THE EFFECTS OF DIANABDL AND ANAEROBIC ENDURANCE EXERCISE 0N MYOCARDIAL DAMAGE IN THE ADULT MALE ALBIND RAT Thesis for the Degree of M. A. MICHIGAN STATE UNIVERSITY DARLEIIE ANN JONES 1973 ._ 2:22):- W LIE 7‘ " " ' 1 ' {'v .. . chmrx u: ‘ L" - L: U'IIVC‘ “Si" -,- t -.:..,v. j if ’6 .Mfl'.‘i"lf1m'“‘1 Y alumna av IIIIAE & SUNS' BBUK BINDERY INC. IBRAFIY SINGERS “ ~nnr nus-un- .2- ' (L1 ”’4‘ ’ ‘ éwdD,}i Z"?- l— "I :L : .. I ". " ‘\ ) r '1 ABSTRACT THE EFFECTS OF DIANABOL AND ANAERGBIC ENDURANCE EXERCISE 0N MHOCARBIAL DAMAGE IN THE ADULT MALE ALBINO RAT By 'Darlene Ann Jones The purpose of this study was to observe the effects of the anabolic steroid, Dianabol, and an anaerobic program.of endurance running on the heart muscle of male albino rats. Some animals were maintained in a sedentary condition while others were trained onra high-intensity, short-duration Controlled Running Wheel (CRW) program established in this laboratory. Myocardial damage was determined A through a histologic technique of staining, examining, and rating different levels of the heart. Anatomical measures were taken on body weight, heart weight, ventricle weight, and ventricle length. Forty-two normal, male, albino rats of the Sprague-Dawley strain were used for the study. All animals were received in the laboratory on the same day. However, they were in three different age groups at the time of their arrival. The differences in the ages of the I animals represented a staggering procedure set up to accommodate other concurrent studies using the same facilities. Within his own age level, each animal was randomly assigned to a training-drug treat- ment group. All animals started treatment at 100 days of age. Dianabol and a placebo were administered subcutaneously at a 1 mg/day dose. The animals received the training and drug treatments Darlene Ann Jones Monday through Friday for eight weeks. All animals were given food and water ad 2111911 tum. The exercise animals were selected for sacrifice on the basis of haVing the highest percent of expected revolution (PER) within their own drug groups. The final sample consisted of 36 animals (six per cell). At sacrifice, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital. The heart was removed, trimmed, and weighed. After flushing and removal of the atria, the ventricular length was measured. The apical sections were dehydrated in graded concentrations of alcohol and then embedded in paraffin blocks. Serial cross sections, seven microns thick, were cut at eight levels spaced equally through the blocks. A Gomori trichrome stain was administered to the serial heart sections. Myocardial damage was determined by a subjective rating scale of one-to-five. A rating of one indicated no myocardial damage while a rating of five meant severe myocardial damage. The results indicated there was a significant drug effect in terms of heart damage. The severity of heart damage was greater within the Dianabol group than in either the placebo or the control groups. Furthermore, training tended to decrease the severity and incidence of heart damage. Exercised animals showed a lesser degree of heart damage than did sedentary animals. Training also appeared to decrease the Dianabol necrotizing effect in that the exercised animals receiving Dianabol had lesser severity of heart damage than did the sedentary animals receiving Dianabol. The exercised animals had smaller body weights than did the sedentary animals. Greater relative heart weights and ventricular *weights were observed in the exercised group than in the sedentary group. There were significant training effects on both absolute and Darlene Ann Jones relative ventricular lengths. The exercised animals had smaller absolute lengths and greater relative lengths than did the sedentary animals. The body weights of the Dianabol and placebo groups were both greater than that of the control group but not different from each other. The relative ventricular lengths of the Dianabol and placebo groups were less than that of the control group but were not different from each other. a «L. ,1 i». ’. ‘Q ~1- A -v‘ it .1. l, .. . e . 73!. .‘ ‘. . ,. .I‘Ju... 0. J .. . ..'. L; N r \ TIT-3 p A ' r. .v . Z . I -- . . . . I. '.0 (.~ .. .. . . .2: .,... THE EFFECTS CF DIANABGL‘AND ANIERCBIC ENDURANCE EXERCISE ON’MYOCARDIAL DAMAGE‘IN TEE . ADULT MALE ALBING RAT By Darlene Ann Jones A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of'Health,“Physical'Education and“Recreation 1973 DEDICATIDN To my Mom and Dad for enduring with me and instilling confidence in me 11 ACKNQWLEDGMENTS Many thanks go to Dr. William Heusner for his continual guidance and help. Gratitude also is extended to Dr. Wayne D. Van Buss for direction and knowledge. Thanks are extended to Mr. Kwok-Wai Ho for his technical assist- ance and friendship. Thanks are also extended to Dr. Rexford E. Carrow,‘Mrs. Barbara Wheaten, and Mrs. JoAnn LaFay. A very special thanks goes to Mr. Robert C. Hickson for his enduring supervision and faith. iii TABLE OF CGNTENTS LIST 0? TABLES O 0 O 0 O O O O O O O I O 0 LIST OF FIGms O O O O O O O O O O O O O 0 Chapter I II III ImeUc T IGN O O O O O O C O O O 0 Statement of the Problem. . Overview of Methods . . . . Rationale . . . . . . . . Significance of the Problem Limitations of the Study. . Definition of Terms . . . . REVIEW OF RELATED LITERATURE. . . Effects of Exercise as Specifically Related Catecholamines . . . . . . . . . . . Hypertrophy. . . . . . . . . . . . . Myocardial Infarction. . . . . . . . Stress in Relation to the Myocardium Preventive Aspects . . . . . . . . . Therapeutic Aspects. . . . . . . . . . Effects of Dianabol and Other Anabolic Steroid Exerc18e0 O O 0 O O O" 0 O O O O O O 0 O O O Myotropic Activity . . . . . . . . . . . . . . . . Properties of Dianabol and Other Anabolic Steroids. Steroids in Relation to the Heart RESEARCH METHDDS Sampling Procedures . . Research Design . . . . Training Groups . . . . Exercise (E) . . Sedentary (S). . Drug Groups . . . . . . Dianabol (D) . . Placebo (P). . . Control (C). . . Experimental Procedures Animal Care . . . . . . Sacrifice Procedures. . iv Its to the Heart. 