THE EFFECT OF PHYSICAL EXERCISE AND ELECTRICAL STRESS ON THE PRODUCTION OF EXPERIMENTAL MYOCARDIAL NECROSES 7 Thesis for the Degree of .Ph. D. MICHIGAN STATE UNIVERSITY RICHARD D. BELL. 1959‘ f , THES" This is to certify that the thesis entitled The Effects of Physical Exercise and Electrical Shock on the Production of Experimental Myocardial Necroses presented by Richard Bell has been accepted towards fulfillment of the requirements for Ldegree in Health, Physical Education, and Recreation €’&./ {6/ W Major professor 7:4 23/757 Date O~169 C. A ‘. 9 P ”H‘r .Al “\I‘ v‘ r}. ‘ hr 3-..A.J“_e .\ “. “If ‘I Pg. a...s “$9: ‘ Q In.“ : a.\:¢‘ze‘ ”Fl . A C. s ’ ‘ J .. «LT. b ~ -~ ABSTRACT THE EFFECT OF PHYSICAL EXERCISE AND ELECTRICAL SHOCK ON THE PRODUCTION OF EXPERIMENTAL MYOCARDIAL NECROSIS By Richard D. Bell Cardiovascular disorders are largely responsible for the high premature mortality rates associated with modern day life. More specifically, coronary heart disease is being increasingly recognized as a predominant threat to individuals in highly industrialized societies such as exist in North America. Among the explanations offered for this phenomenon are lack of physical activity and cholesterol rich diets. Occupational stresses unique to these indus- trialized societies also are thought to play a significant role in the increasing incidence of coronary heart disease. Emotional, sensory, and many other stresses are accompanied by an augmented secretion of adreno-corticoid hormones, and the sensitizing action of these hormones may play an important role in the production of myocardial damage. The danger of increased serum catecholamine concentrations is Richard D. Bell due mainly to the development of a discrepancy between the oxygen supply and the oxygen requirement of the cardiac muscle cells. Physical activity was designed to play a dual role in the present experiment. It was hypothesized that phys- ical activity might have a prophylactic effect on the pro- duction of myocardial damage. But when physical activity and anxiety treatments were administered concurrently, a double stressor situation was simulated. It was postu- lated that such a situation might result in severe myo— cardial damage. Thus, it was thought that a study which incorporated the effects of physical exercise, the effects of stress, and their cumulative effects on the production of myocardial damage would provide important information concerning the etiology of coronary heart disease. One-hundred-six male albino Sprague—Dawley rats 60 days of age were randomly assigned to five treatment groups. These groups were: control, anxiety with no exercise, exercise during anxiety, exercise before anxiety, and exer- cise before and during anxiety. The exercise treatment consisted of two one—half—hour swim periods seven days a week. A weight equal to two per cent of the body weight was attached to the tip of the rats tail during each exercise period. The exercise during anxiety animals were subjected to a two-week exercise program, the exercise before anxiety eer tere 9160 he 9 9. [‘3 with Che»: ‘4‘ Q ¢PCZQ an 0 tilt) s Richard D. Bell animals received a five-week exercise program, and the exercise before and during anxiety animals were adminis— tered a seven-week exercise program. The anxiety treatment consisted of a 0.36 second D.C. electrical shock at 1.5 milliamperes five times per minute nine hours a day for two weeks. The electrical shocks were administered randomly within each minute. All anx- iety treatments began when the animals were 100 days of age. Serum lactate dehydrogenase (LDH) levels were de- termined twice for each animal during the experiment since serum LDH levels have been frequently used as indicators of myocardial damage. Serum LDH levels were used to re- duce the original sample size to 69 animals before sacri— fice. All animals were sacrificed at 116 days of age by an overdose of ether. Immediately after sacrifice, the heart was excised, trimmed, and cut in three equal sections. The apical and basal sections were quick frozen in isopen- tane cooled with liquid nitrogen while the middle section was fixed in ten per cent formalin for A8 hours. At least two serial cross sections from the inner surface of each frozen block of myocardial tissue were taken and stained with a hematoxylin-eosin (H and E) stain and the histo- chemical enzymes mono-amine oxidase (MAO), succinic acid dehydrogenase (SDH), and beta-hydroxy butyrate dehydrogenase Richard D. Bell (B-OHD). The formalin—fixed tissue block was stained via a paraffin—embedded H and E stain. All histologic slides were subjectively analyzed for myocardial damage on the basis of an arbitrary one to five scale. A rating of one meant no myocardial damage while a rating of five indicated severe myocardial damage. A Chi-square contingency statistical analysis was employed to determine differences in heart damage for the various treatment groups as well as for the relationship between serum LDH levels and the degree of myocardial damage. No statistically significant differences were found for any of the above-mentioned statistical analyse. It was concluded that neither physical exercise nor elec— trical shock, as administered in this experiment, had any significant effect on the production of experimental myo— cardial necroses. n . n . flu a: #6 «D b v aJ 1 L h.“ “A A n. .. nu; ,1IIII DEDICATION To my wife Caryl and son Richard. Also to Dr. V. Reggie Edgerton for his interest and guidance during my graduate program. 11 ..e f use of t uuc I'r- ‘ y’i..‘ . ¢ ~ov$ -~--.o'v I Y dcwfi L; 3.. ~ we? I," r. ‘3”. «C a: YIu we 0 ‘vl ‘9; 6 5,? 75 7 7, / ”7 0 ACKNOWLEDGMENTS Appreciation is extended to Dr. W. W. Heusner for his interest and ideas throughout my graduate program and for providing the stimulus for this study. Thanks are due to Dr. Rex Carrow for the unlimited use of the facilities of the Cytology laboratory at Michi— gan State University.' A special thank you is extended to Barbara Wheaton and Trudy Van Huss for their constant help and encourage- ment. Thanks are due to Dick Bowzer, Paul Robinson, Dick Litwhiler, Robin Bohnert, Jinny Wells, and Carol Cook for their help during data collection and for care of the animals throughout the study. A sincere debt of gratitude is due to Dr. V. Reggie Edgerton and Dr. Dee W. Edington for their help during the course of this study and for their constant interest and helpful suggestions without which this study could not have been completed. Lastly a word of appreciation to both Dr. W. W. Heusner and Dr. W. D. Van Huss for proving that both per— fection and idealism are indeed worthy objectives to strive for. iii n--\.A- I- —— -‘ g liefmv I’t uLu'u' ""n AH r-- .r. . '1': L. ‘4... . . J . A -’ H-‘.r. A" Elli . . . K , n n . ‘ l a 9-- . . —-\ ‘*¢. H‘Q“ Ot~~‘ TABLE OF CONTENTS Page ACKNOWLEDGEMENTS . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . vii Chapter I. THE PROBLEM . . . . . . . . . . 1 Introduction . . I . . . 1 Purpose . . . 3 Limitations of the Study . . . A Definition of Terms . . 5 II. REVIEW OF THE RELATED LITERATURE . . . 7 Introduction . . . 7 Catecholamine Concentration 7 Serum Cholesterol, Serum Triglycerides, and Free Fatty Acids . . . . . . 17 Stress . . . . . . . . . . . 27 Physical Activity . . . AO Histochemistry of Myocardial Disease . . A3 III. RESEARCH METHODS . . . . . . . . . 5A Sample . . . . . . . . . . . . . 5A Treatments . . . . . . . . . . . 5A Treatment Croups . . . . . . . . 55 1. Controls . . . . . . 55 2. Anxiety with No Exercise. . . . 56 3. Exercise during Anxiety . . . . 56 A. Exercise Befdre Anxiety . . . . 57 5. Exercise Before and During Anxiety . . . . . . . . 57 Body Weight . . . . . . . . . . 58 Serum Lactate Dehydrogenase Determinations . . . . . . . . 58 Sacrifice Procedure . . . . . . . 59 Histochemical Procedure . . . . . . 59 iv 7V 2:? . at“ II <' I U~ \ ---.’AN-'U~ ‘ ‘ '4 .— . “*UH-UU..1'1. . E:"‘\"\v‘ F i. " - maul n ‘Y A neart Pr VA Chapter Page Methods of Tissue Analysis . . . . . . 61 Statistical Methods‘ . . . . . . . . 66 IV. RESULTS AND DISCUSSION . . . . . . . . 68 Results . . . . . . . . . . . . 68 V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS. . 77 Summary . . . . . . . . . . . . 77 Conclusions . . . . . . . . . . . 79 Recommendations . . . . . . . . . 79 BIBLIOGRAPHY . . . . . . . . . . . . . . 81 APPENDIX A . . . . . . . . . . . . . . 102 Heart Damage Ratings and Serum LDH Concentrations . . . . . . . . . . 10A II . A,.I~ .x .3]~ C~ -: v.1 F u. no . .H» A a. no . {C u {C .. ¢ 0» 1 ¢ - .u» G.» e C S e 0 Cu 1. .0 t .. a .C c.» .1. Au .4 n. n. .nu .ru .ru “No “No C C C e V Ifi I O I 0 I .AU s A. . «L aj 4 CI) Hana ,5 Table LIST OF TABLES Frequency distribution of subjective rating of myocardial damage for ventricular tissue sections of the myocardium . . . . . . Frequency distribution of subjective rating of myocardial damage for atrial tissue sections of the myocardium . . . . . Chi-square test for treatment effects on the degree of myocardial damage for ventricular tissue sections . . . . . . . . . Chi—square test for relationship between the degree of myocardial damage for ventricular tissue sections and serum LDH level . . . Chi-square test for relationship between the degree of myocardial damage and serum LDH levels for atrial tissue sections . . . vi Page 69 70 7O 72 72 .I\ rt ‘4 . A .. _ . . Pu «C I. :C wk n . 7 n1 3 J v . .u. u. rt. «D pa ‘4. .. . a a . J I y . u. I t . . I. u m. ”C .p "n 3 e e r C 01 .5 .C C 5 ~.. ~. 0 I n» e ”I MW e w :; «J O n. e .4 C .. . e L 3; VI“ Qu vlu A N «\U ?..u A5 a U 9. A e .“M I O O v.0 . Is at .(J ..I~.u RN to 7 nine O) FU . d “114 Vi 1 LIST OF FIGURES Figure Page 1. Morphological lesion in heart of rat follow— epinephrine injection. Hematoxylin- phloxine-saffron stain . . . . . . . . 50 2. SDH distribution in control heart . . . . . 50 3. Loss of SDH activity in necrotic area of heart. 50 A. Areas of necrosis and development of granulat- ed tissue in the myocardial tissue of the right ventricle . . . . . . . . . . 50 5. Necrotic foci in myocardium of rats exposed to frustrating and frightening situations. . . 52 6. Sub- endocardial necrosis in rats conditioned by Na2HPOu and Me- CO- COL followed by cold baths 0 o o o o o o o o o o o o 52 7. Loss of SDH activity from 36 hour old human myocardial infarct . . . . . . . . 52 8. Simple stress-cardiopathy in rat subjected to forced restraint for 17 hours. . . . . . 52 9. Decreased SDH activity in right ventricle of exercise before and during anxiety treat- ment animal (SDH, X A00) . . . . . . . 62 10. Loss of B-OH activity in right ventricle of exercise before and during anxiety treat- ment animal (B-OH’ X 1400) c o o o o o o 62 11. Right ventricle from a rat exercised before and during anxiety treatment (H and E, X 1400) o o o o o o o o c o o o o 62 12. Decreased B-OH activity in sub-endocardial region of animal exercised during anxiety (B-OH, X "00) o o o o o o o o o o o 62 vii DA“'l-u-I--$ Figure Figure 'Page 13. Sub-endocardial region of a rat exercised during anxiety (Hand E, XAOO) . . . . . . 62 1A. Loss of MAO activity from epicardial region of left ventricle of animal exercised before anxiety treatment (XAOO) . . . . . . . 6A 15. Epicardial region of left ventricle of animal exercised before anxiety treatment (Hand E, X ”00) o o 0‘ o c o o c o o o o 0 6a 16. Mid ventricular region of control animal (Hand E, x A00). . . . . . . . . . . 6A 17. Endocardial region of left ventricle from animal receiving anxiety with no exercise treatment (SDH, X A00) . . . . . . . . 6A 18. Endocardial region of left ventricle from animal receiving anxiety but no exercise treatment (Hand E, X A00) . . . . . . . _ 6A viii CHAPTER I THE PROBLEM Introduction Cardiovascular disorders have increasingly dominated the mortality statistics of modern day life. In 1962 5A.5 per cent of all deaths in North America were due to cardio- vascular accidents (193). Furthermore, the per cent of cardiovascular deaths, compared to other causes, has been increasing since 1900. Within this picture, coronary heart disease is regarded as a major threat to the life of most adults in highly industrialized societies. Many expla- nations have been offered regarding this phenomena, but low levels of physical activity (123, 12A, 125), socio— economic stresses (5A, 106, 135, 137, 1A1, 1A8), and cholesterol-rich diets (93, 9A) are among the more prom- inent ones. As long as man had to use his muscles to pro- vide for his daily needs in primitive surroundings, a minimum level of physical fitness was automatically guar- anteed by his mode of life. This condition has, of course, changed markedly in our mechanized, overstimulated, and sedentary society. Often such modern modes of life do not provide enough physical exercise to keep the muscles strong and flexible and to keep the cardiovascular system Sufficiently exercised to maintain a minimum degree of phys- ical fitness (102). Many authorities believe that increased levels of physical activity promise to be a major preventative for coronary heart disease. The role of exercise in preventing or modifying the course of coronary heart disease has been, and still is, the subject of much scientific discussion; but, the evidence accumulated to date is still inadequate to warrant unreserved support. Exercise also may act as a Specific stressor agent in the production of myocardial damage (176). In fact, the present experiment was designed to take advantage of this situation. It was thought that physical exercise and conditions of anxiety acting concurrently might effectively simulate a double-stressor situation which, theoretically, should produce severe myocardial damage in those animals receiving such treatment. Ever increasing occupational stresses also play a dominant role in the high incidence of coronary heart dis- ease. Western societies in particular are fraught with stresses which are unique in industrialized societies (16A). Emotional, sensory, and many other stresses are accompanied by an augmented secretion of adreno-corticoid hormones, and the sensitizing action of these hormones may play an important role in the production of myocardial damage (138). The danger of increased serum catechola- mine concentrations probably is due mainly to the develop- ment of a discrepancy between the oxygen supply and the oxygen requirement of the cardiac muscle cells. Any study of heart disease also must mention the effects of serum cholesterol and high fat diets on the pro- duction of myocardial damage. Severe atherosclerosis with its effect on heart disease is closely related to the mean serum cholesterol concentration (70, 9A, 95), since high fat diets and increased serum cholesterol concentrations are thought to sensitize the heart for the subsequent pro- duction of myocardial damage (176). However, neither diet nor serum cholesterol concentrations were part of the present study. Purpose This study was undertaken to determine the effects of various combinations of physical exercise and electrical stress on the production of experimental myocardial damage in male albino rats. Electrical shock was utilized to simulate conditions of socio-economic stress. When administered without prior conditioning it was thought that the stress of electrical shock might result in moderate to severe myocardial damage. Physical exercise (swimming) was included in the present experiment for two reasons. It was postulated that the main effect of forced physical exercise might be prophy- lactic in that it would condition the heart against the production of myocardial damage. But, when electrical shock and forced physical exercise were introduced con- currently, without prior conditioning, it was thought that they would act as a double stressor. It was hypothesized that this situation might result in severe myocardial damage. Limitations of the Study 1. Exercise programs which involve swimming white rats are limited by lack of control of the intensity of muscular activity. The chief factor in the exercise program which can be controlled is the duration of activity. 2. The exercise results of this investigation are based on sixty minutes per day of swimming and cannot be extrapolated to other forms or durations of muscular exercise. 3. The histochemical methods used for determining specific levels of myocardial damage are quan— titatively limited. A. There was no quantitative control over diet as the animals received food and water ad libitum. 5. The results of this experiment cannot be inter- preted as being directly applicable to the human species. Definition of Terms 1. Ischemic Heart Disease Ischemic heart disease refers to a group of myocardial disorders characterized by the presence of angina pectoris and/or myocardial infarction. This term is often used to mean coronary heart disease as well, although some authors (78) say coronary heart disease should be used only in the strict anatomical sense and ischemic heart disease to specify a level of functional impairment (78). 2. Cardiomyopathy Cardiomyopathies are an obscure heterogeneous group of cardiac disorders whose only common feature is that the myocardium is in a state of disorder. They may be patho- logically classified as genetic, inflammatory, metabolic, or vascular, or clinically classified as destructive, con- strictive, or congestive (l). 3. Stress Stress, in a biological sense, is the state mani- fested by a Specific syndrome which consists of all non- specifically induced changes within a biological system. Stress is a condition, a state, which manifests itself by measurable changes in the body's organs. Another simple definition of stress states that stress is the sum of all non-specific changes caused by function or damage (18A). A. Stressor A stressor is that which causes stress. It is a stress-inducing agent. 5. Necrosis A necrotic area of myocardial tissue is characterized by a depletion or lack of enzyme activity. Should the con- ditions causing this necrosis (eg. obstruction of a coro— nary artery) persist, the necrotic area may then be further characterized by the irreversible development of scar tissue. 