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Myocardial ischemic injury in experimental diabetes Bhimji, Shabir 1985

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MYOCARDIAL ISCHEMIC INJURY IN EXPERIMENTAL DIABETES by SHABIR BHIMJI M.Sc, Dalhousie University, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to tfye required standard: THE UNIVERSITY' OF BRITISH COLUMBIA October 1985 ( c ) Shabir Bhimji 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Shabir Bhimji Faculty of Pharmaceutical Sciences Department of Pharmacology and Toxicology The University of British Columbia Vancouver, Canada. V6T IY3. - i i -0 ABSTRACT The nature and extent of myocardial ischemic injury (Mil) produced either by coronary artery ligation/reperfusion or by injection of iso-proterenol (ISO) was studied in the 10-week alloxan-diabetic rabbit. Prior to the induction of ischemic injury, investigation of the left ventricles of the diabetic rabbit after 10-weeks revealed significant magnesium depletion and inhibition of myofibrillar and sarcoplasmic reticulum ATPase a c t i v i t i e s . In addition, the activity of the lyso-somal enzyme, N-acetyl-B-glucosaminidase was significantly increased in diabetic left ventricular homogenates. Ultrastructural studies revealed significant l i p i d and glycogen accumulation, dilatation of the sarcoplasmic reticulum and damage to the mitochondria in left ven-tric l e s of the diabetic animals. Administration of ISO to both control and diabetic animals result-ed in atrial tachycardias and ventricular f i b r i l l a t i o n . The severity of the arrhythmias and the overall mortality was the same in both gro-ups of animals. Serum analyses revealed significantly greater increa-ses in blood glucose, free fatty acids, total cholesterol and creatine kinase activity in the ISO-treated diabetic animals relative to ISO-treated controls. ISO treatment of both control and diabetic animals produced similar increases in heart weight, left ventricular weight and myocardial water content. Analyses of various subcellular orga-nelle marker enzyme activities indicated a significantly greater de-crease in the K ,Ca -stimulated sarcoplasmic reticulum ATPase of ISO-treated diabetic animal hearts. In addition, significantly great-er increases in Ca and hydroxyproline and decreases in the levels of ATP were evident in the ISO-treated diabetic animal hearts. Ultra-structural studies revealed significant damage to the mitochondria in both ISO-treated control and diabetic hearts, the magnitude of the damage being greater in the diabetic animals. Mitochondria from both groups of animals showed swelling and fragmentation, myofibrils appea-red as a homogeneous mass and did not show the characteristic Z-lines. Glycogen depletion and l i p i d accumulation was observed in both groups of animals. In addition, both groups of animals showed amorphous dense bodies in the mitochondria after ISO-treatment. After 40-minutes occlusion of the left circumflex coronary artery followed by 60-minutes of reperfusion, hemodynamic measurements re-vealed significant decreases in the left ventricular and systemic ar-terial pressures in the diabetic animals relative to controls. An-alyses of subcellular organelle enzymes from the ischemic tissue re-+ + vealed that sarcolemmal Na ,K -ATPase, mitochondrial ATPase and sarcoplasmic reticulum ATPase activities were decreased after coronary occlusion in both control and diabetic animals. However, upon reper-fusion, unlike the control, no recovery of the mitochondrial ATPase was observed in the diabetic animals. In addition, a further depre-- i i i i -ssion of both the sarcolemmal and sarcoplasmic reticulum ATPase acti -vities were seen in the diabetic animals compared to controls on re-perfusion. Ion measurements revealed a significant accumulation of calcium in both control and diabetic animals, the magnitude of the increase being greater in the diabetic animals. Similarly, both t i s -sue ATP levels and the abili t y of the mitochondria to generate ATP were depressed in