1 5‘ 0 fl. 0 o e o e 0 g 0 O O O O 0 Relation with Page vi vii I-‘ O‘ hUWNNN 15 18 19 21 26 27 27 30 31 33 34 34 36 36 36 37 37 37 37 38 Chapter Method of Tissue Analysis Statistical Procedures. . . . . . . . . . IV RESULTS AND DISCUSSION. . . . . . . . . . Training Results. . . . . . . . . . . . . Histological Results. . . . . . . . . . . Anatomical Myocardial Results . . . . . . Discussion. 0 O O O O O O O O O O O O O O V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS I smry O O O O O O O O O O O O O O O O 0 Conclusions . . . . . . . . . . . . . . . Recommendations . . . . . . . . . . . . . WCES O O O O O O O O O O O O O O O 0 O O O O APPENDICES O O O O O O O O O O O O O O O O O O O O A Training Program. . . . . . . . . . . . . B Subjective Myocardial Damage Ratings. . . C mtmical Raw Data I O O O O O O O O O O O O O C Page 39 39 47 47 47 51 55 57 57 59 S9 61 68 68 69 70 Table LIST 0F TAELES Design of the prepubertal exercise study. . . . . . . . Staggering procedure for random assignment into training-drug experimental conditions . . . . . . . . . Experimental design with final cell frequency . . . . . Chi-square results for treatment effects on myocardial damage . . . . . . . . . . . . . . . . . . . Analysis of variance and Tukey Test'results for body weight, absolute and relative heart weight, ventricular weight and ventricular length . . . . . . . . . . . . . Standard eightuweek, shorthduration, high-intensity endurance training program:for postpubertai and adult male rats in controllederunning wheels. . . . . . . . . Subjective ratings of myocardial damage according to treatments and by section level . . . . . . . . . . . . Body weight and absolute and relative anatomical. Amyocardial results (Ell cm) presented by animal number, training and drug treatments. . . . . . . . . . vi Page 25 34 35 49 52 68 69 70 Figure LIST OF FIGURES Normal heart damage . . Slight heart damate . . Moderate heart damage . Considerable heart damage . . . . . . . Severe heart damage . . Mean daily percent expected revolutions CRW SHORT program . . . (PER) for vii Page 41 41 43 43 45 48 CHAPTER I INTRODUCTION Heart disease has become one of the major causes of premature fatality. The increased incidence of myocardial disease has been attributed to industrialized society. With the help of mechanical inventions, man does not need to physically exert himself as much as he did in the past. Furthermore, research has indicated that habitual physical inactivity can be a causal factor in myocardial abnormalities (18,20,38,50,53,57,71,72,73,74,75,76,78,82). Stress is another potential necrotizing agent which is prevalent in.our society. Stress can either be beneficial or detrimental to the myocardium, depending on how it is coupled with other factors (7,8,10,55,78,84). Since the myocardium plays such an important role, care should be taken to main— tain proper myocardial function. It would seem that athletes, by means of their training, should enhance the myocardial process, but this may not always be the case. In our competitive society, athletes endeavor to excel using all types of training procedures. Winning, due to social pressure and/or financial betterment, has become the key factor in most sports. In . order to achieve success, many athletes involved in strength and power activities have adopted steroid usage as a means of.extending human potential (14,42,43). Because steroids increase muscle growth (14,15, . 42,43,49,86), the "anaerobic" competitor is attracted to their use with little concern toward the other side of the spectrum-possible harmful l 2 effects. Since the myocardium is muscle tissue, it may be affected by the admdnistration of anabolic steroids. However, muscle hypertrophy, the major benefit claimed for anabolic steroids, may not be advantageous in the myocardium. This certainly would be the case if the hypertrophy were not accompanied by a compensatory increase in capillarization. The result might well be incipient ischemic heart disease. Specifically, the results of steroid usage on the myocardium have not been investigated adequately at this time (15,37,46,47,62). Statement of the Problem The purpose of this study was to observe the effects of the anabolic steroid , Dianabol, and an anaerobic program of endurance running on the myocardium of male albino rats. OvervieW'of‘Methods Some animals were maintained in a sedentary condition while others were trained on a high-intensity, short-duration Controlled Running Wheel (CRW) program established in this laboratory (88). Histochemical tissue analyses of the myocardium at different levels were performed as a means of rating heart damage. The heart weight, ventricular weight, and ventricular length also were measured. Rationale The Controlled Running Wheel (CRW) SHORT program was used because it was the program judged to be most appropriate for this study of those currently available at the Human Energy Research Laboratory, Michigan State University. A review of the literature did not reveal extensive research on the effects of anabolic steroids on the heart. Since the use of steroids 3 is widespread, possible pathological effects need to be pursued. With regard to other anabolic steroids, Dianabol's anaboliceto-androgenic ratio is high, though its anabolic activity is relatively low. The use of Dianabol in this study reflects its wide acceptance by athletes. The rat was selected because this laboratory had the facilities for housing and training small.animals as Opposed to larger ones. Furthermore, most of the research on exercise and the heart has been done on the rat. Significance of the Problem The findings of this study will add to the information on the effects of steroid usage on the heart. Comprehensive knowledge of the effects of steroid usage will help the different sport agencies in defining guidelines for athletic competition. Limitations of the Stu_dy l. The steroid dosage (1 mg/rat/day), chosen upon recommendation by Dr. J. J. Chart of the Department of Endocrinology at CIHA Pharma- ceutical 00., may not have been sufficient to induce maximum anabolic effects in the rat (12). 