6. Infarct An infarct refers to tissue necrosis induced by inter- ference with the blood supply of a particular tissue such as the myocardium. It is usually caused by acute and com- plete occlusion of an artery (176). CHAPTER II REVIEW OF THE RELATED LITERATURE Introduction The causes of myocardial damage have been of interest to medical researches for a great many years. Recently, due to the alarming increase in mortality as a result of "cardiovascular accidents," other related fields, such as physical education, have instigated pertinent studies in the field of cardiovascular disease from their own point of view. This has served the purpose of introducing other possible causal factors into the total picture of cardio- vascular disease. At present, when discussing cardio- vascular disease, it is necessary to consider such diverse factors as circulating catecholamine concentrations, serum cholesterol and triglyceride levels, degree of nervous tension, amount of physical activity, and genetic background of the individual concerned. In order to give the reader an adequate background of the present study, some of the above factors will each be reviewed separately. Catecholamine Concentrations Early investigators have described myocardial necrosis in the absence of coronary artery disease. Reference is made to an article by Josue (87) who demonstrated myocardial lesions produced by catecholamines as early as 1907. Throughout the succeeding years, others have produced simi- lar experimental evidence to confirm this phenomena (7, 160, 176). Starcich (19A) outlined several elementary facts con— cerning the involvement of neuro-hormonal activity in the pathogenesis of acute coronary insufficiency and resulting myocardial hypoxia. First, sympathetic stimulation and the resulting liberated adrenosympathogenic catecholamines such as sympathetic neurogenic norepinephrine and adreno- medullary epinephrine cause a marked augmentation of myo— cardial oxygen consumption. Secondly, the availability of adequate amounts of oxygen to the myocardial tissue depends simultaneously on the vascular oxygen supply which is un- evenly distributed in different areas of the cardiac muscle and on the degree of myocardial oxygen consumption which varies with the degree of sympathetic tone and neuro-hormonal activity (1A6). Thirdly, the myocardial hypoxia-producing effects of sympathetic stimulation and catecholamine action are greatly intensified when the compensatory coronary dilation which normally accompanies augmented cardiac oxygen consumption is impaired by experimental coronary restriction or by atherosclerosis (3, 51). Finally, no clear relation- ship exists between the degree of existing atherosclerotic coronary vascular lesions on the one hand and the occurrence of clinical manifestations of myocardial ischemia on the other except in cases of severe coronary stenosis or occlu- sion. It is no longer possible to ascribe myocardial ischemic structural changes to vascular factors alone with- out consideration of those contributory non-vascular mech- anisms which interfere in myocardial oxygen metabolism (127). Raab (138) states that some forms of human recrotizing card- iomyopathies may be directly attributable to the reflex liberation of catecholamines which, under certain circum- stances, exert a contributory noxious effect on cardiac metabolism. The potential danger of catecholamine over- activity to the functional and structural integrity of the heart muscle may be attributed largely to the development of a discrepancy between the vascular oxygen supply and the oxygen requirement of the cardiac muscle cells. For many years nothing definite was known concerning the precise mechanism through which the catecholamines produced myo- cardial damage, except for the hypoxic effect of these amines on the cardiac muscle. However, recent comparative histochemical studies, such as the one by Bajusz and Jasmin (10), show that early declines in myocardial phosphorylase activity and in the amount of stainable, labile fraction of glycogen are sensitive indices of anoxic myocardial damage and that the behavior of phosphorylase and glycogen during the development of anoxic cardiac lesions is different from that seen during the development of the "metabolic" type of lesion. Differences were also established in the 10 reaction of other enzymes such as cytochrome oxidase (CyT.O) and succinic dehydrogenase (SDH) which implies that dif- ferent histochemical techniques might be applicable for purposes of differentiating between anoxic and metabolic (produced by dietary means or by administration of meta— bolic inhibitors and other cardio—toxic compounds) cardio- myopathies (lO). Bajusz and Raab (8), using white rats, injected epinephrine subcutaneously in a single dose of A50 ug/lOO grams of body weight. The animals were sacrificed at various intervals ranging from five minutes to ninety—six hours after injection. In the hearts affected by epine- phrine, a decline or even a complete loss of phosphorylase Vactivity was observed in some endocardial regions as early as ten to fifteen minutes following the injection of epine- phrine. Furthermore, a depletion of the stainable glycogen reserves and a disturbance in the normal distribution of potassium also were distinguishable ten to fifteen minutes following injection. The foci showing loss of phOSphorylase activity and a parallel depletion of glycogen reserves and potassium content were spotty and mainly located in the sub-endocardium and the apex. A second portion of the ex- perimental series showed that a brief sensitization with fluorocortisol proved extremely potent in enhancing the susceptibility of the heart muscle to the potentially car- diotoxic action of subsequently injected epinephrine. This ll combination of agents resulted in the development of large areas completely devoid of phosphorylase, potassium, and glycogen in one hundred per cent of the hearts studied. The greatly enhanced potassium depleting action of epinephrine, after brief sensitization with a corticoid hormone such as fluorocortisol, may be of clinical significance. Emotional, sensory, and many other stresses are accompanied by an augmented secretion of adrenocorticoid hormones (138), and the sensitizing action of these hormones may play an impor- tant contributory role in catecholamine-induced myocardial injury. In a similar type of experiment, Shimamoto (172) in- jected twenty-six male and female rabbits with one ug/kg. of body weight of epinephrine. Myocardial samples were taken at three, five, fifteen, thirty, and sixty minutes post in- jection. Specimens sampled thirty minutes after the in- jection of epinephrine exhibited a definite change in myo- cardial structure, showing an intracellular edema of the cardiac muscle cells. More specifically, these changes consisted of a definite expansion of the vesicles of the longitudinal system of the sarcoplasmic reticulum, an in- crease of the intracellular clear spaces around the mito- chondria and between the myofibrils, and an appearance of dense bodies in the mitochondria. An analogous expansion of the longitudinal system, an increase of the clear spaces around the mitochondria and myofibrils of the cardiac muscle 12 cells, and an alteration of mitochondria have also been observed in the early stages of anoxia induced by coronary ligation (28). Vendenyeyeva (205) has also reported pro- ducing myocardial necroses by stimulating sympathetic nerve trunks as well as by the injection of epinephrine and norepinephrine. The catecholamine isoproterenol can also produce early myocardial pathology without detectable alterations of the mitochondria (57). However, during conditions of ischemia the mitochondria undergo severe changes in a comparatively short time. A baSic principle postulated by Raab (139), concerning the effects of released catecholamines on the myocardial muscle, is that central stimulation of the sympathetic nervous system exposes the heart to an inten- sified, potentially hypoxiating action of epinephrine and norepinephrine which can produce necroses in the myocardium. It is possible that the cardiotoxicity of various agents depends upon the sudden liberation in the heart of some metabolite that is toxic only in the presence of condition- ing factors and of specifically cardiotoxic agents (15A). If this is true, cardiac damage could be prevented by the stress-induced discharge of this metabolite prior to expo- sure to sensitizing agents. In fact, Raab (1A0) has shown that antiadrenergic drugs protect rats from stress-induced myocardial necroses. ”rt-i I .13 Regan g£_al.(156) infused l-epinephrine into the left coronary artery of male mongrel dogs and observed the metab- olic and hemodynamic effects of sympathetic stimulation. The fact that the infused epinephrine stimulated glycogen- olysis was evidenced by a twenty_per cent decrease in the left ventricular glycogen content after fifteen minutes of infusion. There was also a simultaneous decrease in the extraction of oxygen by_the myocardium. Coronary blood flow was not significantly altered, but the extraction of oxygen by the myocardium was reduced. In addition, the extraction of potassium and phosphate was significantly altered by epinephrine infusion. This pattern of ion and enzyme loss from the myocardium was the same as occurs after destruction of the left coronary artery. Since total coronary blood flow was not significantly decreased, an increase in cardiac activity which was not met by an adequate oxygen supply increase may have been a possible cause of the necrosis observed. In fact, in this particular experiment (156), all measured parameters of left ventricular activity such as stroke output and minute work showed increases of approximately sixty per cent while coronary blood flow increased only fifteen per cent, and oxygen extraction decreased. Richardson (158) agrees that the potentially patho- genic, hypoxiating and cardiotoxic properties of the cate- cholamines have been known for a long time. Furthermore, 1A it was recently discovered that even small doses of cate- cholamines and electrical or reflectory stimulations of the cardiac sympathetic nerves are apt to elicit severe hypoxic and necrotizing manifestations in the heart muscle under certain accessory conditions (158). Examples.of such con- ditions would be atherosclerotic restrictions of the normal compensatory coronary dilatability or the metabolically sensitizing influence of administered adrenal mineralo— corticoids. Richardson further suggests that metabolically and structurally deranged hearts lose their ability for normal production and/or accumulation of neurogenic and blood—borne norepinephrine. He concludes that there is a tendency of patients suffering from angina pectoris to dis— charge abnormally large quantities of catecholamines into the circulation during physical effort and under emotional stress. Furthermore, individuals with coronary athero- sclerosis exhibit an exaggerated sensitivity to the myo- cardial hypoxiating and pain-producing effect of admin- istered and reflex-discharged catecholamines. Animal experiments (1A1, 1A8) suggest that intensive emotional stimuli per se are capable of eliciting myocardial necroses. Both emotional tensions and habitual inactivity share, as their common denominator, a demonstrable augmen- tation of sympathetic adrenergic activity (1A9). In the case of emotional tensions, this is presumably due to ex- aggerated hypothalamic stimulation, in the case of physical 15 inactivity, to a deterioration of sympathoinhibitory inot- ropic and vagal chronotropic antiadrenergic counterregula- tory mechanisms at rest. It is also pointed out (1A9) that another contributory element in hypoxic (ischemic) heart disease, tobacco smoking, is likewise associated with adrenergic overactivity caused by nicotine-induced gan- glionic stimulation. The long-known myocardial oxygen "wasting" and potentially anoxiating cardiotoxic properties of the adrenosympathogenic catecholamines are being increas- ingly appreciated with respect to their clinical pathogenic significance (200, 170). Schimert and Schwalb (173) agree that the concept, proposed by those investigators (138, 156, 19A) who have stressed an unduly augmented oxygen demand in the myocar- dium as a possible accessory functional factor in ischemic heart disease, is being increasingly appreciated. The oxygen requirements of the heart depend on the amount of work performed by the heart muscle and on the specific neurohumoral metabolic influences under which this work takes place. Physical stress, by way of neurohumoral influences which cause a potentially uneconomical mode of action of the heart, also reduces the "coronary reserve quotient" (where a quotient below one is equivalent with myocardial ischemia) unless coronary dilation compensates for the in- creased oxygen demand of the myocardium. An augmentation of myocardial oxygen consumption is caused by adrenergic l6 neurohumoral influences even without alterations of the workload and the aortic pressure. As long as the coronary arteries are intact and normally dilatable, this fact is not of any major pathogenic importance, since any increase in myocardial oxygen consumption is promptly followed by an adequate dilation of the coronary arteries. Nevertheless, it has been shown by numerous investigators (137, 156, 139) that repeated injections of epinephrine, along with stress- induced intensive stimulations of the sympathetic nervous system and the hypothalamus, produce myocardial necroses even in healthy animals. If the coronary vessels are narrow, a rising sympathetic tone will endanger particularly the subendocardium by causing a lack of local oxygenation. In contrast to the effect of the sympathogenic catechola- mines, sympathetic inhibition and vagal stimulation exert an opposite oxygen preserving effect which enables the heart to work with a lesser oxygen consumption. Poor vascularization of the muscles, inadequate coordination of muscular action and of blood distribution during exer- cise, and a reduced effectiveness of muscular effort cause an augmented oxygen requirement of the body as a whole and thus impose an additional strain on the heart to which it responds primarily with an acceleration of the heart rate. The most impressive, and clinically most important, augmen- tation of coronary reserve is achieved by physiological means, namely by physical training. It is known that l7 systematic physical training, or any form of persistent and vigorous muscular activity, reduces the cardiac sympa- thetic tone and excitability and raises the vagal tone. This is manifested by a slow heart rate, a decrease of cardiac output, and a lowering of the systolic blood pres- sure at rest. Other experimental data (52) suggest that, in the presence of stenotic coronary arteries, the develop- rent and dilation of preformed collaterals is favored by physical exercise. Serum Cholesterol,gSerum Triglycerides, and Free Fatty Acids In recent years, experiments have shown the important role of fats as a source of energy for skeletal muscle (99). The previous concept that carboyhydrates were the main source of energy has been changed; and we know now that fats, in the form of free fatty acids, are released from the tri- glycerides of depot fat and then transported through the circulatory system to the muscles where they are split via beta oxidation to meet the energy requirements of the organ- ism (99). One of the significant findings of Konttinen's ex- periment on twenty-six healthy army recruits was the marked rise of plasma free fatty acids during the course of exer- cise: the resting level was found to be 390 u Eq./L. while at the end of three hours of exercise the plasma free fatty acid concentration was found to be 1230 u Eq./L. (100). This l8 pronounced rise of plasma free fatty acids occurred without exception in all men who underwent exercise, whether they had fasted or eaten meals consisting of fats or carbohyrates. It is thought that one of the mechanisms by which free fatty acids are released is mediated through the catechola- mines, as both epinephrine and norepinephrine are released from the tissues during exercise and both cause a prompt increase in plasma free fatty acids. This increase enhances the hydrolysis of neutral fats within the body tissues. The triglyceridemia evoked by the ingestion of fats was cleared from the circulation of active men more rapidly than in the case of sedentary men. It was concluded that, during pro- longed exercise, energy transport in the form of plasma free fatty acids greatly exceeded the utilization of these compounds (35, 36). Nikkila and Torsti (129) agree with Carlson (3A) that the energy needs of working muscles--both myocardial and skeletal--are mainly covered by fatty acids. Because the immediately available mass of body nonesterified fatty acids is quite small, these must be cleaved and mobilized from the different triglyceride or phospholipid pools. While some of the fatty acids may be derived from the muscles' own lipid esters, from the adjacent tissue triglycerides or from plasma triglycerides, it is apparent that the ulti- mate main source must be the triglyceride stores of distant tissue (129). But what about the mechanism of fatty acid l9 mobilization? It might be assumed that the steady state existing during rest is disturbed by the contracting muscle increasing liberation of transcellular and intracellular free fatty acid carriers. This changed free fatty acid gradient across the muscle cell membrane, together with the increased blood flow, leads to an accelerated efflux of free fatty acids from the plasma. When the systemic arteri- !