the diabetic animals as compared to controls follow-ing coronary artery occlusion and reperfusion. Following coronary artery ligation and reperfusion, the diabetic animals showed a signi-ficantly higher incidence of ventricular f i b r i l l a t i o n and cardiogenic shock as compared to controls. Ultrastructural studies revealed myo-cardial damage to both control and diabetic hearts following coronary artery ligation and reperfusion. However, the diabetic myocardium showed a higher incidence and frequency of hypercontraction bands, an increase in the amorphous dense bodies and slightly greater damage to the mitochondria. Coronary artery ligation in conscious control, 6 and 12 week-diabetic rats resulted in post-1igation arrhythmias (especially ven-tricular f i b r i l l a t i o n ) , the incidence of which was much greater in the diabetic animals. The mortality rate of 12-week diabetic rats under-going coronary ligation was 100% within 1-7 minutes following liga-tion. No differences in occluded or infarcted zones of the surviving 6-week diabetic and control rats were detected. Analyses of ionic - V-composition revealed a significant magnesium deficiency in the dia-betic hearts as compared to controls. These data indicate that the diabetic animals show a greater sus-ceptibility of the myocardium to ischemic injury. Although numerous metabolic and chemical alterations are present in the diabetic myoc-ardium, i t is possible that magnesium deficiency may be a factor de-termining the higher incidence of arrhythmias and ischemic injury in diabetic animals. - vi •. -TABLE OF CONTENTS PAGE ABSTRACT i \ ACKNOWLEDGEMENTS j f / i DEDICATION Xvi" TABLE OF CONTENTS vi . LIST OF FIGURES xi LIST OF TABLES x i n 1 . INTRODUCTION 1 1.1 Historical review 1 1.2 Heart Disease and Diabetes 2 1.3 Ischemic Heart Disease in Diabetics 4 1.4 Hyperglycemia, Hyperlipoproteinemia,... 5 1.5 Clinical Aspects of Coronary Artery Disease... 7 1.6 Possible Risk Factors 9 1.7 Intramural Vessels 11 1.8 Subclinical Diabetic Cardiomyopathy 12 1.9 Histopathology in Diabetic Cardiomyopathy 14 1.10 Hemodynamics in Diabetic Cardiomyopathy 16 -vi i 1.11 Catecholamines and Diabetes 17 1.12 Experimental Diabetes and Pathogenesis 20 1.13 Metabolic Alterations in Diabetic Myocardium 21 1.14 Carnitine Metabolism in Myocardial Ischemia 25 1.15 Prevention of Ischemic Heart Disease in Diabetics 26 1.16 Problems with current data 29 1.17 Model of Diabetes 30 1.18 Experimental Approach to the Study of Myocardial.. 31 1.19 Objectives of the Study 33 2 . MATERIALS AND METHODS 35 2.1 Animal Studies 35 2.1.1 Induction of diabetes in the rabbit 35 2.1.2 Induction of diabetes in the rat 35 2.1.3 Myocardial ischemia produced by coronary occlusion 36 2.1.4 Coronary ligation in rats 39 2.1.5 Induction of myocardial ischemic injury by ISO 41 2.2 Isolation and Characterization of subcellular organelles 41 2.2.1 Preparation of mitochondrial and sarcolemmal.. 41 2.2.2 Characterization of sarcolemmal and mitochondrial. 43 2.2.3 Triton X-100 addition 44 2.2.4 Isolation of mitochondria 44 2.2.5 Isolation of lysosomes 44 2.3 Biochemical Assays 45 2.3.1 Nitrophenylphosphatase activity 45 2.3.2 Sarcolemmal Na+,K+-ATPase 46 2.3.3 Mitochondrial azide-sensitive ATPase activity 47 2.3.4 Sarcoplasmic reticulum ATPase activity 47 2.3.5 Mitochondrial ATP generation 48 2.3.6 Serum creatine phosphokinase 48 2.3.7 Myocardial creatine phosphokinase activity 49 2.3.8 Measurement of Myocardial ATP 49 y n \ 2.3.9 Acid phosphatase 50 2.3.10 N-acetyl-e-glucosaminidase 50 2.3.11 Cathepsin D 51 2.4 Chemical Assays 51 2.4.1 Intracellular sodium 51 2.4.2 Intracellular potassium, calcium and magnesium 52 2.4.3 Red blood cell magnesium content 52 2.4.4 Plasma electrolytes 53 2.4.5 Atomic absorption spectroscopy 53 2.4.6 Mitochondrial calcium content 54 2.4.7 Hydroxyproline assay 54 2.4.8 Glycosylated hemoglobin assay 55 2.4.9 Tissue glycogen 56 2.4.10 Tissue lipid extraction 57 2.4.11 Serum triglycerides 57 2.4.12 Cholesterol ^ 58 2.4.13 Free fatty acids 58 2.4.14 Phospholipid analysis 59 2.4.15 