2. The short-duration, high-intensity exercise program represents one form of "anaerobic" exercise. Of the training programs available at the Human Energy Research Laboratory, Michigan State University, it was the most appropriate for the study. Another anaerobic program accentuating a higher expenditure of strength might have produced different results. 3. Only forty-two animals were used in the study due to limita— tions of laboratory facilities and the number of Controlled Running Wheels (CRW) available. 4 4. The staining technique used for determining specific levels of myocardial damage was quantitatively limited and may not have been representative of a complete pathological analysis. 5. The results of this experiment cannot be extrapolated directly to humans. 6. The experimental period was only eight weeks long. It is not known if this duration is optimal for maximizing the exercise and drug effects. 7. The animals were motivated to run by.a shock stimulus. There was no control for the shock in this study. However, unpublished data from this laboratory have shown that the motivational stimuli used are insufficient to produce noticeable myocardial damage. Defigigion of Terms Steroid Hormones. Those hormones possessing the cyclopentanoper- hydrophenanthrene ring system (steroid nucleus) in their molecules. They include the androgens, estrogens, and corticoids. Androgen. A generic term for an agent (usually a hormone, e.g., testosterone) that stimulates the activity of the accessory sex organs of the male, encourages the development of the male sex characteristics, or in special cases prevents the latter. Anabolic Steroid. A compound which relates to or promotes the process of assimilation of nutritive matter and its conversion into living substance. Testosterone. A male steroid hormone with both.androgenic and anabolic effects. It is produced by the Leydig cells of the testes under normal conditions. Dianabol. A synthetic anabolic steroid derivative of testosterone, produced by CIHA Pharmaceutical Co. The pharmacological name of Dianabol 5 is methandrostenolone. The structural name is l7~methyl-l7—hydroxyan- drosta—l, 4Pdien-3-one. Necrosis. The death of an area of tissue. If the condition causing the necrosis persists, the necrotic area may be characterized by irre- versible development of scar tissue. Infarct. An area of tissue in an organ, such as the myocardium, which undergoes necrosis following cessation or interference of the blood supply. Ischemia. Local and temporary anemia due to obstruction of the circulation. If the obstruction continues, an infarct develops. CHAPTER II REVIEW OF RELATED LITERATURE The effects of exercise upon the heart and the effects of exercise in conjunction with anabolic steroids are reviewed separately. subse- quently, the effects of anabolic steroids on the myocardium.are reviewed. Because the literature on anabolic steroids and the heart is scarce, some of the studies included involve.exercised hearts while others do not. Effects of Exercise as Specifically Related to the Heart Catecholamines Contrary to the traditional view emphasizing vascular oxygen supply, myocardial oxygen consumption has been demonstrated to be of equal importance in the myocardial oxygen economy (78). The term "coronary reserve" refers to the ratio of oxygen supply to oxygen demand: Blood (or Oxygen) suflnll, Coronary Reserve - Blood (or Oxygen) demand When this ratio is maintained at one or above, the myocardium receives ample oxygen. Myocardial ischemia can be noted if the ratio falls below one. Although traditional views have focused mainly on diminu- tion of vascular flow and oxygen supply as causal factors in myocardial 7 degeneration, researchers are now realizing the concomitant effects of neurohormonal influences upon oxygen consumption. Regulation of oxygen consumption takes place by way of the autonomic nervous system in the form of sympathogenic catecholamines. These catecholamines (especially epinephrine-—which when liberated from the adrenal medulla infiltrates the heart via the blood streams-and nor- epinephrine--which is intramyocardially liberated at the postganglionic sympathetic nerve terminals) have been shown to play an important role in the myocardial metabolism. Catecholamines have been demonstrated to be tissue-necrotizing agents and have the potentiality of inducing morphological changes in the heart muscle (71,72,7S,76,77,80). Although large doses are neces- sary, administration of epinephrine, norepinephrine, and isoproterenol produce myocardial lesions (72,76). Shimamoto and Hiramoto (80), in their study with albino rabbits, found epinephrine responsible for structural changes in the myocardium. Regan et a1. (77) created myo- cardial lesions in healthy male mongrel dogs through infusion of l-epinephrine. Conditions provoking the pathological potentialities of the adrenergic catecholamines have been reviewed by Raab (71,72): Excessive Catecholamine Action. Through comparative histochemical analyses, Bajusz and Raab (7) studied the cardiac enzymes phosphorylase, cytochrome oxidase, and succinic dehydrogenase and the metabolites glycogen and potassium in one hundred twenty female rats (Sprague- Dawley strain). The rats were divided into two groups, with Group I receiving epinephrine injections and Group II serving as