_w1 a1 plasma free fatty acid level is adequately decreased, a I similar mechanism begins to operate in the opposite direc- tion at and within the adipose tissue fat cell; and ulti- I I mately this results in an increased rate of triglyceride breakdown and of free fatty acid influx into the blood. This concept is supported by the experiments of Havel §£_§l- (80). However, on continued but quantitatively constant muscular work, the free fatty acids do not reach a steady state level. The initial fall in the plasma free fatty acid concentration is followed by a continuous increase up to values above the resting level. The rate of entry of free fatty acids into plasma constantly exceeds their re- moval rate. This would seem to indicate that the "muscular pump" of free fatty acid flow during exercise must induce in man some additional stimulatory mechanism(s) for free fatty acid mobilization in excess of actual needs. An elevated blood catecholamine level is thought to play an important role. 2O Anitchkow (6) has expressed the opinion that myocar- dial infarctions are practically nonexistent without atherosclerosis of the coronary arteries. It is true that, according to statistical data, myocardial infarctions have occurred in the overwhelming majority of instances in the presence of advanced coronary atherosclerosis. However, the exclusive role of atherosclerosis in the origin of myocardial infarction is being increasingly questioned according to recent investigations. In fact, a growing number of investigators have attributed at least equal pathogenic significance to functional disturbances of the coronary circulation and/or to neurogenic and hormonal influences on myocardial and electrolyte metabolism. Necrotic foci of different sizes were observed in the myo- cardium of animals with normal coronary vessels as a result of central nervous and peripheral sympathetic stimulation, emotional stress, and injection of the oxygen—wasting sympathogenic catecholamines, epinephrine and norepine- phrine (Al). Grande (70) states that it has been known for more than fifty years that atherosclerotic lesions can be pro- duced in animals by feeding them on diets rich in fat and cholesterol. The frequencies of severe atherosclerosis and coronary heart disease among various populations are closely related to the mean serum cholesterol levels of the populations. Keys (93, 9A) has shown that the incidence LI 21 rate of myocardial infarction is an exponential function of the "effective cholesterol level." This would seem to in- dicate a close relationship between serum cholesterol levels and the development of the atherosclerotic process. Thus, it would seem reasonable that reduction of the serum choles- terol concentration could result in a degree of prevention or delay in the development of the atheroslerotic process. Present day opinion seems to make it clear that neither coronary thrombosis nor atheroslerosis per se are obligatory prerequisites for the development of myocardial infarctions. Coronary atherosclerosis must be regarded merely as an ex- tremely important predisposing and contributory factor, but not as the exclusive pathogenic factor involved in the origin of myocardial infarction. I Kipshidze (96) concluded that, in the presence of predisposing coronary atherosclerosis, physical stress can serve as an important contributory factor in the patho— genesis of myocardial infarction and that in certain cases both coronary atherosclerosis and superimposed functional over-strain of the heart are jointly responsible for the occurrence of destructive myocardial lesions. Lapiccirella (105) also agrees that views of a sup- posedly purely atherosclerotic origin of ischemic degen- erative heart disease are no longer tenable. Myocardial necrotic foci and infarctions occur frequently in the com- plete absence of vascular occlusions, and coronary athero- 22 sclerosis is frequently found at autopsy in clinically and structurally normal hearts. It has become obvious that some accessory nonvascular factors must be involved. The super-imposition of these factors over widely varying de- grees of coronary atherosclerosis would create the ulti— mately decisive pathogenic background for acute myocardial injury. Common experience has, for some time, suggested a causal role of emotional tensions, anxieties, and excite- ments in the origin of anginal pain as.well as death. Stamler gt_al.(l9l) are of the opinion that a coro- nary prevention program may be undertaken on a scientific basis due to the advances in knowledge concerning the etiology and pathogenesis of atherosclerotic coronary heart disease. Some of these advances pertinent to this study may be summarized as follows: 1. Severe atherosclerosis is the underlying patho- genic process in most cases of clinical athero- sclerotic ischemic heart disease. 2. A several—fold increase in cholesterol——partic— ularly esterfied cholesterol--is the biochemical hallmark of the atherosclerotic plaque. 3. The excess cholesterol in the plaque is derived from the cholesterol-bearing lipoproteins of the circulating plasma. 23 Sustained hypercholesterolemic hyperlipidemia is associated with frequent, premature, severe ather- osclerotic coronary heart disease. In groups of middle-aged patients with clinical coronary heart disease, higher mean serum choles- terol-lipid-B-lipoprotein levels are found than in matched control groups. Sustained ingestion of diets containing increased quantities of cholesterol and fat is a virtual prerequisite for the production of significant atherosclerosis in a wide range of experimental animals. The marked international differences in occurrence rates of premature coronary heart disease are due largely to socioeconomic factors (differences in living habits, principally dietary habits) and not to racial, ethnic, climatic, or geographic factors. Where the mean serum cholesterol levels of popula- tions are low, clinical coronary heart disease and severe coronary atherosclerosis at postmorten are rare, particularly in middle age. High serum cholesterol levels in populations and high rates of middle-age clinical coronary heart 2A disease occur only where the habitual diets are high in calories, total fat, saturated fat, and cholesterol. 10. In populations studied prospectively, risk of premature atherosclerotic disease is increased in the presence of hypercholesterolemia-hyper- lipidemia. 11. In populations with the nutritional-metabolic i prerequisites for severe premature athereclerotic disease, risk is also increased by hypertension, diabetes, overweight, cigarette smoking, and a positive family history of premature vascular disease. Physical inactivity and psychological stress are in all likelihood additional signifi— cant risk factors. 12. Atherosclerosis is, at least in part, a revers- ible disease. 13. The other major coronary risk factors--hyper- tension, diabetes, overweight, cigarette smoking, and physical inactivity-hare amenable to control and correction by nutritional-hygienic-pharma- cologic means. The foregoing compel the conclusion, at least accord- ing to these authors (191), that diet is a key factor in the 25 etiology and pathogenesis of atherosclerotic coronary heart disease. Skinner §£J£;.(190) subjected fifteen middle—aged men to a six-times-per-week program of running and rhythmical calisthenics. During the latter weeks of the program, the exercises were considered to be quite intense. The results showed that exercise capacity, as expected, was signifi— cantly increased. Mean serum cholesterol and phospholipid levels did not change significantly with training, and individual changes in serum cholesterol appeared to be re- lated to changes in diet and/or body weight. Serum tri- glycerides, however, decreased significantly from a pre— training level of 208 mg./100 ml to 125 mg./100 ml at the completion of six months of training. This reduction appeared to be an acute, short-term effect occurring within two to three hours post exercise and lasting only two days. According to Carlson (3A), it is possible that the inhibitory effect of nicotinic acid on the mobilization of free fatty acids from adipose tissue is the immediate cause of a plasma cholesterol-lowering effect. Approxi- mately one-third to one-fourth of the free fatty acids mobilized from the adipose tissue are taken up in the liver. Normally, the major part of these free fatty acids taken up in the liver is oxidized, and the minor part is recirculated into the plasma coupled mainly to the tri- glyceride fatty acids of the plasma lipoproteins. These 26 lipoproteins also contain the plasma cholesterol. Some lines of evidence suggest that inhibition of the rate of mobilization of free fatty acids will decrease the amount of free fatty acids taken up in the liver. This, in turn, would reduce the recirculation of free fatty acids as plasma lipoproteins and hence decrease the concentration of cholesterol and triglycerides in the plasma. I Some investigators (95) have concluded that the I serum triglyceride level is a more reliable indication of future susceptibility to ischemic heart disease than is I the serum cholesterol level. However, in most major pro- spective epidemiological studies on ischemic heart disease, more importance has been attached to serum cholesterol levels (95). Rosenblatt (163) found that serum cholesterol was significantly higher in patients with coronary heart disease than in normal subjects, but the fasting serum triglyceride level was not altered. Carlson (37) concluded that elevated serum triglyceride levels are characteristic of ischemic heart disease in men under fifty years of age, but after this age elevation of the serum cholesterol levels is more prominent. Conversely, Katz 2232, (90) have re- ;ported that hypercholesterolaemia is associated more with ischemic heart disease in men under fifty years of age than .in men over fifty. Obviously, there is no clear agreement annong investigators as to the relative importance of serum cfluolesterol or serum triglycerides in relation to ischemic heart disease. 27 McCabe gt_al. (116) concluded that the increasing occupational stresses unique to industrialized societies play a dominate role in the high incidence of coronary heart disease. However, it should always be kept in mind that the increase in coronary heart disease probably has other causes as well. A good example is a high standard of living involving too much rich food and too little physical activity. The fact that high-fat, high-carbo- hydrate diets sensitize the myocardium for the production of acute necroses by stress has been experimentally demon- strated by Selye (176). Stress It is well established (106) that conditions of stress are accompanied by reactions from the sympatho— adrenomedullary system. If the stress if often repeated or long lasting, it may result in permanent and structural changes of pathogenic significance. The sympathoadreno- medullary system can be activated by a wide variety of stimuli (106, 5A). More specifically, (138), a gross disturbance of the central nervous system causes a massive secretion of epinephrine from the adrenal glands which is absorbed by the heart muscle from the blood. It is probable that the centrally-induced accumulation of large concen- trations of epinephrine in the heart stimulates augmented oxidative metabolic processes in the myocardium and that these hypoxiating metabolic changes in the myocardium, 28 alongside disorders in the coronary blood flow within the heart, give rise to dystrophic lesions of the myocardium. Present opinion indicates there are at least two mechanical factors which are common and important for the development of myocardial infarctions. These factors are atheroscler- osis of the coronary arteries and coronary thromobosis. Raab et_al. (1A1) elicited myocardial necroses in sixty-nine per cent of a series of wild rats exposed to frightening noises such as a tape recording of a cat-rat fight. Myocardial necroses were also demonstrated in fifty per cent of flurocortisol pretreated white rats which had been submitted to prolonged frustrating situa- tions. Civilized competitive living with its socioeconomic emotional tensions and stresses, sensory overstimulation, lack of physical activity, and abuse of nicotine combines several factors which increase sympathetic neurohormonal activity. The result is interference in myocardial metabo- lism both in a sustained fashion and with acute exacerba- tions (63, 11A, 107). Raab and Krzywanek (1A8) among others, state that many studies (59, 202, 207, 208) have demonstrated the sig- nificant contributory roles of socioeconomic emotional factors and lack of physical exercise in the high mortality rate from multicausal ischemic heart disease in industri- alized nations. Psychic stress is said (135) to be caused by rational i “aw 29 conflict of the "drive discharge" in which the "acting out" of problems is prevented. The suggested pathogenetic link occurs via neurohormonal pathways; Sympathetic over- activity with increased production of catecholamines appears to be a major factor causing myocardial oxygen wastage. It has also been shown that the cardiotoxicity of the adreno— sympathogenic catecholamines is greatly potentiated by adrenal corticoids. One might conclude that the civilized pattern of human behavior leads to conflict and stress. Because of rational control, situations of "emotion without action" occur frequently. The neurohormonal repercus— sions adversely affect the cardiovascular system and eventually cause or contribute to ischemic heart disease (135). Kraus (102) is of the same vieWpoint. He argues that the lack of physical activity weakens the heart and skeletal muscles by disuse, but the overstimulation which is inherent in urbanized living keeps man in a state of almost constant alert from which there are few direct or even vicarious outlets. This imbalance in our lives-- namely excesive stimulation combined with lack of exercise-- is built into mechanized society. As a result, many indi- viduals live in a potentially pathogenic environment. Lack of physical exercise and overstimulation are combined in a constant suppression of the "fight or flight" response (32). This constant suppression of an otherwise normal reaction is an additional source of stress and, therefore, 30 in the long run a probable cause of disease. To make matters even worse, this source of stress is compounded by conditioned responses which make mere signals of irritation stressful. Rosenman and Friedman (16A) are among those investi- gators who are convinced that clinical coronary heart dis- ease results from the interaction of multiple causal factors operating within the framework of time. Rosenman's original hypothesis was based on the question of whether the rising incidence of coronary heart disease among middle-aged Amer- ican males might stem from some emotional interplay induced by the stresses imposed by our industrialized civilization acting in conjunction with high fat diets, diminished physical activity, relatively high serum lipids, and so forth. Stress, it is pointed out, has always been an integral part of life, but Western societies in particular are fraught with stresses which are not only restricted to industrialized groups but are uniquely new. To illustrate his point Rosenman (16A) characterized a particular per— sonality structure as Behavior Pattern Type A. Pattern A is characterized by certain personality traits including aggressiveness, ambition, drive, competitiveness, and a profound sense of time urgency. An individual with this particular behavior pattern possesses the above mentioned characteristics to an excessive degree. It was subse- quently found that a population of male and female subjects 31 exhibiting Behavior Pattern A also exhibited a higher than normal prevalence of clinical coronary heart disease. Furthermore, the various physiological and biochemical mechanisms concerned with coronary atherogenesis are sig— nificantly altered by Behavior Pattern A in such a fashion that permanent alterations of the coronary vasculature can ensue. According to Wolffe (212), situational stresses which the individual is unable to cope with are common environ- mental causes of various functional and organic forms of cardiovascular disease. In fact they constitute the pre- dominating factor in many patients of the younger age groups suffering from ischemic heart disease. Wolffe refers to this as the "Nutcracker Syndrome." This term is used to denote a chain of events resulting from suppressed, crushing environmental circumstances from which the patient cannot extricate himself; and, the person with a vulnerable cardiovascular system may develop ischemic myocardial disease if the situational stresses are not relieved. Russek (169) lists several factors in decreasing order of importance in the production of coronary heart disease. These factors are: occupational stress, diet, tobacco, heredity, obesity, and lack of physical exercise. Rosenman (167) states a similar case when he says that the occupa- tional stresses unique to industrialized society play a significant role in the high incidence of clinical coronary 32 heart disease in such societies. However, he also agrees that other factors are of importance and should not be ne- glected. For example, habitual inactivity could also be a potent predisposing agent for myocardial necroses. Thus, the current trend toward an increasingly sedentary exis- tence may be just as noxious as the occupational stresses just mentioned. Selye's demonstration (176) of stress—induced myo- cardial necroses, without coronary artery lesions, provided an impetus for the study of nonvascular hormonal and neuro- hormonal metabolic elements in myocardial pathology. The well-known participation of potentially cardiotoxic adreno- medullary and sympathogenic catecholamines in the response of the autonomic nervous system to all stresses, makes the appearance of severe cardiac disturbances under emotional and other stresses intelligible. This applies particularly to the frequently occurring coincidence of stress-induced hypothalamic and sympathetic stimulations with pre-existing atherosclerotic limitations. Animal experimentation has, in fact, provided indirect evidence that the endocrine and autonomic nervous systems, especially through exaggerated liberation of cardiotoxic adrenomedullary and sympathogenic catecholamines, are fundamentally involved in the develop- ment of emotion-induced lesions of the cardiac muscle. The relationship between occupation and the frequency and course of ischemic heart disease has received increasing 33 attention during the last several years (A6, 27, 123, 203, 12A, 125). Epidemiological studies have received a fair degree of attention; but, at best, they have provided suggestive, ambiguous results. In some instances the re- sults have been contradictory. Studies conducted on people living in Israeli kib- bitzim have added to the growing volume of literature which I agrees that ischemic heart disease is a multietiological I“ disease and physical activity is only one important facet of the disease. Brunner (27) did find that the incidence ‘ I T of anginal pain, myocardial infarction, and mortality due to ischemic heart disease was two and one-half to four times higher in sedentary workers than non-sedentary workers. 