Serum glucose determination 60 2.4.16 Serum T 4 determination 60 2.4.17 Serum T 3 determination 61 2.4.18 Serum insulin 61 2.4.19 Water content 62 2.4.20 Protein assay 62 2.5 Ultrastructural analysis 62 2.5.1 Preparation of samples for electron microscopy 62 2.6 Microsphere Study to Determine Regional Blood Flow 63 2.6.1 Blood flow studies 63 2.6.2 Microsphere preparation 64 3 STATISTICAL ANALYSIS 65 4 MATERIALS 65 5 RESULTS 66 5.1 Model of Alloxan-Induced Diabetes in Rabbit 66 5.1.1 General features 66 5.1.2 Body weight and mortality 66 5.1.3 Left ventricular weight and water content 71 5.1.4 Serum chemical properties 71 5.1.5 Status of atherosclerosis 73 5.1.6 Glucose tolerance test 76 5.1.7 Hemodynamic measurements 76 — I X -5.1.8 Myocardial composition 80 5.1.9 Magnesium metabolism 80 5.1.10 Lysosomal enzymes 87 5.1.11 Myocardial ultrastructural alterations 87 5.2 iSO-Inauced Myocardial Ischemic Injury 94 5.2.1 General features 94 5.2.2 Electrocardiograms 95 5.2.3 Body weight and serum lipids 98 5.2.4 Myocardial chemical and biochemical alterations 101 5.2.5 Lysosomal enzymes 104 5.2.6 Hemodynamics 104 5.2.7 Myocardial ultrastructural alterations 107 5.3 ISO-Induced Myocardial Damage in Diabetes 112 5.3.1 General considerations 112 5.3.2 Serum chemical properties 113 5.3.3 Myocardial enzymatic and compositional properties 116 5.3.4 Lysosomal enzymes 119 5.3.5 Hemodynamics 119 5.3.6 Ultrastructural alterations 122 5.4 Coronary Artery Ligation and Reperfusion in Control 122 and Diabetic Rabbits 5.4.1 Occluded zone and ECG alterations 127 5.4.2 Effect of coronary ligation on ATP 130 5.4.3 Ions and water content 130 5.4.4 Subcellular organelle ATPases 133 5.4.5 Hemodynamics 137 5.4.6 Mitochondrial ATP generation 138 5.4.7 Ultrastructural alterations 141 5.4.8 Blood flow following occlusion and reperfusion 149 5.5 Coronary Artery Ligation in Conscious Control and 152 and Diabetic rats 6 DISCUSSION 157 6.1 Aim 157 6.2 The Choice of the Animal Model 157 - X -6.3 The Diabetic Rabbit Model 158 6.4 Diabetic Cardiomyopathy 160 6.5 ISO-Induced Mil 169 6.6 ISO-Induced Mil in Alloxan Diabetic Rabbits 178 6.7 Coronary Artery Ligation and Reperfusion in Control 182 and Diabetic Rabbits 6.8 Coronary Artery Ligation in Diabetic Rats 192 6.9 Summary and Conclusion 194 7.0 BIBLIOGRAPHY 198 - xi -LIST OF FIGURES FIGURE PAGE 1 Blood glucose levels immediately following alloxan 67 2 Oral glucose tolerance test in 10 week diabetic rabbits 77 - glucose levels 3 Oral glucose tolerance test in 10 week diabetic rabbits 78 - insulin levels 4 Subcellular organelle ATPase activities in control and 82 diabetic animal hearts 5 Red blood cell magnesium content at various times in 83 control and diabetic animals 6 Effect of increasing blood glucose concentration on 84 plasma and red blood cell magnesium content 7 Release of lysosomal enzymes in the left ventricles . 88 of control and diabetic animals 8 Electron micrograph of control rabbit myocardium 90 9 Electron micrograph of 4-week diabetic myocardium 91 10 Electron micrograph of 10-week diabetic rabbit myocardium 92 11 Electron micrograph of mitochondria from hearts 93 of 10-week diabetic rabbit 12 The effects of ISO treatment on left ventricular 105 lysosomal enzymes 13 Ultrastructure of rabbit heart after 4 days of 108 ISO treatment 14 Ultrastructure of rabbit heart after 7 days of ISO 109 treatment 15 Ultrastructure of rabbit hearts after 15 days of 110 ISO treatment 16 Ultrastructural alterations in mitochondria from 111 15 day ISO treated rabbits - x i i -17 Effects of ISO treatment on diabetic left ventricular 120 lysosomal enzymes 18 Ultrastructure of 10-week diabetic myocardium treated 123 with ISO for 5 days 19 Effect of 15 day ISO treatment on 10-week diabetic 124 myocardi urn 20 Mitochondria from diabetic myocardium after 15 day ISO 125 treatment 21 Effect of coronary ligation and reperfusion on 131 myocardial ATP levels in control and diabetic animals 22 Effects of coronary ligation and reperfusion on 134 mitochondrial ATPase activity in control and diabetic animals 