controls. Subcutaneously injected epinephrine was given in a single dose of 450 8 mg/lOO g body weight in 0.2 ml of physiologic saline. An injection of 0.2 ml of physiologic saline served as the control treatment. Following injection, sacrifices were conducted at intervals which ranged from 5 minutes to 96 hours. While the control animals did not show abnormal enzyme or metabolite activity, spotty decreases of phosphorylase, deple- tion of stainable glycogen reserves, and a disturbance of potassium distribution followed in some of the experimental rats. ‘Most of the above activity was noted in the necrotically susceptible subendocardium apex. These prenecrotic changes could be used as possible indications of myocardial abnormalities before the morphological signs are apparent. Bajusz and Jasmin (6) also suggested the decrease or loss of phosphorylase activity as a possible indication of early anoxic myo- cardial damage. Cardiomyopathies were defined as either primary (non- occlusive or metabolic) or secondary (occlusive or anoxic). With three hundred sixty female rats, three primary necrotizing cardiomyopathy groups were produced by: (a) plasmocid (a metabolic inhibitor), (b) dihydrolachysterol (DHT) [a steroid of the vitamin D group], and (c) a Redeficient diet. Myocardial lesions were induced in three secondary groups by: (a) ligature of the left main coronary artery, (b) methoxamine (a synthetic vasopressor amine), and (c) metaraminol (a strong vaso- pressor). With respect to the primary cardiomyopathies, phosphorylase activity not only both increased and decreased but often was still present in the fibers after degeneration had started. An inverse relationship between phosphorylase activity and glycogen reserves was found in the plasmocid and DHT groups. Rats on the Redeficient diet demonstrated decreased glycogen content with complete loss before degenerative myocardial structures appeared. Secondary cardiomyopathies were accompanied by loss of phosphorylase and glycogen within 1 to 5 9 ‘minutes after coronary ligature and as soon as 15 to 30 minutes after administration of the vasopressors. Although Bajusz and Jasmin admit phosphorylase activity to be different between the two groups, they suggested phosphorylase to be an early index of anoxic myocardial damage. . Nickerson (64) mentioned epinephrine potentialities with regard to phosphorylase but did not deal with phosphorylase activity as an early index of myocardial abnormalities. Upon injecting albino male and female.rabbits with epinephrine, Shimamoto and Hiramoto (80) observed the response of.the heart muscle. Noted aberrations were intracellular edema, an increase of the clear spaces around the mitochondria and myofibrils of the cardiac muscle cells, dense bodies in the mitochondria, an expansion of the longi- tudinal sarcoplasmic reticulum, and fat droplet increases in the cyto- plasm. Although Bryant (16) dealt with rats that had their coronary arteries ligated, he observed similar responses in the myocardial is chemic areas. Excretion of urinary catecholamines reveals their liberation in the body. An increase in both epinephrine and norepinephrine occurs during muscular work, suggesting the increased secretion is from the adrenal medulla and the adrenergic nervous system. Catecholamine secretion appears to be related to the intensity of the work and to stresses. In a ski-run competition, the most successful skiers exhibited the highest excretion values (23,24). Raab (71) argued that urinary catecholamine excretion does not reflect the action of circulating catecholamines on the heart and therefore is of little use. Instead, he suggested using as a more accurate measure of catecholamine turnover the excretion of vanillin 10 mandolic acid (VMA) or free fatty acids (FFA) which have been liberated in the blood by catecholamines. Raab based his rationale for using these measures on the fact that the myocardium absorbs circulating catecholamines, thus more valid conclusions could be drawn from abnormal occurrences of VMA or FFA. Raab (71,72) summarized the potentially dangerous pathway of excessive catecholamines. By augmenting oxygen consumption, local hypoxia proceeds in cellular structures with depleted oxygen availa- bility. Impaired oxygen availability.eventually leads to anaerobic energy production where glycogen depletion occurs, followed by pyruvate accumulation. Lactate, formed from the pyruvate, becomes excessive. When the glycogen stores are depleted, ATP formation becomes inhibited, thereby hindering myocardial contractility. From this, depressed actinrmyosin interaction causes inefficient external cardiac work performance. Uneconomic work performance and unbalanced internal metabolism result from excessive catecholamines. Coronary Vascular Constriction. Excessive catecholamine action, along with sympathetic over-stimulation, results in focal necrotizing hypoxia due to cellular oxygen utilization surpassing oxygen availa- bility. However, upon increased myocardial oxygen consumption, coronary vascular dilatation operates as the hypoxia avoidance mechanism (71, 72,78). When dilatation becomes impaired, hypoxia follows due to coronary vascular constriction. Impaired Vagal Counterrggulation. When Raab and erywanek (74) submitted two hundred normal male subjects averaging 44 years of age to varied sensory and mental stresses, increased heart rate and a shortening of the left ventricular isometric period ensued, representing ll augmented cardiac sympathetic tone and adrenergic.reaction to stresses. Several researchers have confirmed these responses (71.72.75.78). These investigators also have noted that the same responses occur with habitual physical inactivity. Training promotes the opposite effects: a prolonged isometric period and elevated cardiac vagal tone leading to a slower heart rate. In conjunction with an increased