1 Selye (175) says that the belief is common, not only among physicians but also among laymen, that sudden expo- sure to a particularly stressful experience may elicit a cardiac infarct, at least in predisposed individuals. Yet for various reasons, many cardiologists seriously doubt that stress plays any role in the pathogenesis of cardio— vascular disease in general and acute cardiac necroses in particular (78). First of all, it is often impossible to identify a particularly stressful experience in the imme- diate past of a patient who died of cardiac infarction. Secondly, until quite recently, it has not been possible to produce any parallel of a cardiac infarct in experimental 3A animals by exposure to even lethal stress. Thirdly, there is considerable evidence in support of the view that certain stressful experiences such as exercise or cold baths for ex- ample can actually protect the heart against infarction (175). Under normal conditions, exposure to stress produces no serious cardiac damage in healthy young people. However, in unconditioned animals treated with certain electrolytes and certain steroids, such as corticoids in doses ineffec- tive by themselves, subsequent exposure to stress invari— ably elicits massive infarct-like cardiac necroses. This electrolyte-steroid-induced cardiopathy, characterized by necrosis, fails to occur if the animals are exposed to stress prior to the electrolyte-steroid treatment. For ex- ample, rats pretreated with sodium acetate plus fluorocort- isol for a few days, and then exposed to the stress of forced muscular exercise in a revolving drum, all died with massive infarctoid myocardial necroses within twenty-four hours after the exercise period. Another group of animals survived when similarly treated with sodium acetate and fluorocortisol but were forced to exercise both before and after the conditioning treatment was given (175). Many observations have been cited in support of the view that stress can elicit myocardial infarcts in man (5, 169, 208). In an excellent publication, Selye (176) approaches the subject of cardiac disease from.a different point of view. As the title The Pluricausal Cardiopathies suggests, 35 he has concerned himself with the experimental production of different types of cardiac lesions by various types of experimental procedures. According to Selye (176, p. 315) "many structurally distinct cardiopathies can be produced or prevented at will by varying combinations of electro- lytes, steroids, and stress." Furthermore, "stress, de- pending upon circumstances, can both produce and prevent the same cardiopathy." However, as a rule electrolytes, corticoids, or stressors produce no consistent cardiac changes by themselves as only certain combinations of them are cardiotoxic. For example, necroses associated with simple stress-cardiopathy only develop after very severe exposure to stress alone and then occur only under excep- tional circumstances. One of the major experimental cardiopathies classi- fied by Selye is the Electrolyte—Steroid—Cardiopathy with Hyalinization (ESCH). This cardiopathy is produced by combined treatment with the mineralocorticoids and certain sodium salts. Stress, apparently, is not necessary for production of cardiac lesions of this type. The ESCH cardi- opath is characterized by the formation of hyaline deposits within the myocardium and the coronary arteries. The hyaline material appears partly in the disintegrating muscle fibers and partly in the stroma around the muscles. As this lesion becomes more chronic, the hyaline material is replaced by connective tissue. 36 Another major experimental cardiopath is the Electro- lyte-Steroid-Cardiopathy with Necrosis (ESCH). This lesion is produced by the administration of sensitizing electro- lytes (NaCl or Na2HPOu) and corticoids with or without sub- sequent exposure to stressors. The ESCN is characterized by large infarctoid necroses. Histologically, the first detectable change is a necrosis of the muscle fibers. The I coronary arteries are not affected. A notable subdivision _M! of the ESCN cardiopathy is the Ardenergic Cardiopathy pro- duced by heavy overdosage with adrenaline, noradrenalin or other catecholamines such as isoproterenol. Histologically, these lesions are recognizable in the form of a spotty myolysis especially in the subendocardial layers near the apex of the heart. Another subdivision of the ESCN is directly related to the work in this investigation. The Simple Stress-Cardiopathy is produced by exposure to sudden intense stress without any special conditioning. The re- sulting cardiac lesions manifest themselves histologically as scattered necroses of individual muscle fibers or small fiber groups. However, these individual lesions are so small that they do not tend to terminate in permanent scar formation and thus are difficult to detect. Microscopic foci of necrosis tend to heal within a few days without leaving a trace. During exposure to a stressor there is an increase in the cardiac lipid content and a decrease in the quantities of essential metabolities of oxidative phosphorylation. 37 Selye also attempts to point out a relationship be- tween clinical and experimental necrotizing cardiopathies (176). Possibly, microscopic chronic coronary lesions often act merely as conditioning factors that predispose the myocardium to the induction of massive necroses by metabolic derangements. Even the development of a thrombus within a vessel damaged by chronic atheromatosis may depend upon metabolic changes. Thus, thrombus formation may be of secondary importance in the precipitation of acute myo— cardial ischemia. Flame-photometric studies indicate that, in clinical cardiac insufficiency and especially in myo- cardial infarction, the fall in potassium and the rise in sodium concentration of the heart are of sufficient magni- tude to account for a derangement in cardiac energy pro- duction and utilization that could be the responsible pathogenic factor. Whether a stressful situation produces or prevents a cardiac infarct depends on the circumstances that condi- tion the body'sreactivity. For example, forced muscular exercise can, if applied before the animal is humorally conditioned for the development of a cardiac necrosis, act as a reliable preventative agent against the same necrotiz- ing cardiac lesions which are elicited, when a similar ex- ercise stressor is applied after humoral conditioning. Another major experimental cardiopathy is the Elec- trolyte-Steroid-Cardiopathy with Calcification or ESCC. 38 It is characterized by extensive calcification of the myo- cardium and the coronary arteries. This.calcification occurs in patches which are irregularly distributed throughout the heart. The ESCC is produced by combined treat- ment with certain steroids and calcium salts. Concurrent exposure to stress greatly facilitates the production of the ESCC type of lesion. In general, the stress—related myocardial necroses in corticoid-pretreated rats are thought to be produced by catecholamine discharges which accumulate in the myocardium under stressful conditions (15A). Raab (153) is also of the opinion that there is a fundamental causal involvement of metabolic catecholamine action in the origin of stress— induced myocardial damage. In contrast, the protective action of stress is thought to be due to increased glu- cocorticoid activity (153). Even though he did not feel justified in presenting a unified hypothesis of the pathogenesis of the pluricausal cardiopathies, Selye (176) did advance some plausible theo- ries. According to his experiments, Selye is convinced that potassium is the decisive pathogenic factor in the elec- trolyte steroid cardiopathies because: 1. Mineralocorticoids, which cause the loss of potassium, are indispensable for the production of both ESCH and ESCN. 39 2. In experimental animals, the ESCN.can be dupli- cated by feeding a potassium deficient diet without the treatment of corticoids, sodium salts, or stressors. 3. The classical ESCN can be prevented by oral admin— istration of potassium salts. I h. Acute stress causes a pronounced sudden loss of potassium and can precipitate electrolyte- steroid cardiopathies. r W Other observations suggest that a "potassium servomechanism" regulates cardiac work and that potassium efflux from the heart is increased when the cardiac.work load and heart rate =are increased. For example, in coronary disease myocardial intracellular pottassium loss is accentuated by ischemia and extracellular potassium is elevated. Not only myo- cardial ischemia, but various stressors such as muscular work and adrenalin deplete myocardial potassium and increase coronary venous potassium. Thus, it seems that potassium is a pivotal factor in the pathology and normal physiology of the myocardium (164). If there were to be a unified interpretation of his Texperiments, Selye would classify it as changes in ionic «equilibrium. This concept states that the: precipitous influx of sodium and/or efflux of potassium renders the cell vulnerable to various potentially pathogenic agents that can produce NO the diverse structural lesions previously dis- cussed. Both mineralocorticoids and.stressors enhance the replacement of potassium by sodium in the cell so the precipitation of lesions could be due to the effect of these agents upon ionic equilibrium. Conversely, the protective action of antimineralocorticoids may depend on their ability to prevent such an electrolyte shift. The prophylactic effect of pretreatment with stressors or with mineralocorticoids may find its explanation in the fact that they in- duce potentially pathogenic changes in ionic equilibrium at a time when the cell is not yet exposed to a potential pathogen and, thereby, .. diminish the gradient with which such a sen- sitizing shift could occur during a subsequent critical exposure (176, p. 345). ; Physical Activity The benefits of physical activity in preventing heart disease and in improving cardiac function in patients with heart disease have been accepted for many years even though proof of such benefits was difficult to find. The values of physical activity in coronary heart disease might result from: (a) possible prevention or delay in the development of atherosclerosis, (b) changes in clotting tendency with the possible prevention of thrombotic complications of coro- nary atherosclerosis, or (c) conditioning the body of stress via exercise which, in turn, could reduce the possibility of ionic imbalance due to the action of either mineral- ocorticoids or other stressors. Furthermore, improved cardiac function and/or coronary collaterol circulation may result from increased physical activity so that impaired coronary flow may be better tolerated (N7). 1:1 The biological principle of Roux that all organs are maintained and developed by function also applies to the heart. Lack of function and movement leads to atrophy and disease, especially hypokinetic disease (103). Owing to lack of movement, physical work, and exercise in mechanized civilization, coronary insufficiency has become one of the most common diseases. The observation is born out by the fact that those who regularly engage in physical exercise suffer less frequently from coronary insufficiency (61, 83, 118). I Hernberg (81) was concerned with the correlation be- tween physical working capacity and serum cholesterol in business men. The results seem to indicate that persons with a high physical working capacity have lower serum cholesterol values in the age group of 30 to #9 years. However, other factors such as mode of activity, dietary differences, and degree of business stress also play an important role (#0). Many studies have been carried out comparing the incif dence of myocardial disease as well as death due to myocar- dial disease in physically active and sedentary occupations. The same general conclusions have been reached in most of these studies, and there is general agreement that men and women in physically active occupations appear to have lower rates of fatal coronary disease than those in sedentary occupations. Habitual physical inactivity is associated 42 with a progressing deficiency of the sympathoinhibitory and vagal mechanisms which normally influence cardiac sympa- thetic chronotropic and inotropic activities at rest and during exercise (26, 149). According to Raab (155), the characteristics of a "loafer's heart" such as high pulse rate, short isometric contraction period, proneness to de- velop hypoxic ECG changes during exercise, and low overall _ I efficiency are opposite to those characteristics of the "* well-trained athlete's heart with its reduced sympathetic tone and high overall efficiency. Physical training re- duces the excess oxygen utilization of the myocardium by decreasing the sympathetic activity of the heart (146). Morris' work on employees of the London transport system (123, 125) showed that sedentary London bus drivers had twice as many fatal heart attacks as did the conductors who were required to climb stairs many times per day. Sed- entary postal clerks also showed a higher rate of heart attacks when compared to postmen who were physically active most of the day (124). Similarly, two and one-half million skilled, semi-skilled, and unskilled workers were studied, and higher incidences of fatal heart attacks were observed in those occupations requiring little physical activity. Furthermore, the proportion of men who survived a first heart attack was twice as great among heavy duty laborers. Autopsies on four thousand coronary deaths revealed more extensive cardiac damage in those who had been engaged in 43 sedentary occupations (125). A study of the relationship between mortality, resulting from an initial myocardial infarction, and physical activity revealed that death for those who pursued a sedentary existence.characterized by habitual lack of activity was almost three times that of those individuals who were classified as most physically active (60). E Histochemistry of Myocardial Disease # The heart muscle is extremely rich in a number of oxidative and hydrolytic enzymes; and it is reasonable to assume that alterations in the normal metabolic scheme, which lead to irreversible myocardial damage, can be rec— ognized early via studies on enzyme activities. Histo- chemical studies are, in fact, of value in demonstrating the onset of damage to the myocardial fibers. Histochemical methods for demonstrating the activity of certain enzymes reveal the presence of myocardial infarctions long before conventional staining methods show.convincing structural changes in the damaged fibers (8). The decline in most enzyme activities is probably due to leakage of the enzyme in question from the dying cell (1). However, the decline in monoamine oxidase action "may possibly be a homeostatic response to prevent oxidation of the scanty catecholamines that remain in the infarcted myocardium" (1, p. 234). According to Niles et_al. (131), the use of precise histochemical techniques helps elucidate the biochemical 44 reactions involved in myocardial lesions. Histochemical techniques can achieve this because they show not only the amount of activity, as disclosed.by the intensity of the stain, but also the location of that activity within the tissue and cellular components. Myocardial fibers from hearts treated with isoproterenol or subjected to condi— tions of hypoxia show predominantly a granular type of staining rather than a characteristic.fibrillar staining (130). As early as two hours after the onset of hypoxic conditions, small foci of abnormal granularity are evident with SDH. By fourteen hours, this condition is prevalent; but, a standard hematoxylin-eosin stain shows evidence of scar tissue formation only after approximately eighteen hours. Following experimental coronary artery ligature, the reactions for the oxidative enzymes start to decline in two to three hours (8). At an early stage of ischemia, the activity of succinic dehydrogenase (SDH) declines more readily than that of Cyt.O. The normal myocardial fibers surrounding the necrotic area retain normal SDH and Cyt.O. activities. Monamine oxidase activity is somewhat different. It becomes rapidly depleted in the affected areas but is also depleted or absent in the remaining parts of the coronary artery ligated heart. In the case of metabolically induced necroses (i.e. injection of plasmocid) the activity and distribution of the 45 various oxidative enzymes remain normal for about eight hours following injection. Later, as could be expected, a decrease in reaction intensity is noted but only in those fibers that are obviously degenerating. Again the reaction for SDH declines more readily than that for Cyt.O. The myocardial fibers surrounding the necrotic area are found to be hyperactive as far as histochemical demonstration I of the enzymes under consideration are concerned. The T“ earliest histochemically demonstrable alteration than can i be detected in plasmocid treated animals is the initial decrease in 5-nucleotidase activity in the capillary walls. This is evident as early as ten minutes following the in- jection of plasmocid (8). Schnitka (174) reported a reduced mitochondrial Cyt.O.