23 Effects of coronary ligation and reperfusion on the 135 sarcolemmal ATPase activity in control and diabetic animals 24 Effects of coronary ligation and reperfusion on the 136 sarcoplasmic reticulum ATPase activity in control and diabetic animals 25 Effects of 20 minute occlusion on ultrastructure of 142 control myocardium 26 Effects of 40 minutes occlusion on ultrastructure of 143 control myocardium 27 Effects of 60 minutes reperfusion on ultrastructure 144 of control myocardium 28 Effects of 20 minutes occlusion on ultrastructure of 145 diabetic myocardium 29 Effects of 40 minutes occlusion on ultrastructure of 146 diabetic myocardium 30 Effects of 60 minutes reperfusion on -ultrastructure 147 of diabetic myocardium - x i M — LIST OF TABLES TABLE PAGE 1 General features of alloxan-induced diabetics rabbits 68 after 10 weeks 2 Effects of 10 weeks of alloxan-induced diabetes on 69 rabbit body and heart weight 3 Effects of 10 weeks of diabetes on blood glucose and 72 serum lipids 4 Effects of 10 weeks of diabetes on serum T3, T4, 74 blood urea nitrogen, electrolytes and creatine phosphokinase activity 5 Extent of l i p i d accumulation in aortae from 10-week 75 alloxan-diabetic rabbits 6 Effect of 10 weeks-of diabetes on various cardiovascular 79 parameters 7 Effect of 10-weeks of diabetes on left ventricular 81 content of ions and biochemical substances 8 Plasma and urinary magnesium concentration in 10-week 85 alloxan diabetic rabbits 9 Magnesium content in tissues of 10-week alloxan- 86 induced diabetic animals 10 Heart rate, PR-interval, QRS complex, QT-interval 96 and ST-segment elevation in control and ISO-treated rabbits 11 Time-course of body, heart and left ventricular 99 weight after 5 and 15 days of ISO treatment 12 Changes in blood glucose and serum lipids after 100 15 days of ISO-treatment 13 Effect of 5 and 15 day ISO-treatment on subcellular 102 organelle ATPase activities 14 Changes in intracellular ions in the left ventricles 103 after 15 day ISO-treatment - X i V -15 Hemodynamic alterations in rabbits treated with 106 ISO for 15 days 16 Effects of ISO treatment on serum biochemical 114 changes in control and diabetic animals 17 Body, heart and left ventricular weight in control, 115 diabetic and ISO-treated animals 18 Subcellular organelle ATPase activities in left 117 ventricles of control and diabetic animals treated with ISO 19 Myocardial ATP, glycogen, hydroxyproline and 118 intracellular ions in control and diabetic animals treated with ISO 20 Hemodynamic alterations in control and diabetic 121 animals treated with ISO 21 Objective assessement of ultrastructural features 126 of control, diabetic and ISO-treated animals 22 Incidence of ventricular f i b r i l l a t i o n , cardiogenic 129 shock and appearance of Q-waves in control and diabetic animals following occlusion and reperfusion 23 Ionic alterations in hearts of control and diabetic 132 animals following occlusion and reperfusion 24 Hemodynamics in control and diabetic aniamals 139 following occlusion and reperfusion 25 Mitochondrial ATP generation in control and diabetic 140 animals following coronary occlusion and reperfusion 26 Objective assessment of ultrastructural features in 150 control and diabetic animals following coronary occlusion and reperfusion 27 Blood flow in control and diabetic animals following 151 coronary occlusion and reperfusion 28 General features of 6 and 12 week diabetic rats 153 29 Effect of coronary artery ligation in control and 155 6-week diabetic rats on mortality, infarcted and occluded zones Effect of coronary artery occlusion in control and 12-week diabetic rats in mortality, infarcted and occluded zones ACKNOWLEDGEMENTS I am immensely grateful to Drs. David Godin and John McNeill for their support and guidance during the course of my graduate studies. My interactions with them has earned me a very rewarding and meaning-ful learning experience. I am particularly thankful to Dr David Godin for the stimulating discussions and useful suggestions he provided during the preparation of my