vagal tone, training lowers the sympathetic tone. Evidence suggesting sympatho- inhibitory and antiadrenergic mechanisms has been observed through sympathetic preponderance brought about by sedentary living. Physical training promotes the opposite. Following a training regimen of 90¢minute sessions three times a week for three months, De Schryver et a2. (20) obtained significant decreases in both mean and total heart catecholamine concentrations in exercised rats. Although the concentrations remained low after three days of rest, on the sixth day a slight recovery was observed. After 21 days of rest, the catecholamines had increased almost to con- trol levels. This indicates a reversible phenomenon. De Schryver's study showed that training must be maintained in order to sustain the catecholamine inhibitory effect of exercise. In addition, it demon- strated that the ordinary individual concerned with cardiovascular health may be able to maintain low myocardial.catecholamine levels through a regular program of intermittent exercise. This is an important consideration for the "time-pressed" man of today. In attempts to elicit the potentialities of antiadrenergic agents as protective mechanisms in catecholamine cardiotoxicity, Raab et a1. (76) experimented with female rats. Before producing stress (restraint on a board, cold water immersion, or nicotine), pretreatment consisted of hormone injections of flurocortisol, dihydrotachysterol or 12 thyroxine. Neither stress nor pretreatment separately promoted myo- cardial lesions. However, when stress and pretreatment were combined, cardiac necroses did occur. With the administration of the anti- adrenergic factors, diminished myocardial lesions were noted. 0f the varied types of antiadrenergic agents used (catecholamine-depleting, adrenergic-blocking, ganglionic-blocking, and centrally-inhibiting), the best results occurred with the ganglionic blockers and the catecholamine-depleting agents. The others failed to inhibit both the circulating epinephrine and the intramyocardial norepinephrine. From these results, the importance of physical exercise begins to become apparent. Exercise, especially when considered as a form of stress, does not cause excess cortisol activity as occurs with other types of exaggerated stresses. Instead exercise stimulates the adrenergic agents (72). Nickerson (64) briefly reviewed the functions of the adrenergic receptors (u and B). For antiadrenergic purposes, it is of importance to be aware that catecholamines affect the heart by way of the B receptors. Therefore, 8 receptor blocking agents may be useful as myocardial-catecholamine inhibitory agents. Opie (68) points out the fact that antiadrenergic drugs are contra- indicated in heart failure because of the existing catecholamine deple- tion in the heart muscle. Conversely, a normal heart absorbs and stores circulating catecholamines while also storing intramyocardial cate- cholamines (68,71,75,76). Raab (72) and Nickerson (64) have both discussed the ability of the normal sedentary heart to function in the total absence of catecholamines. However, the concept probably would not hold true under exercise conditions. 13 Electrolyte Imbalance. As has been shown, catecholamines stimulate myocardial necrosis when certain conditions prevail. Qne condition not yet mentioned is the cardiotoxicity of adrenocortical overactivity. The corticoids act as sensitizing agents for the potentially dangerous catecholamine activity of epinephrine (7,71,73,76). Becker and Kreuzer (9) pointed out that both the adrenocorticoid system and the sympatho- adrenal medullary system function under stress conditions. In a study by Raab et a1. (76), rats received corticoid hormone treatments and/or stress. Histologic analyses showed that neither the corticoidvtreated animals nor the stress-treated animals suffered structural lesions. However, when animals were treated with corticoids prior to stress, extreme cardiac necrosis was demonstrated. A second study by Bajusz and Raab (7) yielded the same results. The corticoid pretreatment sensitized the myocardium to the cardiotoxicity of epinephrine. When the "ionic pump", which is needed to maintain the electrolyte balance, is inactivated by focal hypoxia, myocardial structural damage paralleled by cell necrosis results. Intracellular potassium is displaced to the extracellular space and there is a.depletion‘of glycogen and magnesium. Excessive sodium is housed within the myocardial cells (72). Regan at al. (77), Shimamoto and Hiramoto (89), and Bajusz and Raab (7) found a decreased glycogen content and dislocation of potas- sium after epinephrine injection. Despite the myocardial structural changes in Shimamoto's and Hiramotols study with rabbits, the electrolyte changes disappeared within one hour after epinephrine injection. Because the amount of displaced potassium was equivalent to the gain of sodium, intercellular replacement was postulated (68,72). 14 Factors known to be responsible for electrolyte derangement are: l. Emotional and Sensory Stresses. Investigators agree that stress situations cause catecholamines to be elicited. Becker and Kreuzer (9) attempted to show that although both the adrenal medulla and the sympathetic nervous system discharge catecholamines, one can prevail over the other depending on the nature of the stress factors involved. In one experiment with a well-trained man.who had been adapted to treadmill running, norepinephrine excretion increased sig- nificantly whereas epinephrine excretion did not. Becker and Kreuzer attributed their results to the fact that the individual was primarily under physical stress and not emotional stress, since his previous experience with treadmill running eliminated mental tension. Further studies on hypoxia (9) were conducted on an expedition to Monte Rosa (4,560 m). The urinary norepinephrine