‘ activity six hours following induced experimental lesions. On the other hand, the activity of LDH was sometimes main- tained for a while in the middle of infarcts (110). It is now known (30) from biochemical subcellular fractionation studies that the mitochondria contain Kreb cycle enzymes such as SDH and most of the cells Cyt.O. activity. Lushnikov (110) reported that B—OHBD and iso-citric dehydrogenase are the first enzymes to decline in activity. Reduced activity has been reported as early as one and one- half to four hours after experimental cardiac infarction of the rat myocardium. In fact, B-OHBD is one of the most sensitive histochemical indicators of early human myocardial infarction (110). 46 Fine gt_al. (58) agree that the various enzyme changes are obvious before there is histologic evidence of cardiac infarction; and, twenty-four hours following myocardial infarction, histochemical reactions for SDH and nucleotide- tetrazolium reductases may be nearly absent from experi- mental lesions. These histochemically demonstrable reduc— tions in enzyme activity in early necrotic myocardial I fibers are an indication of an enzyme leakage that occurs i‘" from the infarcted myocardium. This, then, would be the reason for the elevated plasma levels of the enzymes in f “ question soon after the onset of the necrotic condition. According to Maller and Pearse (126), MAO activity is indicative of adrenergic function. MAO deaminates pri- mary aliphatic amines to aldehydes as shown in the follow- ing reaction: MAO RCH NH + O > RCHO + NH + H O 2 2 2 3 2 2 However, the exact function of MAO in the animal body is not known. Bajusz and Jasmin (16) report that MAO activity also disappears early and is also considered to be a sensitive indicator of early myocardial infarction. MAO activity has been identified in the sarcoplasm, especially in the mito- chondria of myocardial fibers in biopsies of the human right ventricle (134). It has also been observed that MAO activ- ity is strongly active in the rat heart particularly in the 147 region of the sino—atrial and atrio—ventricular nodes (126). In fact, in the prenecrotic stages of the development of experimental cardiac lesions, MAO activity declines sub— stantially while the activities of SDH and Cyt.O. remain normal until the actual onset of cellular degeneration. In another article Bajusz (7) reiterates the same facts when he says that during the early stages of myocardial damage, I the reactions for such oxidative enzymes as SDH and Cyt.O. “ remain normal or are even somewhat elevated within the areas devoid of phosphorylase, glycogen, and potassium. ( w When a conventional HPS stain is used, morphologic changes do not oCcur in the myocardium until five to eight hours following injection with epinephrine, and the HPS stain is found to be informative only when dealing with lesions of at least twenty-four hours duration. In the case of an obstructive cardiomyopathy, there is a marked increase in SDH in the mitochondria of the affected myocardial fibers. In a similar manner, the activity of MAO is also increased. Possibly, "the prolif- eration of mitochondria in the cardiomyopathic fibers re— flects the increased production of MAO in the tissue as a homeostatic response to the focally high concentration of catecholamines" (l, p. 243). In cardiac infarcts produced by coronary ligature in the rat, the SDH activity begins to diminish about four hours after the operation and disappears completely within 48 forty—eight hours. Cyt.O. activity disappears at a slower rate according to comparable histochemical observations (91). However, according to Kaufman §t_§l, (91) the most extensive studies on myocardial changes that follow coro— nary ligature have been made on dogs. In this species, the evidence has been accumulating that chemical and histo- chemical alterations (such as glycogen, lactic acid, PAS- tingible material, electrolytes, and enzyme changes) occur from the first few minutes to hours after vascular occlu- sion. Loss of SDH activity from infarcted myocardial fibers has been detected histochemically as early as two hours after coronary occlusion in man and as early as four to six hours after coronary ligation in rats (174). LDH is present in the cardiac muscle and its activity is dependent upon the co-enzyme nicotonamide-adenine— dinucleotide (NAD). Under anerobic conditions, the inter- conversion of pyruvate and lactate shifts toward lactate which necessarily decreases the relative amounts of NADH available to the cell. The increase of LDH activity in both normal and de- generating cardiac fibers following the injection of plas- mocid may reflect an adoptive alteration in enzyme pathways (8). Both Vessels (206) and Wrobewski (213) say that cases of myocardial infarction are accompanied by an increased concentration of LDH. Similarly, Stuart (198) found that LDH concentrations rose immediately after myocardial in- _.l__‘ _ 7 49 farction and remained elevated for seven to ten days. Garbus (65) found that untrained male albino rats showed a marked rise in serum LDH concentrating following sixteen hours of exercise. However, in a trained group of rats, the LDH level remained normal. Blatt gt_al. (22) found no change in the LDH concentration of the liver or skeletal muscles after three to four weeks of exposure to a cold ! environment. After the first week, the LDH level in the '_ heart increased significantly; but, after the animal adopted to the stress of the cold environment, the LDH " concentration in the heart returned to normal. Presumably a similar reaction to other forms of stress could be expected. ‘ 'T'fil U Figure Figure Figure Figure .-—A morphologic lesion in the heart muscle of a rat following injection of epinephrine. The lesion is shown at 48 hours following injec- tion and is fully developed and characterized by more or less confluent sub-endocardial areas of myolysis with inflammatory infil- tration (Hematoxylin-phloxime—saffron (HPS) stain). (7) .--The activity and distribution of SDH in a normal control heart. (7) .--Partial decrease and loss of SDH activity in myocardial fibers that are already in various stages of degeneration. Note the increased SDH activity in fibers surrounding the necrotic area. (7) .--Confluent areas of necrosis and development of granulated tissue in the rat myocardial tissue of the right ventricle. (185) 50 51 Figure 2 Figure l Figure 4 Figure 3 . Figure 44 Figure Figure Figure .--Degenerating and necrotic foci in the myo- cardium of rats which had been exposed to frustrating (obstacles in access to food) and frightening (tape recorded cat-rat fights) situations (141). .--Subendocardial infarctoid necrosis in left septum and wall of right ventricle in two rats conditioned by Na HPO and Me-CL—Col followed by exposure t8 co d baths. (176 p. 64) .-—Nearly complete loss of succinic dehydr— ogenase from necrotic myocardial fibers of a 36-hour old human myocardial infarct. Note preservation of SDH activity in normal fibers at left. SDH x 160. (l p. 228) .--Simple stress-cardiopathy in rat restrain- ed for 17 hours. Focus with edema, necrotic muscle fibers, and round cell infiltration under endocardium. Fuchson x 125. (176 p- 125) 52 53 Figure 6 Figure 5 .43. w. .6. ... .fifl‘wgfipfi i o . .. 7 .H o u l... 2% .m .w. Figure 8 Figure 7 CHAPTER III RESEARCH METHODS Sample The sample consisted of 106 male albino rats of the Sprague-Dawley strain. The animals were 60 days of age when they were delivered to the Human Energy Research Lab— oratory. Treatments Two experimental treatments were used during the course of this investigation: an anxiety treatment and an exercise treatment. These treatments were used separately and in various combinations. The anxiety treatment consisted of seven shock peri- ods each day for two weeks. During each shock period, the animals received a disturbing, but noninjurious, 36 second electrical shock of 1.5 milliamperes five times per minute. In order to present a random pattern of shock to the animals, the time between shocks ranged from seven to sixteen seconds within each shock period, and the du- ration of the shock periods themselves ranged from twenty- eight minutes to two hours and eight minutes. 54 5’- 55 The exercise treatment consisted of a one-half hour swim, twice daily, seven days a week for two, five, or seven weeks. For the first three days of the experiment, each animal completed the exercise program without the attachment of additional weight. Thereafter, each animal had a weight equal to two per cent of his body weight attached to the tip of his tail for all exercise periods. The weights were attached near the tip of the rat's rail with miniature plastic Clothespins. White adhesive tape was inserted be- tween the prongs of the Clothespins to reduce trauma. The animals were swum in individual cylindrical metal tanks measuring 28 cm. in diameter and having a depth of 75 cm. Immediately after each animals was placed in its individual swimming cylinder, it was lifted out and the air stripped from its fur. It was then replaced in its respective cylinder. If an animal was obviously having difficulty during the exercise period, it was removed from the water and given a brief rest before being returned to the water to complete the one—half hour swim without its tail weight. Treatment Groups The animals were randomly assigned to five experi— mental treatment groups. The five treatment groups used in this experiment were as follows: 1. Controls (Con) Ten animals were housed in sedentary cages throughout the duration of the experiment. They 56 received neither the exercise nor the anxiety treatment and were removed from their cages only once weekly for body weight determinations. It was expected that these animals would show little or no myocardial damage. Anxiety with no Exercise (ANE) Twenty-four animals were housed in sedentary cages until they were moved to the anxiety cages for the two-week anxiety treatment. This group received no exercise treatment and, on completion of the two-week anxiety treatment, they were returned to their sedentary cages. It was ex- pected that this group of animals would show moderate to severe myocardial damage. Exercise during Anxiety (EDA) Twenty-four animals were housed in sedentary cages until they were moved to the anxiety cages for the anxiety treatment. They were also sub- jected to a two-week exercise program concur- rently with the anxiety treatment. After com- pletion of the exercise-anxiety treatment, the animals were returned to their sedentary cages until sacrifice.. It was expected that this group .of animals would show severe myocardial damage. 57 Exercise before Anxiety (EBA) Twenty—four animals were housed in sedentary cages for one week prior to the start of the ex— ercise treatment and in voluntary cages through- out the five-week exercise period. The exercise treatment was terminated at the start of the anxiety treatment at which time the animals were I moved to the anxiety cages for the anxiety treat— d ment. It was expected that this group of animals would show no myocardial damage due to the f“ hypothesized prophylactic effect of exercise. Exercise before and during Anxiety (EBD) Twenty—four animals were housed in voluntary cages during the five—week pre-anxiety exercise period. Following the five-week exercise period, the animals were moved to the anxiety cages for the anxiety treatment. They also received an additional two-week exercise treatment concur- rently with the anxiety treatment. At the com- pletion of the anxiety-exercise treatment, the animals were returned to the involuntary cages until sacrifice. It was expected that this group of animals would show minimal myocardial damage. 58 Body Weight All animals were weighed every Saturday at 12:00 noon, and the weights used during the exercise periods were ad- Justed weekly according to the animal's most recent body weight. Serum Lactate Dehydrogenase Determinations Serum lactate dehydrogenase (LDH) concentrations were E determined using the standard spectrophotometer technique described by Cabaud and Wroblewski (31) and by the Sigma Chemical Company (189) which supplied an assay kit for serum LDH determinations. The selected wave length on the spectrophotometer was 540 mu. Basically, the serum LDH level was used in an attempt to detect heart damage before sacrifice. Animals were chosen for study, which according to their LDH level, showed some evidence of myocardial damage. The original sample size of 106 was thus reduced to only 69 animals. The four groups of animals receiving the exercise and/or anxiety treatments were reduced to 15 animals per group while the control group was reduced to nine animals due to the accidental death of one control animal prior to sacrifice. Serum LDH determinations were not made prior to the start of the anxiety treatment nor were they made on the control animals as previous studies in the Human Energy Research Laboratory had established serum LDH levels for control animals. Serum LDH deter— minations were made only on those animals receiving the 59 anxiety treatment and only five and twelve days after the start of the anxiety treatment. Sacrifice Procedure Each animal was sacrificed at 116 days of age with an overdose of ether. Next, a midabdominal incision was made and continued cranially through the sternum. Loose fascia was removed and the heart exposed and quickly excised. The E heart was trimmed at both the apex and the base. Three approximately equal blocks of myocardial tissue were ob- tained by a transverse ablation of the atria and ventricles. The outer two of these sections were mounted on metal chucks with five per cent gum tragacanth and quick forzen immediately in isopentane-cooled with liquid nitrogen. This method allowed the tissue to be cooled to approx- imately «1600 C within ten seconds. The middle section of myocardial tissue was placed in ten per cent formalin solution for 48 hours prior to staining with a paraffin embedded H and E stain. Serial tissue sections were obtain- ed from the inner surfaces of the frozen blocks of myo- cardial tissue. Histochemical Procedures At least two fresh-frozen serial cross sections approx- imately 10 microns thick, were cut from each frozen block of myocardial tissue on an Ames-Lab Tek cryostat. The tissue sections were briefly fan dried immediately after 60 they were thawed at room temperature and then they were placed on 22 mm. sq. Corning cover glasses. One tissue section from each block of myocardial tissue was stained with a standard H and E stain. Other sections from each tissue block were subjected to specific histochemical pro- cedures--SDH (17), MAO (133), and B—OHBD (133). Tissue sections from all animals of each sacrifice were processed s;multaneously in order to ensure the use of identical histochemical techniques and to ensure equivalent staining reactions within the cell tissues being processed. Succinic dehydrogenase (SDH) activity was studied using NBT (2,2'-di-p-nitropheny1 -5,5'-diphenyl -3,3'- (3,3'-dimethoxy -4,4'-dipheny1ene) ditetrazolium chloride) as an election acceptor, as described by Barka and Anderson (17). Monamine oxidase (MAO) activity was demonstrated by the method of Glenner e£_al. (133) with NBT again used as an election acceptor. Beta-hydroxybutyrate (B-OH) activ- ity was studied according to the method as described by Pearse (133). - Incubation times varied slightly according to the histochemical procedure being followed. For example, SDH was allowed to incubate foronly seven minutes while MAO was incubated for at least 40 minutes. B-OHBD also was incu- bated for 40 minutes. Glycerin Jelly was used as a mount- ing medium for all histochemical enzyme procedures. Per— mount was the mounting medium used for the H and E procedure. 61 Method of Tissue Analysis After the histological slides from each sacrifice had been prepared, each slide was individually examined for evidence of myocardial damage.' The method of evaluation was subjective and myocardial damage was rated on an arbi— trary one to five scale in a manner similar to that of Niles et_a1. (130). Within this arbitrary scale, a rating of one was used to indicate no myocardial damage. A rating of two indicat- ed questionable myocardial damage, while a rating of three indicated the presence of a slight degree of myocardial damage. A rating of four indicated moderate myocardial damage, and a rating of five indicated severe myocardial damage. Both the atria ("a" tissue sections) and ventricles ("b" tissue sections) were examined, but most attention was focused on the left and right ventricles as little myo— cardial damage was evident in the atrial muscle. With the histochemical enzymes, granulomatous tissue areas produced by deposition of diformazan granules were considered to indicate sites of decreasing enzyme activity and thus of myocardial damage (Figure 10). The pattern and intensity of enzyme activity were also studied. These variables are indicators of anoxic damage. With H and E slides, small eosinophilic areas characterized by darkened foci indicated areas of myocardial necrosis (Figures 11, 15). Figure Figure Figure Figure Figure 10 ll l2 13 .--Right ventricular region of the myocardium show- ing decreased SDH activity. Animal is from the exercise before and during anxiety treatment group. (X 400). .—-Same region as shown in Figure 9 but showing the relatively complete loss of B-OHBD activ— ity. The diformazan granules are plainly visible. (X 400). .