thesis. I express my deep appreciation to the Staff in the Department of Pharmacology and Therapeutics, Faculty of Medicine for allowing me not only to use their f a c i l i t i e s but also for their suggestions during my work. I am indebted to the B.C heart Foundation and the University of British Columbia for the financial support during my studies. I also wish to express my gratitude to Maureen Garnnett, Theresa Ng , Saleh Wohaieb and my fellow colleagues who provided me with assi-stance during my thesis. Finally, I am deeply grateful to Dr John McNeill for giving me the opportunity to work with him and am thankful to him for his tolerance, patience and understanding. DEDICATION To my wife for a l l she has done 1 1 . INTRODUCTION 1.1 Historical Review In accounts of the history of diabetes i t is stated that in the Ebers papyrus there was mention of polyuria and that 'honey urine' was noted by Sushruthra in India in 400 B.C. The f i r s t good clinical de-scription of the disease was made by Celsus, and the name 'diabetes' was introduced by Aretaeus. In the f i r s t century, Avicenna, an Arab physician, gave a remarkably good description of diabetes, including certain of its complications such as gangrene. Thomas Willis (died 1675) observed that the urine of diabetics was sweet, and Dobson (1775) demonstrated that the sweetness was, indeed, due to sugar. In 1869, Langerhans discovered the islets which were later given his name by Laguese. In addition, the 19th century brought many other major additions to knowledge in biochemistry and physiology of the pancreas by C. Bernard and others including Bou-chardat, Cantani, Kulz, Kussmaul, Naunyn, Joslin and Allen, to mention a few (1,2). The greatest step forward came in 1921 when Banting and Best succeeded in extracting from the pancreas a substance with hypo-glycemic properties (3). This epoch-making achievement, which completely altered the out-look for persons with diabetes, stimulated research immeasurably. The introduction of oral hypoglycemics in 1955 provided another stimulus because studies designed to elucidate the mode of action of these drugs led investigators to a closer examination of the pathophysiology of diabetes. In the last 2 or 3 decades, interest in diabetes and 2 related problems has increased greatly and has been remarkably well sustained. Diabetes is now recognized as a common and widespread disease. It has been estimated that about 20-25%of the population either have diabetes, will develop diabetes or have a diabetic relative. The im-portance of the disease as a public health problem becomes even more apparent when one considers the complications or sequelae of long-term diabetes. Chiefly vascular in nature, these complications lead to marked disability due to involvement of both large vessels in the brain, heart, kidneys and extremities and of small vessels, especially in the eyes and the kidneys (4). Diabetes, therefore, poses a problem for large numbers of persons and furthermore, i t provides for both the basic scientist and the cli n i c a l investigator a f e r t i l e and fascinating area of research endeavour. 1.2 Heart Disease and Diabetes Whereas diabetic nephropathy has become the chief cause of death among diabetic patients with onset of diabetes in childhood, cardiac disease is the primary cause of death among all diabetics with onset of the disease after the age of 30. Cardiac disease continues to be the outstanding factor in determining overall diabetic mortality and morbidity (5). It occurs most commonly in individuals with maturity onset type of diabetes diagnosed in the sixth to seventh decade of l i f e and in whom.the date of onset is uncertain with "chemical" diabe-tes having possibly been present for years (6,7). Diabetic patients 3 in whom the diagnosis was made early in l i f e (20-30 years of age) are subject to a vascular disorder now known as diabetic microangiopathy with venular, capillary and arteriolar changes leading to c l i n i c a l l y significant involvement of the retina and kidney (8). Follow-up