excretion increased to almost twice the amount at sea level, while epinephrine excretion varied little. Attributing the adrenergic nerve ending excretion to low oxygen tension, the experiment was simulated in a barometric Chamber. This time, the result was an increased epinephrine excretion. To explain the varied catecholamine excretion pattern under what seemed similar conditions, the researchers concluded that the Monte Rosa expedition dealt primarily with physical activity as the stress factor. In the chamber experiment, the subjects were not exposed to physical work. In both situations, emotional stress factors existed but of different types. Aggressive exhilaration prevailed in the mountain climbing situation; whereas individuals involved in the chamber situa- tion experienced passiveness, anxiety, and unpleasant feelings of confinement. This led Becker and Kreuzer to conclude that the person's mood may be partly responsible for the mode of catecholamine excretion. 15 Catecholamine increases correlate more closely with the intensity of work than with the quantity of work. Nowacki, Schmid, and Weist (67) showed the catecholamine metabolization with training by measuring the MHMA (vanilmandelic acid) excretion in basketball players. Although 'MHMA levels increased significantly during training, as compared to pretraining levels, the levels practically doubled following competition. 2. Lack of Physical Activity. As already mentioned, the vagal and sympathoinhibitory mechanisms are hindered by sedentary living. ’Adren- ergic preponderance can be overcome by these counterregulatory mechanisms. 3. Nicotine. Cigarette smoking augments myocardial oxygen con- sumption, causes cardiac acceleration, and induces shortening of the left ventricular isometric period (71,72,75). Raab at al. (75) found that although nicotine stimulates the sympathetic ganglia and the adrenal medulla, the effects are not lasting. Hypertrophy In 1956, Whitehorn and Grimmenga (89) chronically exercised rats through swimming. They found bradycardia but no hypertrophy. 0n the other hand, according to De Schryver et a1. (20), adaptation to con- tinuous exercise consists of bradycardia and hypertrophyb-the so-called "athletic heart." Many researchers support this line of thought (3,4,17,52,54,85). Though knowledge of cardiomegaly has existed for years, researchers still do not agree on whether it is useful (physiologic) or detrimental (pathologic). In terms of general cardiomegaly, two schools of thought have evolved. The traditional view is that the fiber thickening is an adaptation to increased work load or to a weakened chamber. Thus hypertrophy may be looked upon as a useful physiologic mechanism. The 16 deleterious school of thought views hypertrophy as essentially patho- logic in the sense that the increased muscle size is in response to actual injury. Upon exhaustion of the energy reserves, hypertrophy occurs. Grant (35) pointed out deleterious aspects of hypertrophy to stimulate clinicians to investigate and possibly revise their attitudes about hypertrophy as a natural consequence of increased work. In terms of strength of contraction, the superiority of the experimentally hypertrophied heart, in the sedentary animal, has not been shown over the normal heart. However, Crews (17), by direct measurement with a strain-gauge lever system, showed that myocardial contractility is increased when hypertrophied hearts are produced in rats through swim- ming. Crews suggested that hearts hypertrophied by other means might not react in the same manner. Badeer (3) proposed that the two views on hypertrophy, physiologic versus pathologic need not necessarily be in opposition. Though physiologic adaptation increases work performance by improving con- tractile force, early stages of pathologic hypertrophy may also be useful in that increased energy expenditure per unit mass of myocardial tissue is not needed. However, in advanced stages of pathologic cardiomegaly, the wall thickens toss point where nutrient and metabolite diffusion are restricted and the result is a deleterious cardiomegaly. The quantitative aspects of physiologic and pathologic hypertrophy were reviewed by Linzbach (54). Physiologic hypertrophy develops by increasing the work of the heart. The normal human heart weight of 300 gm may be increased to as high as 500 gm, the critical heart weight. The muscle fibers increase in size, become thicker and longer, but do not increase in number. The number of capillaries is increased, but 17 the nuclei remain unchanged. With pathologic hypertrophy, the heart is continually at work and there is an increase in the number of fibers and nuclei as well as capillaries. In such an instance, the weight of the heart not only exceeds the critical heart weight but can reach 1,000 gm or more. The mechanisms responsible for the onset of hypertrophy have not yet been determined (4,17,35). Badeer (4).postulated a "common stimulus" could be responsible. In attempting to pin point the stimulus, he reviewed some of the possibilities. Though nutritional deficiency might be a factor in pathologic myocardial hypertrophy, other conditions offer a better chance of explaining the phenomenon. Hormones such as those produced by the anterior pituitary, the thyroid, and the adrenal cortex have been shown to influence hypertrophy of the heart. Increased work has been suggested as a simple mechanical stimulus for physiologic hypertrophy, but.a major fault with the theory lies in the fact that the mechanical work and the hypertrophy are not proportional. The "acute-dilatation-injury" hypothesis is not supported by recent studies. Chronic dilatation, or increased tension in the cardiac chamber walls during systole, does seem to be highly plausible as the stimulus in many clinical and experimental myocardial hypertrophies. Increased metabolic rate per