--Hematoxylin and eosin stain of the same region as shown in Figures 9 and 10. Necrotic voci and scar tissue formation are evident. (Hand E, X 400). .--Sub-endocardia1 region of rat from exercise during anxiety group showing a decreased B-OHBD activity, expecially on the right edge of the photograph. (X 400). .--Same area shown in Figure 12. Scar tissue formation is evident as are the normal cardiac fibers to the left. (Hand E, X 400). 62 A” u:- “6‘ Figure 12 63 Figure 10 Figure 13 Figure Figure Figure Figure Figure 14 15 16 17 18 .--Epicardia1 region of the left ventricle of animal receiving the exercise before anxiety treatment. The almost complete loss of MAO activity is evident. Note the degenerating fibers on the left. (X 400). .--Same region as shown in Figure 14. Scar tissue formation is obvious. (Hand E, X400). .--Mid-ventricular region of control animal. (H and E stain, X 400). .—-Small endocardial region of left ventricle showing loss of SDH activity for animal receiving the anxiety with no exercise treat- ment. (X 400). .--Same region as depicted in Figure 17. Note the fibers on the right are in a degenerative state. (Hand E, X 400). 64 55 Figure 15 Figure 14 : e if 3W“ 1v- Figure 16 Figure 18 Figure 17 66 Thus, myocardial damage was easily defined if, in fact, it was present. The first demonstrable change with the H and E stain is the presence of edema which usually is exaggerated by the formation of ice-crystal artifacts. This was avoided somewhat in this investigation by the quick-freeze tech— nique. Approximately twenty-four hours later, the ede- matous fibers degenerate to form necrotic areas (1). Each slide was evaluated at least twice in order to increase the reliability of the subjective evaluations. A11 ratings were made without prior knowledge of the treatment groups. Statistical Methods Because of the nature of the data, statistical anal- ysis was limited to a non—parametric Chi-square test. A contingency Chi-square was calculated to test the hypoth- esis of no difference in the various treatment groups as far as the production of myocardial damage was concerned. This analysis was completed only on the ventricular tissue sections as the requirements for the Chi-square test could not be met when using the atrial tissue sections. A second Chi-square analysis tested the hypothesis that there was no relationship between the serum LDH levels and the degree of myocardial damage. This analysis was completed for both atrial and ventricular tissue sections. 67 The .05 level of probability was chosen to determine statistical significance for the contingency Chi—square analyses. Because the incidence of myocardial necrosis was sub- jectively determined, some variability naturally existed. In this particular analysis, apparent evidence of myocardial damage was ignored if it occurred on the tissue perimeter or in the immediate vicinity of a large blood vessel, since in these areas it was often difficult to differentiate damage from technique artifact. CHAPTER IV RESULTS AND DISCUSSION Results The purpose of this study was to determine the effects of a specific exercise program and/or electric shock on the production of myocardial damage in male albino rats. Myo— cardial damage was determined subjectively by using both histochemical stains and a standard hematoxylin—eosin stain. Table 1 gives the subjective ratings for myocardial damage for all five treatment groups based on ventricular tissue sections only. Ratings were based on a scale of one to five, with a rating of one meaning no myocardial damage and a rating of five indicating severe myocardial damage. This rating procedure is similar to the one used by Niles _e_t_g_1_. (130). Evidence of myocardial damage was determined largely with the H and E stain. The histochemical stains B-OHD, MAO, and SDH were used to corroborate the evidence of myo- cardial damage as indicated by the H and E stain. The H and E stain and the histochemical stains had to show agreement, as to the degree of myocardial damage, before the final subjective rating was assigned to each animal. 68 69 TABLE 1.—-Frequency distribution of subjective rating of myocardial damage for ventricular tissue sections of the myocardium. Group Rating Con. ANE EDA EBA EBD n l 2 0 0 0 0 2 2 2 6 6 5 5 24 3 4 7 8 8 8 35 4 l 1 l 2 1 6 5 0 l 0 0 1 2 n 9 15 15 15 15 69 Table 2 gives the subjective ratings for myocardial damage for atrial tissue sections. Statistical analysis, be means of a contingency Chi- square, showed no significant differences between the five treatment groups as far as degree of myocardial damage for ventricular tissue sections was concerned (Table 3). During this particular analysis, it was necessary to group the data into fewer than five rankings in order to meet the requirements of the contingency Chi-square test. It seemed logical to group the animals with the ranks of one or two together as these ranks indicated no myo- cardial damage or questionable myocardial damage. The animals ranked three, four, or five were grouped together as these groups indicated at least some definite degree of 70 TABLE 2.--Frequency distribution of subjective rating of myocardial damage for atrial tissue sections of the myocardium. Group Rating Con. ANE EDA EBA EBD n 1 7 7 2 0 19 2 3 6 13 11 37 3 5 2 0 3 12 4 0 0 0 0 0 5 0 0 0 0 0 n 15 15 15 14 68 TABLE 3.-—Chi-square test for treatment effects on the degree of myocardial damage for ventricular tissue sections. Myocardial Damage Group Rank Control ANE EDA EBA EBD n 1-2 4 6 6 5 5 26 3-5 5 9 9 10 10 43 n 9 15 15 15 15 69 Note: Chi-square of .489 (N.S. at the .05 level). 71 myocardial damage. However, one should not be misled by the apparent high incidence of heart damage rated three to five, as most of these cases had a rank of only three in- dicating slight myocardial damage (see Table 1). Table 4 gives the results of the contingency Chi—square test for the relationship between the degree of myocardial damage and the serum LDH levels. The degree of myocardial damage in Table 4 was grouped differently from that in Table 3, as the requirements of the Chi-square test could now be met with three groupings. It should be noted that the total number of animals used in this statistical analysis was only 60, as no serum LDH determinations were made on the control animals. Table 5 gives the results of the contingency Chi- square test for the relationship between the degree of myo- cardial damage and the level of serum lactate dehydro- genase for the atrial tissue sections. Thus, statistical analysis indicates that electric shock and/or forced muscular exercise, as administered in this experiment, were not of sufficient duration or inten— sity to significantly affect myocardial damage. According to the rationale of this study, it had been expected that the exercise during anxiety group of animals would show the most severe heart damage due to the appli- cation of a double stressor. Logically, the anxiety with no exercise group should have shown at least moderate to 72 TABLE 4. -—Chi- -square test for relationship between the degree of myocardial damage for ventricular tissue sections and serum LDH level. Myocardial Damage LDH Level n Rank 0 - .39 .40 - .69 1-2 - 8 15 23 3 9 21 30 4-5 3 4 7 n 20 40 60 Note: Chi—square of .5101 (N.S. at the .05 level). TABLE 5.--Chi-square test for relationship between the degree of myocardial damage and serum LDH levels for atrial tissue sections. Myocardial Damage LDH Level Rank 0 - .39 .40 - .69 1-2 17 ' 33 50 3-5 3 7 10 n 20 ‘ 40 60 Note: Chi—square of .4595 (N.S. at the .05 level). 73 severe myocardial damage. The group of animals subjected to the exercise before and during the anxiety treatment should have shown some minimal amounts of myocardial damage, while both the controls and the exercise before anxiety animals should have shown no myocardial damage. There are several possible explanations for the myo- cardial damage which was observed in this study. The cardiac myopathies might be accounted for by one or a combination of contributory factors. The anxiety treatment probably caused, at least in the beginning stages, some increased catecholamine release from the adrenal glands. The harmful effects of large amounts of catecholamines on the cardiac muscle have been well documented in previous studies (7, 57, 137, 138). Thus, if the anxiety treatment was of sufficient intensity to cause massive secretion of catecholamines, the resulting myocardial infarcts could, in part, be attributable to the direct effect of catecholamines on the heart. It is doubtful, however, that sufficient quantities of catech- olamines were released in this study to produce such effects. A subjective evaluation of adrenal glad weights showed no apparent differences between the various treatment-groups means. Even if the secretion of catecholamines elicited in this study was small, it might have been sufficient to cause cardiac necroses if certain accessory conditions were 74 met. The most obvious of these would be an advanced degree of atherosclerosis in the coronary arteries (94, 127, 138, 191). However, the degree of atherosclerosis in the animals (involved in this experiment was not measured, so there was no way of determining whether or not atherosclerosis was a contributing factor. In contrast, the relative lack of consistent and extensive myocardial damage also could be attributed to one or several factors. If coronary artery atherosclerosis was non-existant or minimal, then the increased myocardial oxygen demand caused by any moderate elevation of cate- cholamines would be of little pathologic significance. Under normal circumstances, moderate stress produces no serious cardiac damage in healthy animals (175). In fact, electrolytes, corticoids, or stressors, as a rule, produce no consistent cardiac change in themselves. The myocardial necroses of the simple stress cardiopathy only develop after severe exposure to stress alone and occur only under exceptional circumstances. Periodic examination of the animals receiving the anxiety treatment revealed that, except for the first day or two, the amount of electric shock received by the animals provided little stress. Many, in fact, slept through much of the anxiety treatment. The simple stress cardiopathy categorized by Selye (176) is produced by exposure to sudden intense stress without any special conditioning. The resulting cardiac 75 lesions manifest themselves histologically as scattered _necroses of individual muscle fibers or fiber groups. How— ever, these lesions are so small that they do not tend to terminate in permanent scar tissue formation. In addition, such microscopic foci tend to heal within a few days without leaving a trace. Thus, it is possible the anxiety treatment did, in fact, produce small foci of necrosis, but any trace of these had vanished by the time the animals were sacri- ficed. Finally, the effects of the five-week or a seven— week training program may have been sufficient to condition the hearts of some of the animals against the production of myocardial damage. Physical training improves general cardiac efficiency which results in several possible benefits. It could cause improved coronary collateral circulation (97) so that impaired coronary artery flow due to atherosclerosis is better tolerated. Physical training reduces cardiac sympathetic tone and increases cardiac parasympathetic tone which is manifested by a decreased heart rate and decreased cardiac output for a given level of work (77). This, of course, results in a reduced excess oxygen utilization of the myocardium, and conditions of myocardial hypoxia or anoxia are less likely to develop. It should be noted, however, that if these possible benefits of physical activity had been fully operative in this study, significant groups differences should have been observed. 76 In summary then, myocardial damage could have been elicited by the combined actions of the catecholamines, coronary artery atherosclerosis, and electrical stress. Some transient myocardial damage may have been missed by the techniques used. Finally, myocardial damage could have been prevented by the effects of the physical ex- ercise program and a lack of coronary artery atheros- clerosis. The scarcity of a large number of high LDH readings, a supposed indicator of conditions of myocardial ischemia, provided additional evidence that, in general, the animals had adapted to the anxiety treatment. In any case, no sig- nificant results were obtained. wt '1 ‘n' {'33P .x CHAPTER V SUMMARY, CONCLUSIONS, RECOMMENDATIONS Summary The purpose of this study was to determine the effects of physical exercise and/or electrical stress on the pro- duction of experimental myocardial damage. One hundred and six male albino Sprague-Dawley rats 60 days of age were randomly assigned to five treatment groups. These groups were control, anxiety with no exer- cise, exercise during anxiety, exercise before anxiety, and exercise before and during anxiety. The last three treat- ment groups were subjected to two thirty-minute swim periods per day, seven days a week, with a weight equal to two per cent of the body weight attached to the tip of the tail. All animals were fed ad libitum with commercial laboratory blockfeed. Ambient air temperatures in the animal quarters were kept between 21 to 25 C. Those animals subjected to the anxiety treatment re- ceived a .36 sec D.C. electrical shock of 1.5 M.A. five times a minute, nine hours a day, for two weeks. The elec- trical shocks were administered in a random pattern. All anxiety animals began their treatments at one hundred days of age. 77 78 All animals were sacrificed at 116 days of age with an overdose of ether. The heart was excised immediately, trimmed and cut in three equal sections. The apical vent- ricular section and the basal auricular section were imme- diately quick-forzen in isopentave cooled with liquid nitrogen. The middle section was fixed in a ten per cent formalin solution for forty-eight hours. At least two fresh-frozen serial cross sections approximately ten micra thick were cut and stained with a standard H and E stain as well as with the histochemical enzymes SDH, MAO, and B-OHBD. Formalin-fixed tissue sections were stained with a paraffin embedded H and E stain. Heart damage was evaluated subjectively using a one to five rating scale. A rating of one indicated no myo- cardial damage, while a rating of five indicated severe myocardial damage. All ratings were made without know- ledge of the treatment groups. Serum LDH levels were determined by a standard photo- metric method, and LDH levels were used to select those animals for sacrifice which, according to the LDH level, showed some evidence of myocardial damage. The data were evaluated statistically using non- parametric contingency Chi-square analyses. It was found that a chronic swimming program and/or electrical shock did not significantly affect heart damage. Because of the lack of severe heart damage, Serum LDH levels 79 were not useful indicators of myocardial cardiopathies. The heart damage which was elicited by the experimental treat- ments and detected with the H and E stain was confirmed in each case by decreased histochemical enzyme activity in a Similar section of cardiac tissue. Conclusions The results of this study have led to the following conclusions: 1. A .36 sec. D.C. electrical shock of 1.5 M.A. lasting 9 hours a day for 2 weeks is not a suf- ficient stressor to induce extensive myocardial damage. Exercise treatments, as used in this study, did not significantly affect myocardial damage. Serum LDH did not accurately predict the minimal amounts of myocardial damage found in this ex- periment. Recommendations A similar study should be done which involves greater exercise intensities as well as different types of exercise. A more systematic guideline for the rating of myocardial damage should be established. This 80 would be of benefit to future investigators. Animals participating in such a study should be "sensitized" during the study by the use of either a high cholesterol or a high fat diet. Although MAO is a good early indicator of myo- cardial damage, more work needs to be done with this particular enzyme as uniform results were difficult to obtain. Serum LDH determinations Should be made more fre- quently. Serum catecholamine concentrations should be de- termined before, during, and after stress. Cross sections of the aorta should be made, and histochemical analysis of the extent of athero— sclerosis determined. This should also be carried out on the coronary arteries. A sequence of sacrifice times should be considered in order to take advantage of the predictive qualities of the various histochemical enzymes. A quicker, more administrable method of stressing the animals would be advisable. An example would be 15 to 20 hours of forced restraint which has been proven to yield excellent results (176). BIBLIOGRAPHY 81 BIBLIOGRAPHY Adams, C. W. M. Vascular Histochemistry. Year Book Medical Publishers. Chicago, Illinois. 1967. Ahrens, E. H.; Hirsch, J.; Insull, W.; Tsaltes, T. T.; Blomstrand, R.; and Peterson, M. L. The influence of dietary fats on serum lipid levels in man. Lancet. 1:943, 1957. .:_4 Allela, A.; Williams, F. L.; Bolen-Williams, C.; and . Katz, L. N. Interrelation between cardiac oxygen L' consumption and coronary blood flow. American “1 Journal Physiology. 183:570, 1955. American Heart Association. Dietary fat and its relation to heart attacks and strokes. Circ. 33:133, 1961. Andreev, I., and Doskov, I. Coronary disease with myocardial infarction following electrotrauma. Abstract Bulgariam Scient. Lit. 1:50, 1958. Anitchkov, N. N.; Walter, A. V.; Volkova, K. G.; and Sinitsina, I. A. Morphological fundamentals of the origin of the myocardial infarct. In Trudy Usyesoyuznoy Konterentsii Patologoanatomov. Moscow, 1956. Page 246. Bajusz, E., and Raab, W. Early metabolic aberrations through which epinephrine elicits myocardial necrosis. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas Publisher, Springfield, Illinois. 