stud-ies of this group after 20 or more years indicate that the typical severe sequelae may be postponed or prevented with persistent and con-tinuous control of diabetes with proper diet and optimal insulin ther-apy (9). However, even those patients sufficiently well-treated to prevent severe retinal and renal lesions frequently develop premature ischemic heart disease after 20-30 years of diabetes. As yet, an in-sufficient number of rigorously treated early onset diabetic patients have been followed for longer periods to determine the incidence with which occlusive vascular disease develops in such individuals (9). In individuals with maturity-onset diabetes, particularly those with l i t t l e or no glycosuria and few i f any symptoms, the diagnosis of diabetes is often not made until the appearance of vascular lesions. According to the Joslin C l i n i c , c l i n i c a l l y significant atherosclerotic lesions commonly, and microangiopathic or neuropathic sequelae not infrequently, have been noted early in the course of maturity-onset diabetes (10). This raises the obvious and as yet incompletely an-swered question: Are these vascular changes related to insulin defi-cit and to the biochemical abnormalities of diabetes including hyper-glycemia or do they represent a specific vascular defect progressing independently of the biochemical abnormalities controllable with insulin ? 4 1.3 Ischemic Heart Disease in Diabetics It is well known that diabetics are more prone than the general population to ischemic heart disease (11). The preservation of the ischemic myocardium, the reduction of infarct size and the effective modification of substrate delivery to the injured heart are all thera-peutic aims being actively pursued in the current management of acute ischemic heart diasease. By contrast, in the chronic stages of i s -chemic heart disease in diabetics, prognosis for l i f e relates more to the residual amount of muscle surviving the acute myocardial infarc-tion. The remaining viable areas of myocardium are sustained by the magnitude of coronary collateral flow and determine the competence of the heart as a pump which modulates the quality of l i f e , through its s k i l l in adapting cardiac output to a wide range of physiological demands (12). The physician, therefore, is faced with many dilemmas. How thorough is his knowledge of basic metabolic and physiological mechan-isms for correct decision making; have pathological concepts of disease processes been clearly defined, and what r e l i a b i l i t y can be placed on an ever increasing number of highly technical non-invasive diagnostic procedures* The prognosis is equally confusing. Symptoms need not bear a direct quantitative correlation with pathological pro-cesses; the electrical action of the heart may pursue bizarre, impon-derable, and lethal courses, while the mechanical competence of the heart, a good predictor of future events, may be hard to evaluate. If these problems in ischemic heart disease are complex, they are 5 many times compounded in diabetes mellitus, where the likelihood of premature arterial disease, altered myocardial metabolism, and de-pressed ventricular function may exist as additional complicating variables in determining the nature and severity of c l i n i c a l heart disease. 1.4 Hyperglycemia, Hyperlipoproteinemia, Hypertriglyceridemia,  Obesity and Coronary Artery Disease Extensive reviews concerning diabetes and the heart have recently been published (13,14). The complex inter-relationships between hyperglycemia, hyperlipoproteinemia, obesity and peripheral tissue insulin resistance are s t i l l without a precise etiological understand-ing. That these factors have significance in premature coronary art-ery disease seems increasingly evident, because diabetic subjects who have these biochemical derangements are at increased risk of develop-ing coronary artery disease. The increased death rate in the diabetic population has been attributed to coronary atherosclerosis, which oc-curs far more frequently and is more