beat, sustained over a long period, may constitute an effective stimulus. Lutenov (52) discussed the development of hypertrophy as a result of anaerobic ATP resynthesis, which causes an increased phosphorylation' oxidation in the sarcosomes and, thus, increases in nucleic acid and protein synthesis. Also detecting protein synthesis in hypertrophied hearts, Fanburg (27) attributed it to increased work loads. It should be noted that RNA synthesis was one of the first biochemical changes to 18 be found with an increased overload (27). Another source of protein synthesis within the hypertrophied heart exists in the increased ribosomes. Following the swimming of one hundred twenty-five young male rats, Leon (51) noted hypertrophy in animals exercised daily but not in an intermittent group trained twice a week. In accord with previous literature (4,85), cardiac hypertrophy regressed upon cessation of training. Both Kitamura (44) and Bloor (11) have discussed the occur— rence of atrophy when excessive training has been discontinued. Myocardial Infarction Bryant (16) studied myocardial ischemia in 12 adult male rats and found that gross changes in the infarcted areas could not be detected in less than 5 hours. He stated that the ischemic changes, regardless of how soon they occurred, were probably nonreversible. Enlarged mitochondria, swelled sarcoplasm and sarcoplasm reticulum, and increased lipid bodies were the earliest alterations. Bajusz and Homburger (5) described the sarcoplasmic reticulum, the nucleus, and the mitochondria as being especially sensitive to injury which substantiates Bryant's findings. In Fine's (29) work 'with rats and Morales' (59) study with humans, myocardial infarcted areas showed irregularly increased lipid bodies. Klionsky (45) reported that one of the earliest changes in the ischemic rabbit heart could be seen as a rapid decrease.in glycogen. Diminution of glycogen in ischemic hearts also appears to be an early alteration in other animals (6,29). [Some researchers have shown the demonstration of decreased enzyme activity to be superior to histologic methods in determining early myocardial damage (29,33,59). 19 Stress in Relation to_the Myocardium Stress has been shown to have the quality of being either a pro- vocative or a preventive factor in cardiac damage. Selye's (78) concept of "simple resistance" is that insensitivity to a stressor can be achieved through pretreatment with the same agent. "Cross resistance", using a pretreatment stressor different from the one eliciting the damage, also has been shown to provide effective protection against a necrotic inducing stressor. Fifty female rats, sensitized with Na-acetate and 9u-fluorohydro- chlorocortisol (F-CoL), developed severe cardiac necroses upon sudden exposure to muscular exercise or forced restraint (8). Under the same circumstances, few, if any, cardiac lesions could be seen in animals gradually exposed to exercise or restraint. In a second experiment, 100 female rats received combined Na-acetate and F-COL pretreatments. After the animals had become adapted to one stressor, a second necrotic inducing stressor was administered. Those groups-not receiving the pre- treatment showed marked cardiac necrosis while the groups adapted to one stressor and then subjected to a different stressor showed little or no incidence of cardiac damage. In both simple resistance and cross resistance, adaptation to a pretreatment proved to be an effective protective mechanism against cardiac necrosis. Litwhiler (55) summarized the early research undertaken on the effects of selected stressors on the heart at the Human Energy Research Laboratory, Michigan State University. In one study of the relationships between anxiety, activity and the genesis of heart disease, eight anxiety-activity treatment groups were used. The exercise was a 30- minute swim per day, five days per week, with two percent of the rat's body weight attached to his tail. Anxiety consisted of electrical 20 shock administered every 15 seconds, 30 minutes per day, five days per week. The results from the study revealed no myocardial damage beyond that considered as routine variations. Because the findings were in contradiction to similar investigations, it was believed that the dura- tion and/or intensity of the electrical stress was not sufficient to produce pathological changes. Furthermore, the detection of heart damage was limited to histologic procedures. To overcome these two problems, a series of pilot studies evolved. By converting the stress cages to "live-in" cages, nine hours per day of the shock treatment were found to produce sufficient anxiety to stimulate mild to moderate heart damage within a two-week period. The accuracy of the histochemical techniques was checked by injecting 30 animals with epinephrine to elicit myocardial necrosis. Histochemdcal procedures included analyses with hematoxylin and eosin, succinic dehydrogenase, mitochondrial o- glycerophosphate, cytochrome oxidase, monoamine oxidase, and B- hydroxybuterate dehydrogenase. Findings corresponded to those obtained by Bajusz and Raab (7). Bell (10) attempted to demonstrate the role of physical activity by using exercise and anxiety in various combinations. The exercise treatment consisted of two one-half-hour swim periods seven days a week. Two percent of the animal's body weight was attached to his tail during each swimming period. The anxiety treatment consisted of a 0.36-second D.C. electrical shock of 1.5 milliamperes, five times per minute, nine hours a day for two weeks. Sacrifice took place at 116 days of age. Serial heart sections were stained with hemaxotylin-eosin, succinic dehydrogenase, monoamine oxidase, and beta-hydroxybuterate. Using a Chi-square contingency analysis, no significant differences between five treatment groups were detected. Bell concluded that 21 neither the exercise nor the electrical shock were of adequate duration or intensity to produce myocardial damage. ”However, later reanalysis of Bell's data showed that the sample size was inadequate for the statistical technique used. In a follow-up study, Thomas (84) did find significant differences in heart-damage.ratings between control animals and rats receiving both the exercise and the electrical-stress treat- ments. ‘More recent unpublished data, from this laboratory, have shown that aerobic exercise has a protective effect on the myocardium.when it is administered for a period of eight weeks prior to the anxiety period; but that the same exercise routine is detrimental when initiated with the anxiety treatment. Preventive.Aspects Many researchers believe exercise: an; o _ c. _ m _ m _ w _ m _ N _ . 