1966. Bajusz, E., and Jasmin, G. Comparative morphogenesis and enzyme histogenesis of some occlusive and metabolic cardiac necroses. Rev. Canad. de Biol. 22:181, 1963. Bajusz, E., and Jasmin, G. Observations on histo- chemical differential diagnfisis between primary and secondary cardiomyopathies. Amer. Heart. Journ. 69:83, 1965. 82 10. ll. 12. l3. 14. 15. 16. 17. 18. 19. 20. 83 Bajusz, E., and Jasmin, G. Histochemically demonstrable phosphorylase as an early index of anoxic myocardial damage. Experientia 29:373, 1964. Bajusz, E. (editor). Electrolytes and Cardiovascular Diseases. Physiology-Pathology-Therapy. Vol. 1: Fundamental Aspects; Vol. 2: Clinical Aspects. Basel-New York. S. Karger, 1965. Bajusz, E. Unspecific systemic stress reactions and necrotizing cardiopathies. Amer. Journ. Phys. Med. 39:153, 1960. Bajusz, E., and Selye, H. Conditioning factors for cardiac necroses. Tr. New York Acad. Sc. 21:659. 1959. Bajusz, E., and Selye, H. The chemical prevention of cardiac necroses following occlusion of coronary vessels. Canad. Med. Assoc. Journ. 82:212, 1960. Bajusz, E., and Selye, H. Adaptation to the cardiac necrosis-eliciting effect of stress. Am. Journ. Physiol. 199:153, 1960. Bajusz, E., and Jasmin, G. Histochemical studies on the myocardium following experimental interference with the coronary circulation in the rat. I Occlusion of coronary artery. Acta Histochem. 18:222-237, 1964. Barka, T., and Anderson, P. J. Histochemistry Theory, Practice and Bibliography. Harper and Row, New York, 1963. Basu, A.; Passmore, R.; and Strong, J. A. The effect of exercise on the level of non-esterified fatty acids in the blood. Quart. Jour. Exper. Physiol. 65:312, 1960. Bell, G. H.; Davidson, J. N.; and Scarborough, H. Textbook of Physiology and Biochemistry. Williams and Wilkins Co. Baltimore, 1965. page 137. Beveridge, J. M. R.; Connell, W. F.; Haust, H. L.; and Mayer, G. A. Dietary cholesterol and plasma chole- sterol levels in man. Canad. Journ. Biochem. Physiol. 37:575, 1959. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 84 Bing, R. J.; Danforth, W. H.; and Ballard, F. B. Physiology of the myocardium. J. A. M. A. 172:438, 1960. Blatt, W.; Walker, J.; and Mayer, M. Tissue lactic dehydrogenase isozymesz‘ Variation in rats during prolonged cold exposure. Amer. Journ. Physiol. 209:785, 1965. Boas, E. P. Angina pectoris and cardiac infarction from trauma or unusual effort. J. A. M. A. 112:1887, 1939. Bois, P.; Jasmin, G.; and Band, P. R81e de la cortico- surrénale et du rein dans la production de necroses du myocarde. Ann. A.C.F.A.S. 25:63, 1957-58. Bogdonoff, M. D.; Estes, E.H.; Harlan, W. R.; Trout, D. A.; and Kushner, N. Metabolic and cardiovascular changes during a state of acute central nervous system arousal. Journ. Clin. Endocrin. 20:1333, 1960. Brouha, L. Effect of work on the heart. In Work and the Heart. Ed. by Rosenbaum, F. F. and Belknop, E. L. New York, P. B. Heeber, Inc., 1958. page 108. Brunner, D. The influence of physical activity on incidence and prognosis of ischemic heart disease. In Prevention of Ischemic Heart Disease. Ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Bryant, R. E.; Thomas, W. A.; and O'Neal, R. M. An electron microscopic study of myocardial ischemia in the rat. Circ. Res. 6:699, 1958. Burstone, M. S. Histochemical demonstration of cyto- chrome oxidase with new amine reagents. Journ. Histochem. Cytochem. 8:63, 1960. Burstone, M. S., and Miller, E. N. Histochemical demon— stration of changes in cytochrome oxidase activity in human myocardial infarctions. Amer. Journ. Clin. Path. 35:118, 1961. Cabaud, P. G. and Wroblewski, F. Colorimetric measure- ment of lactic dehydrogenase activity of body fluids. Amer. Journ. Clin. Path. 30:234, 1958. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 85 Cannon, W. B. The mechanism-of emotional disturbance of bodily functions. New Eng. Journ. Med. "198:877, 1928. Carlson, L. A., and Wadstrom, L. B. Determination of glycerides in blood serum. Clin. Chem. Acta 4:197, 1959. Carlson, L. A. Acute effects of exercise on lipid and carbohydrate metabolism in some animals. In Physical Activity and the Heart. Chas. C. Thomas Publisher. Springfield, Illinois, 1967. Carlson, L. A., and Pernow, B. Studies on blood lipids during exercise. I. Arterial and venous plasma concentrations of unesterified fatty acids. Journ. Lab. Clin. Med. 53:833, 1959. Carlson, L. A., and Pernow, B. Studies on blood lipids during exercise. 11. The arterial plasma free fatty acid concentration during and after exercise and its regulation. Journ. Lab. Clin. Med. 58:673, 1961. Carlson, L. A. Serum lipids in men with myocardial infarctions. Acta. Med. Scand. 167:399, 1960. Casanegra, P.; Pacifico, A.; Hellens, H. K.; and Lehan, P. H. Catecholamine action on phasic coronary flow patterns. Clin. Res. 11:392, 1963. Caulfield, J., and Klionsky, B. Myocardial ischemia and early infarction. An electron microscopic study. Amer. Journ. Path. 35:489, 1959. Chapman, J., and Massey, F. The interrelationship of serum cholesterol, hypertension, body weight, and risk of coronary disease. Journ. Chron. Dis. 17:933, 1964. Cherkovich, G. M. Micronecroses in the heart of the monkey as a result of experimental neurosis. Patalogeecheskaya fiziolguja y eksperimental nayaterapiya. Moscow, Medioiz, 1959. Chidsey, D.; Harrison, C.; and Braunwold, E. Augmen— tation of plasma norepinephrine response to exer- cise in patients with congestive heart failure. New Eng. Journ. Med. 267:650, 1962. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 86 Cox, J. L.; McLaughlin, V. W.; Flowers, N. C.; and Horan, L. G. Changes in cellular dehydrogenase enzymes in acute myocardial infarction. Circ. 34 (suppl. III) 80, 1966. Cureton, T. K., Jr. Anatomical, physiological, and psychological changes induced by exercise programs in adults. In Exercise and Fitness. Chicago. The Athletic Inst. Univ. of Illinois. Colloguuim Report. 1960. Dawber, T. R., and Kannel, W. B. Susceptibility to coronary heart disease. . Med. Conc. Cardiov. Dist. 30:671, 1961. Dawber, T. R.; Kannel, W. B.; Revotskie, N.; Stokes, J.; Kagan, A.; and Gordon, T. Some factors asso- ciated with development of coronary heart disease. Amer. Journ. Pub. Health. 49:1349, 1959. Dawber, T. R.; Kannel, W. B.; and Friedman, G. D. Vital capacity, physical activity, and coronary heart disease. In Prevention of Ischemic Heart Disease. Ed. by Raab, W. Chas. C. Thomas, Pub- lisher, Springfield, Illinois, 1966. Dawber, T. R.; Meadors, G. F.; and Moore, F. E. Epidemiological approaches.to heart disease: The Framingham Study. , Amer. Journ. Pub. Health. 41:279, 1951. Diculesco, I.; Onicesco, D.; and Mischui, L. A histo- chemical analysis of dehydrogenase variety in the different types of muscular tissue. Journ. Histochem. Cytochem. 12:145, 1964. Du Ruisseau, J. P., and Mori, K. Biochemical studies on electrolyte-steroid cardiopathy : electrolytes in rat tissues. Br. Journ. Exper. Path. 40:250, 1959. Eckstein, R. W.; Stroud, M.; Dowling, C. V.; Eckel, R.; and Pritchard, W. H. Response of coronary blood flow following sympathetic nerve stimulation. Amer. Journ. Physiol. 162:266, 1950. Eckstein, R. W. Effect of exercise and coronary artery narrowing on coronary collaterol circula- tion. Circ. Res. 5:230, 1957. 53. 54. 55. 57. 58. 59. 60. 61. 62. 53. 87 Ellis, S. The Metabolic effects of epinephrine and related amines. Pharm. Rev.- 8:485, 1956. Elmadjian, F.; Hope, J. M.; and Lamson,.E. T. Ex- cretion of epinephrine and norepinephrine in various emotional states._ Journ. Clin. Endocr. ‘17:608, 1957. Elmadjian, F.; Hope, J. M.; and Lamson, E. T. EX- cretion of epinephrine and norepinephrine under stress. Recent Progr. Hormone Res. 14:513, 1958. Euler, V. 8. von; Gemzell, C. A.; Levi, L.; and Strom, A G. Cortical and medullary adrenal activity in = emotional stress. ‘ Acta. Endocr. 30:567, 1959. . Ferrans, V. J.; Hibbs, R. G.; Black, W. C.; and Weilbaecher, D. B. Isoprotereriol-induced myo- cardial necrosis. A histochemical and electron microscopic study. Amer. Heart Journ. 68:78, 1964. Fine, G.; Merales, A.; and Scarpella, J. R. Experi- mental myocardial necrosis. Arch. Path. 82:4, 1966. Fox, J. M. III., and Skinner, J. S. Physical activity and cardiovascular health. Amer. Journ. Cardiol. 14:731, 1964. Frank, C. W.; Weinblatt, E.; Shapiro, S.;.and Sager, R. V. Physical inactivity as a lethal factor in myocardial infarction among men. 533. 34:1022, 1966. Frick, M. H.; Konttinen, A.; and Sarajas, H. S. S. Effects of physical training on circulation at rest and during exercise. Amer. Journ. Cardiol. 12:142, 1963. Friedling, C. K., and Horn, H. Acute myocardial in- farction not due to coronary artery occlusion. J.A.M.A. 112:1675, 1939. Friedman, M.; St. George, S.; Byers, S. O.; and Rosenman, R. H. Excretion of catecholamines, l7 - ketosteroids, 17 - hydroxy corticoids, and 5 - hydroxyindole in men exhibiting a particular behavior pattern associated with high incidence of 64. 65. 670 68. 69. 70. 71. 72. 73. 74. 88 clinical coronary artery disease. Journ. Clin. Invest. 39:758, 1960. Friedman, M.; Rosenman, R. H.; and Byers, S. 0. De- ranged cholesterol metabolism and its possible relationship to.atherosclerosis : a review. Journ. Geront. .10:60, 1955. Garbus, J.; Highman, B.; and Altland, P. D. Serum enzyme and lactic dehydrogenase isozymes after exercise in rats. Amer. Journ. Physiol. 207:467, 1964. Gazes, P. C.; Richardson, J. A.; and Woods, E. F. Plasma catecholamine concentrations in myocardial infarction and angina pectoris. Circ. 19:657, 1959. Gorlin, R. Physiologic studies in coronary athero- sclerosis. Fed. Proc. 21:93, 1962. Gertler, M. M.; Garn, S. M.; and Lerman, J. The inter-relationship of serum cholesterol, choles- terol esters and phospholipids in health and in coronary artery disease. Circ. 2:205, 1950. Golding, L. Effect of physical training upon total serum cholesterol levels. Res. Quart. 32:499, 1961. Grande, F. Dietary factors and serum cholesterol in man. In Prevention of Ischemic Heart Disease. Grey, J., and Beetham, W. P. Changes in plasma con- centration of epinephrine and norepinephrine with muscular work. Proc. Soc. Exp. Biol. Med. 96:636, 1957. Groover, M. E. Myocardial infarction without athero- sclerosis in the Kenya baboon. Circ. 26:645, 1962. Groover, M. E., and Stout, C. Neurogenic.myocardial necrosis. In Prevention of Ischemic Heart Disease. Grunbaum, B. W.; Geary, J. R.; Grande, F.; Anderson, J. T.; and Glick, D. Effect of dietary lipids on rat serum cholesterol and tissue mast cells. Proc. Soc. Exp. Biol. Med. 94:613, 1957. 75. 76. 77. 79. 80. 81. 82. 83. 84. 89 Grundy, S. M., and Griffin, A. C. Relationship of periodic mental stress to serum lipoprotein and cholesterol levels. J.A.M.A. 171:1794, 1959. Gubner, R. S. Potassium servomechanism regulating cardiac work. Role of potassium in myocardial disease and angina pectoris. Clin. Res. 6:209, 1958. Guyton, A. Textbook of Medical Physiology, Third Edition. W. B. Saunders Co., Philadelphia and London, 1966. Harrison, T. R., and Reeves, T. J. Principles and Problems of Ischemic Heart Disease. Year Book Medical Publishers. Chicago, Illinois, 1968. Havel, R. J., and Goldfien, A. The role of the sympathetic nervous system in the metabolism of free fatty acids. Journ. Lipid Res. 1:102, 1959. Havel, R. J.; Naimark, A.; and Borschgrevink, C. F. Turnover rate and oxidation of free fatty acids of blood plasma in man during exercise: Studies during continuous infusion of palmitate - l - Cl”. Journ. Clin. Invest. 42:1054, 1963. Hernberg, Sven. Correlation between physical working capacity and serum cholesterol in leading business- men. In Physical Activity and the Heart. Chas. C. Thomas, Publisher, Springfield, Illinois, 1967. Herrlich, H. C.; Raab, W.; and Gigee, W. Influence of muscular training and of catecholamines on cardiac acetylcholine and cholinesterase. Arch. Int. Pharmacodyn. 129:201, 1960. Holloszy, J. P.; Skinner, J. 8.; Barry, A. J.; and Cureton, T. K., Jr. Effect of physical condition- ing on cardiovascular function. Amer. Journ. Cardiol. 14:761, 1964. Holoszy, J. O. The epidemiology of coronary heart disease : National differences and the role of physical activity. Journ. Amer. Geriat. Soc. 11:718, 1903. 850 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 90 Jasmin, G., and Bajusz, E. International.symposium on Progress in Muscle Research. II. Cardiac Muscle. From Rev. Canad. de Biol. Volm 22, No. 2, 1963. Jennings, R. B., and Wartman, W. B. Production of an area of homogeneous myocardial infarction in the dog. A.M.A. Arch. Path. 63:580, 1957. Josue, O. Hypertrophic cardiogue causee par 1'ardena- line et la toxine typhigue.- .. Compt. Pend. Soc. Biol. 63:285, 1907. Karr, G. W.; Nickerson, M.; and Drese1,.P..E. Role of potassium in the.electrolyte—steroid.cardio- pathy. Proc. Canad. Fed. Biol. Soc. 3rd Ann. Meeting, June 8 - 10, Winnipeg, Man. 1960, page 31. Katz, L. N.; Williams, F. L.; Laurent, D.; Bolene— Williams, C.; and Feinberg, H. Effects of l—nor- epinephrine and l—epinephrine on coronary flow and oxygen consumption of the intact open Chest dog. Fed. Proc.. 15:106, 1956. Katz, L. N. Recent concepts of the performance of the heart. Circ. 28:117, 1963. Kaufman, N.; Gavan, T. C.; and Hill, R. W. Experi- mental myocardial infarction in the rat. A.M.A. Arch. Path. 67:482, 1959. Kent, S. P., and Diseker, M. Early myocardial ische- mia study of histochemical changes in dogs. Lab. Invest. 4:398, 1955. Keys, A. Cholesterol and anticholesterol agents in regard to arteriosclerosis and its complications. Mal. Cardiov. 4:93, 1903. Keys, A. Diet and the epidemiology of coronary heart disease. J.A.M.A. 164:1912, 1957. Keys, A.; Taylor, H. L.; Blackburn, H.; Brozek, J.; Anderson, J. T.; and Simonson, E. Coronary heart disease among Minnesota business and professional men followed fifteen years. Circ. 28:381, 1963. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 91 Kipshidze, N. N. Role of functional factor in the pathogenesis of myocardial infarction. In Pre- vention of Ischemic Heart Disease. Ed. by Raab, W. Chgz. C. Thomas Publisher, Springfield, Illinois. 19 . Kitamura, K. The role of sport activities in the pre- vention of cardiovascular malfunction. In Pro— ceedings of International Congress of Sports Sciences, 1964. University of Tokyo Press, Tokyo, Japan. 1966. Klionsky, B. Myocardial ischemia and early infarc- tion. A histochemical study. Amer. Journ. Path. 36:575, 1960. Konttinen, A., and Nikkila, E. A. The effects or acute exercise on serum triglycerides and free fatty acids. In Physical Activity and the Heart. Chgs. C. Thomas, Publisher. Springfield, Illinois, 19 7. Konttinen, A.; Sarajas, H. S. S.; Frick, M. H.; and Rajasalona, M. Arteriovenous relationship of non- esterified fatty acids, triglycerides, cholesterol, and phospholipids in exercise. Ann. Med. Experim. Fenn. 40:250, 1962. Karvonen, M. J., and Barry, A. J. Physical Activity and the Heart. Chas. C. Thomas, Publisher. Springfield, Illinois, 1967. Kraus, Hans. Preventive aspects of physical fitness. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Kraus, H., and Raab, W. Hypokinetic Disease -— Dis— eases Produced by Lack of Exercise. Chas. C. Thomas, Publisher. Springfield, Illinois, 1961. Kroman, H.; Nodine, J.; Bender, S.; and Brest, A. Lipids in normals and patients with coronary artery disease. Amer. Journ. Med. Sc. 248:571, 1964. Lapiccirella, V. Emotion-induced cardiac disturbances and possible benefits from tranquil living. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois. 1966. 106. 107. 108. 109. 110. 111. 112 O 113. 114. 115. 116. 92 Levi, L. Life stress and urinary excretion of adrena- line and nor-adrenaline. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Levi, L. The urinary putput of adrenaline and nor- adrenaline during experimentally induced emotional stress in clinically different groups. Acta Psychother. 11:218, 1963. Lowry, O. H.; Gilligan, D. R.; and Hastings, A. B. Histochemical changes in the myocardium of dogs following experimental temporary coronary arterial occlusion. Amer. Journ. Physiol. 136:474, 1942. Luongo, E. P. 'Health habits and heart disease -- challenge in preventive medicine. J.A.M.A. 162:1021, 1956. Lushnikov, E. F. Histochemical study of experimentally produced myocardial infarction. Fed. Proc. 22: Trans. Supple. l. 906, 1963. Maling, H. M. - ,Highman, B.; and Thompson, E. C. Some similar effects after large doses of catecholamines and myocardial infarction in dogs. Amer. Journ. Card. 5: 628, 1960. Marchetti, G.; Maccari, M.; and Merlo, L. Récherches expérimentales sur les effets de 1' adrenaline et de la 1 - nordrénaline sur la circulation coronarienne. Cardiologia. 42:1, 1963. Mascitella-Coriandoli, E.; Boidrini, R.; and Citterio, C. Cardiac damage caused by experimental stress. Nature. 181:1215, 1958. Mason, J. W.; Mangan, G.; Brady, J. V.; Concrad, D.; and Rioch, D. Concurrent plasma epinephrine, nor- epinephrine and l7—hydroxy cortico—steroid during conditioned emotional disturbances in monkeys. Psychosom. Med. 23:344, 1961. McAllen, P. M. Myocardial changes occuring in potassium deficiency. Br. Heart. Journ. 17:5,1955. McCabe, W.; Hammarsten, J.; Schottstaedt, W.; Adsett, C. A.; Yamamoto, J.; and Wolf, S. Elevation of .serum cholesterol in man in association with life stress and independent of diet and exercise. Journ. Lab. Clin. Med. 54:922, 1959. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 93 Melcher, G. W., and Walcott, W. W. Myocardial changes following shock. Amer. Journ. Physiol. 194:832, 1951. Mellorowicz, H. The effect of training on heart and circulation and its importance in preventive. cardiology. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Pub- lisher. Springfield, Illinois, 1966. Mellorowicz, H. Verfeichende Untersuchungen fiber das Oekonomie prinzip in Arbeit and Leistung des traimerten kreiolaufs und seine bedeutung ffir die praventive und rehabilitative medizin. Arch. Kreislaufforsch. 