severe in diabetics than in the general population (15). In addition, the premenopausal diabetic fem-ale has a prevalence of ischemic heart disease equal to or even ex-ceeding that of the diabetic male of comparable age (16). Hyperten-sion is an additional risk factor. It is more prevalent in the dia-betic subject than in the general population and coronary artery disease is twice as common in the diabetic hypertensive subject (17). Once established, coronary artery disease worsens the prognosis for the diabetic subject. Risks of early mortality from acute myocar-6 dial infarction is increased approximately twofold and repeated heart attacks have a less favorable prognosis in these patients. Five year survival rates in diabetic subjects are sobering; 38%for those with i n i t i a l discrete epsiodes of myocardial infarction and only 25% survi-val for those patients with repeated attacks (18). A high incidence of cardiogenic shock and congestive heart failure occurs in this popu-lation (19). Acute myocardial infarction may precipitate ketoacidosis and diabetic coma in diabetic subjects, and the associated symptoms and signs of abdominal and/or chest pain, nausea, hypotension, glyco-suria and ketonuria may cause the cardiac problem to be overlooked. Even when infarction is promptly recognized, its outcome under these circumstances is frequently fatal (20,21). Painless infarction is an additional feature of this group (22). Hyperglycemia is well recognized as a risk factor in premature coronary artery disease, there being a high prevalence of c l i n i c a l l y significant ischemic heart disease in overt diabetics. A high preval-ence of preclinical (latent) diabetes exists in patients with angio-cardiographically demonstrable premature coronary artery disease (23). Among survivors from acute myocardial infarction, at least one-third have hyperlipoproteinemia, with hypertriglyceridemia elevation being three times as common as hypercholesterolemia (24). Obese patients have a significant number of metabolic abnormalit-ies, including maturity-onset hyperglycemia, glycosuria, increased insulin production and increased insulin resistance, hypertriglyceri-demia (usually type 4 hyperlipoproteinemia) and hypertension (25). 7 The influence of these factors and of obesity in general on the c l i n i -cal evolution of heart disease is not f u l l y known, but theoretical considerations suggest that they are all likely to exaggerate the eff-ects of established diabetes on the heart. 1.5 Clinical Aspects of Coronary Artery Disease in Diabetes Turning now to the c l i n i c a l manifestations of coronary artery dis-ease in diabetic patients, one might f i r s t inquire as to whether there are any c l i n i c a l patterns of angina pectoris peculiar to the diabetic in view of the unusual pathologic changes that may occur in the coro-nary circulation of such patients. The answer is unequivocally no ; among a l l patients with coronary artery disease i t has not been possi-ble to identify pain patterns corresponding to specific types of coro-nary lesions. Neither has there been any consistent correlation of particular pain patterns with the presence or absence of collateral vessels or a correlation of the site of ischemia in the myocardium with the somatic site to which the pain has been refered (26). The contribution of occlusive disease due to atherosclerosis of the coronary arteries based on angiographic or pathologic c r i t e r i a has been d i f f i c u l t to establish in the diabetic population. A review of autopsy studies indicates that the risk ratio for coronary atheroscle-rosis in diabetics was approximately 2:1 without correction for other cardiac risk factors (27). More recent analyses reveal that insulin-dependent diabetics have substantially greater narrowing of coronary vessels than do age-matched controls and the extent of coronary 8 involvement is also greater (28). Several recent studies of adult-on-set diabetes provide more equivocal results. In patients who succumb-ed after long-term follow-up in a