.3468 <0.0005
Duration 7.6310 .001
Sacrifice Time .0022 .963
Treatment x Duration 4.9869 .002
Treatment x Sacrifice Time .9269 .402
Duration x Sacrifice Time .0385 .962

Treatment x Duration x Sacrifice Time 1.0807 .375

 

99

APPENDIX B--F-Ratios and P-Values for Atrial Weight/Body Weight.

 

 

 

 

 

 

 

 

 

 

 

Effect F-Ratio P
Treatment 4.6884 .013
Duration 38.3905 <0.0005
Sacrifice Time .1942 .661
Treatment x Duration 3.4041 .015
Treatment x Sacrifice Time 1.6904 .194
Duration x Sacrifice Time .7981 .455
Treatment x Duration x Sacrifice Time 2.4985 .053
APPENDIX B--F-Ratios and P-Values for SDH.

-Effect » F-Ratio P
Treatment 4.2564 .019
Duration 7.9315 .001
Sacrifice Time .7746 .383
Treatment x Duration 2.7905 .035
Treatment x Sacrifice Time 2.4658 .094
Duration x Sacrifice Time 1.3549 .267
Treatment x Duration x Sacrifice Time .5964 .667
APPENDIX B--F-Ratios and P-Values for FA.

Effect F-Ratio P
Treatment 1.2705 .289
Duration , .3928 .677
Sacrifice Time .3700 .546
Treatment x Duration .9747 .429
Treatment x Sacrifice Time .1885 .829
Duration x Sacrifice Time 2.1245 .129
Treatment x Duration x Sacrifice Time 1.2458 .303

 

100

APPENDIX B--F-Ratios and P-Values for PAS.

 

 

 

Effect F-Ratio P

Treatment .4750 .624
Duration 28.5153 <0.0005
Sacrifice Time 1.8098 .184
Treatment x Duration .8905 .476
Treatment x Sacrifice Time 1.0064 .372
Duration x Sacrifice Time .5632 .573
Treatment x Duration x Sacrifice Time 2.0314 .103

 

APPENDIX B--F-Ratios and P-Values for LDH.

 

 

 

Effect F-Ratio P

Treatment 1.3542 .267
Duration 303.7086 <0.0005
Sacrifice Time 10.1504 .002

Treatment x Duration 4.1457 .005
Treatment x Sacrifice Time .0133 .987
Duration x Sacrifice Time 64.9390 <0.0005

Treatment x Duration x Sacrifice Time 10.8390 <0.0005

 

APPENDIX C
MORPHOLOGICAL AND HISTOCHEMICAL RAW DATA

101

102

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103

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104

 

 

 

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nxcwronw STATE UNIV. LIBRRRIES
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