9.3 .225 ow on on mu 8 m. o. m :3 .225 P P p n - — n p r n P P n h P % — b _ p — h n u b b — b L — h p _ _ — P — — _ - H £1 00 \fi q wk» /9 bk?» x .‘. Id, \IFP \/ Joe. Om 83:! a,” Om 00. 0.. ON _ Jomhzoo Ollio omwodqm I 484245 I On _ . L 03 Table 4. Chi-square results for treatment effects on myocardial damage Drug and heart damage Table 4.1. Training and heart Table 4.2. damage Training Drug Heart Seden— Exer- Heart Con- Pla- Diana- Damage tary cise Damage trol cebo bol None 111 198 Home 77 82 60 Slight 20 31 Slight 19 12 20 Definite 13 5 Definite 6 2 16 Chi-square of 5.969 Chi-square of 31.212 Table 4.3. Drug and heart damage Table 4.4. Drug and heart damage within the sedentary 'within the exercise training group training group Dru Dru Heart Con- Pla- Diana- Heart Con- Pla- Diana- Damage trol cebo bol Damage trol cebo bol None 40 41 30 None 37 41 30 Slight 8 5 7 Slight ll 7 13 Definite D 2 11 Definite 0 0 5 Chi-square of 18.546 Chi-square of 13.529 Table 4 (cont'd.) 50 Table 4.5. Training and heart Table 4.6. Training and heart damage within the damage within the control drug group placebo drug group Train Train Heart Sedenv Exer- Heart Seden- Exer- Damage tary cise Damage tary cise None 40 37 None 41 41 Slight 8 11 Slight 5 7 Definite 9 0 Definite 2 Chi-square of 0.591 Chi-square of 2.333 T‘ble 4079 Training and heart damage within the Dianabol drug group Training Heart Damage Seden- tary Exer- cise None Slight Definite 30 13 5 Chi-square of 4.050 51 Heart damage was less severe in the exercised group than in the sedentary group. When looking at the training factor alone. 9.0 percent of the sedentary animals had definite heart damage; whereas. only 3.5 percent of the exercise group showed definite heart damage. In terms of frequency of heart damage, 72.2 percent of the animals with identi- fiable damage were sedentary animals while only 27.8 percent were exercised animals. The training effect on heart damage could.not be explained in terms of the control or placebo drug groups. Though the training effect was not statistically significant, Table 4.7 shows that the difference in the severity of heart damage with the training factor seems to be concentrated in the Dianabol drug group. The drug factor produced a significant effect on heart damage. The severity of heart damage in the control and placebo animals was less than that in the Dianabol group. ‘Nearly 90 percent of the definite heart damage was found in the Dianabol group. Tables 4.3 and 4.4 show the occurrence of heart damage for the three levels of the drug factor and within specific training groups. There was a nonsignificant tendency for a greater degree of heart damage to occur in the Dianabol-sedentary group than in the Dianabol-exercise group. Anatomical myocardial Results Body weights. absolute and relative total heart'weights, total ventricular weights and total ventricular lengths are tabulated flu Appendix C. The analysis of variance results and appropriate Tukey Test comparisons are presented in Table 5. Body weights of the exercised animals were smaller than those of the sedentary animals. There also was a significant effect among the drug groups with respect to body weight. Both the Dianabol and placebo 52 Table 5. Analysis of variance and Tukey Test results for body weight, absolute and relative heart weight, ventricular weight and ventricular length ANDVA Tukey Trainigg Row Results Results Exercise Sedentary means by Rows by Rows Table 5.1. Body weight Drug Dianabol 436 493 464 F-5.69 D > C Placebo 430 507 468 P-0.008 Control 411 483 447 P > C Column Means 426 494 460* ANOVA Results P-155.38 by Columns P<0.0005 Interaction F-l. 13; P-0.336 Table 5.2. Absolute heart weight M Dianabol 1.35 1.34 1.35 F-2.96 Placebo 1.41 1.39 1.40 2-0.067 Control 1.30 1.37 1.34 Column Means 1.35 1.37 1.36* AHDVA Results F-0.46 by Columns P-0.502 Interaction F-1.29; P-D.289 Table 5.3. Absolute ventricle weight Drug Dianabol 1.24 1.22 1.23 F-2.63 Placebo 1.28 1.28 1.28 P-0.088 Control 1.19 1.26 1.22 Column Heans 1.23 1.25 1.24* ANDVA Results F-0.52 by Columns P-0.477 Interaction F-l.65; P-0.209 Table 5 (cont'd.) 53 Train Exercise Sedentary ANDVA Row' Results Means ‘by Rows Tukey Results by Rows Drug Dianabol Placebo Control Column Means ANGVA Results by Columns Interaction PEER Dianabol Placebo Control Column‘Means ANDVA Results by Columns Interaction Drug Dianabol Placebo Control Column Means ANOVA Results by Columns Interaction Table 5.4. 0.65 0.67 0.67 0.67 F-5.43 P-0.027 0.67 0.69 0.68 0.68 F-0.63; P-0.541 Tab1e5.5. 'Relative 3.10 3.27 3.17 3.18 F-67.57 P<0.0005 2.72 2.75 2.84 2.77 F-1.33; P-0.280 Table 5.6. _R£1ative ventricle weight SXl0-32 2.84 2.98 2.89 2.90 F-60.35 P<0.0005 2.47 2.52 2.61 2.53 F-1.20; P-0.317 Absolute ventricle leggth 0.66 P-2.69 0.68 P-0.084 0.68 0.67* ' heart weight (£0.32 2.91 P-l.67 3.01 P-0.205 3.01 2.98* 2.66 P-1.67 2.75 P-0.206 2.75 2.72* 54 Table 5 (cont‘d.) ANDVA Tukey Trainigg Row Results Results Exercise Sedentary' Means by Rows by Rows Table 5.7. 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