24:70, 1956. Melville, K. I., and Shister, H. E. Cardiac responses to epinephrine and norepinephrine during prolonged cholesterol and high fat feeding in rabbits. Amer. Journ. Cardiol. 4:391, 1959. Miller, R. D.; Burchell, H. B.; and Edwards, J. E. Myocardial infarction with.and without coronary occlusion. Arch. Int. Med. 88:597, 1951. Minc, S. Civilized pattern of activity, cardiac adaptation, and ischemic heart disease. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Publisher, Springfield, Illinois, 1966. - Morris, J. N.; Heady, J.; and Raffle, P. A. B. Physique of London busmen. 'Lancet 2:569, 1956. Morris, J. N.; Heady, J.; Raffle, P. A. B.; Roberts, C. G.; and Parks, J. W. Coronary heart disease and physical activity of work._ Lancet 2:1053, 1953. Morris, J. N., and Crawford, M. D. Coronary heart disease and physical activity of work. Br. Med. Journ. 2:1485, 1958. Miller, E., and Pearse, A. G. E. Localization of monoamine oxidase in mammalian and reptilian heart. Br. Heart. Journ. 27:116, 1965. Myasnikov, A. L. Myocardial necrosis of coronary and non-coronary genesis. Amer. Journ. Cardiol. 13:435, 1964. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 94 Nasmyth, P. A. Effect of cortico-steroids on isolated mammalian heart and its response to adrenaline. Journ. Physiol. 139:323, 1957. Nikkila, E. A., and Torsti, P. Adjustment of fat metabolism to exercise as studied in vitro. In Physical Activity and the Heart. Chas. C. Thomas, Publisher. Springfield, Illinois, 1967. Niles, N. R.; Zavin, J. D.; and Monikado, R. N. Histochemical study of effects of hypoxia and isoproterenol on rat myocardium. Amer. Journ. Cardiol. 22:381, 1968. Niles, N. R.; Bitensky, L.; Chayen, J.; Brainbudge, M. V.; and Cunningham, G. J. The value of histo- chemistry in the analysis of myocardial dysfunction. Lancet. 1:963, 1964. Nuzum, F. R., and Bischoff, F. The urinary putput of catechol derviatives including adrenaline in normal individuals, in essential hypertension, and in myocardial infarction. Circ. 7:96, 1953. ‘Pearse, A. G. E. Histochemistry. Theoretical and Applied. London. Churchill Ltd. 1960. Pearse, A. G. E. The histochemistry and electron microscopy of destructive cardiomyopathies. In Cardiomyopathies page 132-164. ed. by G. E. W. Wolstenholme and M. O'Connor. London:Churchill. Pearson, N. E. S., and Joseph, J. Stress and occlusive coronary artery disease. Lancet. 1:415, 1963. Poppen, K. J.; Green, D. M.; and Wrenn, H. T. The histochemical localization of potassium and glycogen. ' Journ. Histochem. Cytochem. 1:160, 1953. Raab, W. The nonvascular metabolic myocardial vulner— ability factor in coronary heart disease. Amer. Heart Journ. 66:685, 1963. Raab, W. Neurogenic multifocal destruction of myo- cardial tissue. Rev. Canad. de. Biol. 22:217, 1963. Raab, W., and Gigee, W. Specific avidity of the heart muscle to absorb and store epinephrine and nor- epinephrine. Circ. Res. 3:553, 1955. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 95 Raab, W.; Stark, E.; MacMillan, W. H.; and Gigee, W. R. Sympathogenic origin and antiadrenergic.prevention of stress induced myocardial lesions. Amer. Journ. Cardiol. 8:203, 1961. Raab, W.; Chaplin, J. P.; and Bajusz, E. Myocardial necrosis produced in domesticated rats.and in wild rats by sensory and emotional stress. Proc. Soc. Biol. Med. 116:665, 1964. Raab, W.; vanLith, P.; Lopeschkin, F.; and Herrlich, H. C. Catecholamine-induced myocardial hypoxia in the presence of impaired coronary dilatability independent of external cardiac work. Amer. Journ. Cardiol. 9:455, 1962. Raab, w. The pathogenic significance of adrenalin and related substances in the heart muscle. Exp. Med. Supg. 1:188, 1943. Raab, W. Hormonal andLNeurogenic Cardiovascular Disorders. Williams and Wilkins Co. Baltimore, 1953. Raab, W., and Gigee, W. R. Norepinephrine and epin— ephrine content of normal and diseased human hearts. Circ. 11:593, 1955. Raab, W. The neurogenic metabolic factor in ischemic heart disease. Pathogenesis and prevention. Dis. Chest 46:150, 1964. Raab, W. The sympathogenic biochemical trigger mechanism of angina pectoris. Its therapeutic suppression and long-range prevention. Amer. Journ. Cardiol. 9:576, 1962. Raab, W., and Krzywanek, H..J. Cardiac-sympathetic tone and stress response related to personality patterns and exercise habits. In Prevention of Ischemic Heart Disease ed. by Raab, W.. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Raab, W.; DePaula, E.; Silva, P.; Marchet, H.; Kimura, E.; and Starcheska, Y. K. Cardiac adrenergic overactivity due to lack of physical exercise and its pathogenic implications. Amer. Journ. Cardiol. 5:300, 1960. -l. -'A “am ‘ . ~. . 1 I 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 96 Raab, W.; DePaula, E.; Silva, P.; and Starcheska, Y.K. Adrenergic and cholinergic influences on the. dynamic cycle of the normal human heart. Cardiologia 33:350, 1958. Raab, W. Neurohormonal atherogenesis. Amer. Journ. Cardiol. 1:113, 1958. Raab, W. Metabolic protection and reconditioning of the heart muscle through habitual physical exercise. Ann. Intern. Med. 53:87, 1960. Raab, W.; Stark, B.; and Gigee, W. R. Role of catecho- lamines in the origin of stress induced myocardial necrosis. Circ. 20:754, 1959. Raab, W. The catecholamines-in.cardiovascular pathology. Cardiologia 36:181, 1960. Raab, W. LOafer's heart. A.M.A. Arch. Int. Med. 101:194, 1958. Regan, T. J.; Mosches, C. B.; Oldewurtel, H. A.; and Heelems, H. K. Metabolic role of catecholamines and the production of myocardial necrosis. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Regan, T. J.; Lehan, P. H.; Henneman, D. H.; Behar, A.; and Hellems, H. K. Myocardial metabolic and contractile response to glucagon and epinephrine. Journ. Lab. Clin. Med. 63:638, 1964. Richardson, J. A. Plasma catecholamines in angina pectoris and myocardial infarction. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chgz. C. Thomas, Publisher. Springfield, Illinois, 19 . Richardson, J. A.; Woods, E. F.; and Bagwell, E. E. Circulating epinephrine and nor-epinephrine in coronary occlusion. Amer. Journ. Cardiol. 5:613, 1960. Richardson, J. A. Circulating levels of catecholamines in acute myocardial infarction and angina pectoris. Prog. Cardiov. Dis. 6:56, 1963. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 97 Rochelle, R. Blood plasma cholesterol changes during a physical training program. Res. Quart. 32:538, 1961. Rona, G.; Chappel, C. I.; and Kahn, D. S. Experimental production of chronic cardiac aneurysm and con- gestive heart failure.in the rat. Exp. Melec. Path. 2:40, 1963. Rosenblatt, G.; Stokes, J.; and Bassett, D. R. Whole blood viscosity, hematocrit, and serum lipid levels in normal subjects and patients with coro- nary heart disease. Journ. Lab. Clin. Med. 65:202, 1965. Rosenman, R. H. The role of a specific overt behavior pattern in the genesis of coronary heart disease. In Prevention of Ischemic Heart Disease. .ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Rosenman, R. H., and Friedman, M. The role of a specific behavior pattern in the occurrence of ischemic heart disease. ' Cardiol. Prot. 13:42, 1962. Rosenman, R. H., and Friedman, M. Behavior patterns, blood lipids, and coronary heart disease. J.A.M.A. 184:934, 1963. Rosenman, R. H., and Friedman, M. The possible re- lationship of occupational stress to clinical coronary heart disease. . Calif. Med. 89:169, 1958. Russek, H. J., and Zohman, B. C. Relative signifi— cance of heredity, diet, and occupational stress in coronary heart disease of young adults. Amer. Journ. Med. Sc. 235:266, 1958. Russek, H. J. Role of heredity, diet and emotional stress in coronary heart disease. J.A.M.A. 171:503, 1959. Russek, H. J. Emotional stress and the etiology of coronary artery disease. ' Amer. Journ. Cardiol. 2:129, 1958. Schopiro, S., and Marmorston, J. Response to stress differing responses of atherosclerotic and normal dog to electric shock or cold.‘ Proc. Soc. Exp. Biol. Med. 103:520, 1960. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 98 Shimamoto, T., and Hiramoto, Y. Electron microscopic observations on edematous myocardial response.to epinephrine. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Pub- lisher. -Springfield, Illinois, 1966. Schinert, G. C., and Schwalb, H. Functional and metabolic factors in the origin and.prevention of myocardial ischemia. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Schnitka, T. K., and Nachlas, M. M. Histochemical alterations in ischemic heart muscle and early myocardial infarction. Amer. Journ. Path. 42:507, 1963. Selye, H. The role of stress in the production and prevention of experimental.cardiopathies. .In Prevention of Ischemic Heart Disease. ed. by ‘ Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois,'1966. Selye, H. The Pluricausal Cardiopathies. Chas. C. Thomas, Publisher. Springfield, Illinois, 1961. Selye, H. Conditioning by cortisol for the pro- duction of acute massive myocardial necroses during neuromuscular exertion. . Circ. Res. 6:168, 1958. Selye, H. The humoral production of cardiac infarcts. Brit. Med. Journ. Mar. 15, 1958. page 599. Selye, H; Le stress et l'infarctus du myocarde. Journees endocrinol. Marseille-med. No. 7 575, 1958. Selye, H. Stress and myocardial necroses. New Phys. 8:11, 1959. Selye, H. Stress and cardiac necroses. Clin. Med. 7:133l,1960. Selye, H., and Bajusz, E. Effect of various electro- lytes upon cardiac and skeletal musculature. Br. Journ. Pharmacol. 14:83, 1959. Selye, H., and Bajusz, E. Sensitization by potassium deficiency for the production of myocardial necrosis by stress. Amer. Journ. Path. 35:525, 1959. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 99 Selye, H. The Stress of Life. McGraw-Hill Co. New York, 1956. Shkhvatsabaya, I. K. Experimental production of myocardial lesions by disturbing-the central. nervous system. .In Prevention of Ischemic Heart Disease. ed. by Raab,.W. Chas. C. Thomas,.- Publisher. Springfield, Illinois, 1966. Shkhvatsabaya, I. K., and Menschikov, V. V.. The significance of the catecholamine in the patho— genesis of neurogenic lesions of the myocardium. Kardeologuja. 2:27, 1962. Siegel, S. Nonparametric Statistics for the Be- havioral Sciences. McGraw-Hill Book Co. Inc. New York, Toronto, London, 1956. Siegel, J. H.; Gilmore, J. P.; and Sarnoff, S. J. Myocardial extraction and production of catecho- lamines. Circ. Res. 9:1336, 1961. Sigma Chemical Company. Sigma Technical Bulletin No. 500 Lactic Dehydrogenase. Published by Sigma Chem. Co., St. Louis, Mo. Pg. 1. Skinner, J. S.; Holloszy, J. 0.; Toro, G.; Barry, A. J.; and Cureton, T. K., Jr. Effects of a six month program of endurance exercise on work tolerance, serum lipids, and ULF-Ballistocardio- grams of fifteen middle aged men. In Physical Activity and the Heart. Chas. C. Thomas, Pub— lisher. Springfield, Illinois, 1967. Stamler, J.; Lindberg, H. A.; Berkson, D. M.; Hall, Y.; Miller, W. A.; Mojonnier, L.; Epstein, M.B.; Burkey, F.; Cohen, D. B.; Levinson, M.; and Young, Q. D. Approaches.of the coronary preven- tion evaluation programs to the primary prevention of clinical coronary heart disease in middle—aged American men. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Pub- lisher. Springfield, Illinois, 1966. Stamler, J. Current status of the dietary prevention and treatment of atherosclerotic coronary heart disease. Progr. Cardiov. Dis. 3:56, 1960. Stamler, J. Preventive Cardiology. New York. Grune and Stratton, 1967. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 100 Starcich, R. Plasma catecholamines and urinary vanillyl mandelic acid in clinical.ischemic.heart disease. In Prevention of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Publisher. Springfield, Illinois, 1966. Steinberg, D.; Nestel, P. H.; Buskuk, E. R.; and Thompson, R. H. Calorigenic effect of nore- pinephrine correlated with plasma free fatty acid turnover and oxidation. Journ. Clin. Invest. 43:167, 1964. Steinhaus, A. H. Chronic effects of exercise. Physiol. Rev. 13:103, 1933. Stevenson, J. A. F. Exercise, food intake, and health in experimental animals.. In Proceedings of the International Symppsium on Physical Activity and Cardiovascular Health. Reprinted from the Canadian Megical Association Journal. Vol. 96, NO. 12. 19 7. Stuart, J. Diagnostic enzymology in myocardial infarction. Amer. Heart Journ. 70:717, 1965. Szakacs, J. E., and Mehlam, B. Pathologic changes induced by 1 - norepinephrine : quantitative aspects. Amer. Journ. Cardiol. 5:619, 1960. Szakacs, J. E., and Carnon, A. l — norepinephrine- myocarditis. Amer. Journ. Clin. Path. 30:425, 1958. Takeuchi, T., and Kuriaki, H. Histochemical detection of phosphorylase in anima1_tissues. Journ. Histochem. Cytochem. 3:153, 1955. Taylor, H. L. Coronary heart disease in physically active and sedentary populations. Journ. Sports. Med. 2:73, 1962. Taylor, H. L. The mortality and morbidity of coronary heart disease of men in sedentary and physically active occupations. In Exercise and Fitness. Univ. of Illinois, 1959. The Athletic Inst. Taylor, H. L.; Anderson, J. T.; and Keys, A. Physical activity, serum cholesterol and other lipids in man. Proc. Soc. Exp. Biol. Med. 95:383, 1957. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 101 Vendenyeyeva, Z. I. Myocardial lesions produced by sympathomimetic anines of endogenous and exogenous origin and their analysis. Farmakologiya y Toksikelogiya. 3:286, 1963. Vessels, E. S., and Bearn, A. G. Localization of lactic acid dehydrogenase activity in serum fractions. Proc. Soc. Exp. Biol. Med. 94:96, 1957. Wardwell, W. I. Stress and coronary heart disease in three field studies. Journ. Chron. Dis. 17:73, 1964. Weiss, E.; Dlin, B.; Rollin, H. R.; Fischer, K.; and Bepler, C. R. Emotional factors in coronary occlusion. Arch. Int. Med. 99:628, 1957. Wertlake, P. T.; Wilcox, A. A.; Haley, M. J.; and Peterson, J. E. Relationship of mental and emotional stress to serum cholesterol levels. Proc. Soc. Exp. Biol. Med. 97:163, 1958. Welgram, G. F. Experimental atherosclerosis and cardiac infarcts in rats. Journ. Exp, Med. 109:293, 1959. Williamson, J. R. Metabolic effects of epinephrine in the isolated, perfused rat heart. Journ. Biol. Chem. 239:2721, 1964. Wolffe, J. B. Situational stresses as cause of cardio— vascular disease. In Prevention Of Ischemic Heart Disease. ed. by Raab, W. Chas. C. Thomas, Pub— lisher. Springfield, Illinois, 1966. Wroblewski, F., and LaDuc, J. S. Lactic dehydro- genase activity in blood. Proc. Sec. Exp. Biol. Med. 90:210, 1955. Yokayama, H. 0.; Jennings, K. B.; Clahaugh, G. F.; and Wartman, W. B. Histochemical studies of early experimental myocardial infarction. A.M.A. Arch. Path. 59:347, 1955. APPENDIX 102 APPENDIX A HEART DAMAGE RATINGS AND SERUM LDH CONCENTRATIONS 103 104 APPENDIX A sgyg:pgggp Afifljlflfl§~flflu S a;4__paggonpgNTRATIONS Animal Number reatment Group Heart Damage Heart Damage LDH LDH (Ventricles) (Atria) (early) (later) 1 Control 2 1 No LDH NO LDH 2 Control 2 1 NO LDH NO LDH 3 Control Not Analyzed Not Analyzed No LDH No LDH 4 ANE 2 1 .43 .44 5 ANE Not Analyzed Hot Analyzed .58 .55 6 ALB Not Analyzed Not Analyzed No LDH No LDH 7 ANE 3 l .46 .53 8 ANE Hot Analyzed Not Analyzed .49 .50 ) ANE 4 l .5 .46 ll A35 2 2 .45 .42 11 ALL 3 l .52 .51 12 AXE 3 3 53 .44 13 EDA Hot Analyze: Not Analyzed 56 .58 14 EDA 2 2 55 .43 15 EMA 3 1 .39 .43 16 EDA 3 1 .48 .40 17 EDA dot Analyzed Not Analyzed 41 .58 18 EDA 3 3 44 .50 l} E.n 3 l 55 .52 2d ELA Not Aralyzed Wet Analyzed .55 .57 21 EDA 3 l .53 .49 22 Control 3 2 No LDH NO LDH 23 Control 1 1 No LDH No LDH 24 Control Hot Analyzed Hot Analyzed No LDH No LDH 25 ANE 3 3 .58 .56 26 AN5 2 2 .51 .59 27 Ann 3 1 .63 .53 28 AXE 3 3 .56 .57 2) AXE 2 l .53 .59 30 ASP 2 1 .37 .62 31 AM» Hot Anal; oi Hot Analyzed .53 .63 32 AM Hot Analyz 1 hot Analy/ed .56 .62 35 AM; Hot Analyzed Hot Analyzed .53 .63 34 ELA 2 l .47 55 35 53A 2 2 .58 .51 3e EJA 3 2 .63 .50 37 EDA 2 1 .60 .56 38 ErA Net Analy 31 Not Analyzed No LDH NO LDH -j FLA 2 1 .53 -53 40 EDA Hot Analyzed Not Analyzed .53 .61 41 EDA Not Analyzed Not Analyzed .54 .64 42 EDA 3 2 .52 .48 43 Control 2 2 No LDH No LDH 44 Control 3 2 No LDH NO LDH 5 Control Not Analyzed Not Analyzed NO LDH NO LDH 46 EPA 3 2 .59 .51 47 EBA 2 2 40 .23 48 EBA 3 2 .51 .29 49 EPA Not Analyzed Not Analyzed .40 .56 50 EBA Not Analyzed Not Analyzed .50 .60 51 EBA 2 2 .45 .28 52 EBA Not Analyzed Not Analyzed .52 .52 53 BEA 3 2 .24 .31 54 EBA 3 2 .30 .53 55 E08 Not Analyzed Not Analyzed .44 .47 W: A! uh "‘ 105 HEART DAMAGE RATINGS AND SERUM LDH CONCENTRATIONS Animal Number Treatment Group Heart Damage Heart Damage LDH LDH (Ventrieles) (Atria) (early) (later) 56 E08 3 2 .41 .33 57 E08 Not Analyzed Not Analyzed .33 .39 58 E08 3 2 .58 .35 59 E08 3 2 .54 .29 60 EDB 3 2 .67 .34 61 E08 2 2 .48 .37 62 E08 3 2 No LDH .41 63 E08 Not Analyzed Not Analyzed 46 .41 64 Control 4 3 No LDH No LDH 65 Control 3 2 No LDH No LDH 66 Control Not Analyzed Not Analyzed No LDH No LDH 67 EBA 4 2 .54 .12 68 EBA 3 l .24 .53 69 EBA 4 1 .62 .26 70 EBA Not Analyzed Not Analyzed .52 .58 71 56A 3 2 .63 .08 72 EBA 2 2 .61 .18 73 EHA Not Analyzed Not Analyzed .32 .58 74 EBA Not Analyzed Net Analyzed .61 .60 75 EBA 3 2 .49 .55 76 EDH 4 2 .41 .41 77 EDB 2 .54 .38 78 EDH Not Analyzed Not Analyzed .57 .60 79 EDH 5 3 .59 -55 80 E08 3 3 .60 .38 81 EDB 3 3 .57 2O 82 E08 Not Analyzed Not Analyzed .56 No LDH 83 E03 2 2 .55 .43 84 EDD Not Analyzed Not Analyzed .59 .55 85 Control 3 3 No LDH No LDH 86 Control Not Analyzed Hat Analyzed No LDH Ho LDH 87 Control Not Analyzed Net Analyzed Nu LDH No LDH 88 ANE 3 3 .33 .45 89 ANE Net Analyzed Net Analyzed .46 .58 90 ANE 5 3 .33 .29 91 ANE 2 2 .64 .28 92 EDA Not Analyzed Not Analyzed No LDH No LD 93 EDA 3 2 .56 .25 94 EDA 4 3 .41 .53 95 EDA 2 2 .57 .38 J6 EDA Not Analyzed Not Analyzed No LDH No LDH 97 EEA Not Analyzed Not Analyzed .60 .51 98 EBA 2 2 .57 .46 99 EBA 2 2 .76 .58 100 EBA 3 2 .53 .47 101 EBD 2 2 .63 .04 102 EBD 2 2 .47 .40 103 EBD Not Analyzed Not Analyzed .58 .58 104 EBD Not Analyzed Not Analyzed .51 .60 105 EBD 3 2 .28 .57 BBBBB lulu Wll'filfllllijfllllfltfllflifll{71111111 7 699