diabetes c l i n i c , autopsy revealed a similar narrowing of the major coronary arteries by atherosclerosis in diabetics with and without cl i n i c a l evidence of coronary heart disease (29). However, the study did not include a control group of non-dia-betics with mortality from causes other than coronary artery disease. A European study revealed at most only a modest increase in the extent of coronary atherosclerosis in diabetics compared with age- and sex-matched controls (30). It is noteworthy that in a recent large-scale cine-angiographic study of patients suspected of having coronary artery disease, diabe-tes per se was not associated with significantly greater occlusive disease in men when corrected for the co-existence of other risk fac-tors. However, coronary disease was significant in one sub-group, namely women aged 40-60 years (31). Finally, two studies of non-human male primates in which atherosclerosis was produced over an 18-month period by feeding an atherogenic diet indicated that, at equivalent degrees of hypercholesterolemia, diabetic rhesus monkeys manifested no greater degree of fatty streak lesions in coronary vessels than to non-diabetic animals (32). In examining the effects of alloxan diabe-tes upon the development of plaque lesions in the Cynomolyus monkey, neither the degree nor the distribution of atherosclerosis or the phy-siologic response to coronary vasodilatation were different in diabe-t i c and non-diabetic animals suggesting no basic differences in the 9 vasculature of diabetics as compared to controls (32,33). 1.6 Possible Risk Factors Some of the factors which have been suggested to account for the increased risk of coronary artery disease in diabetics are : a_) insulin antagonists: Bornstein and Hyde proposed that a circulating insulin antagonist was present in juvenile diabetics and might account for the complications of diabetes (34). b_) Growth hormone has been suggested as a causative agent of diabetic angiopathy by Lundback (35). In arterial myomedial cell cultures from normal rabbits, greater amounts of procollagen (type 1) were produced in the presence of dia-betic serum or normal serum containing small amounts of growth hormone (36). Although well-controlled diabetics appear to have normal serum growth hormone levels, when related to the level of hyperglycemia the hormone level is significantly higher than in normals with equivalent hyperglycemia induced by glucose infusion (37). Merimee et al have also presented similar findings, showing that vascular complications were minimal in the presence of anterior pituitary insufficiency (38). c) Brunner et al (39) have proposed that elevated renin activity could be related to angiopathy. In diabetics, plasma renin activity has been found to be elevated in long-term juvenile diabetics. Whether or not these and other hormones are important in the development of CAD remains to be determined, dj With regard to genetics, sex may be a risk factor. Diabetic women are at a greater or equal risk to diabet-ic men to develop CAD until middle age, and from that time they may possibly be at lesser r i s k . This is in contrast to the non-diabetic 10 woman, who is at a lower risk than the non-diabetic man until after menopause (40). The complexity of genetic versus environmental com-ponents in the etiology of atherosclerosis is exemplified in the re-port of Sidd et a l , where identical twins both had CAD, but their sib-lings were unaffected (41). e) Studies of altered blood properties and of mucopolysaccharide metabolism in relation to vascular disease have also been made. In regard to mucopolysaccharide metabolism, Spiro has proposed that thickening of basement membrane in diabetics may alter the vasculature and thus make diabetics more prone to atherosclerosis (42). f) McMilland found increased serum viscosity in relation to microangiopathy and a decreased rate of fibrinolysis in diabetics (43). _g_) Elevated serum free fatty acids, common in uncon-trolled diabetics, have also been shown to increase platelet adhesive-ness (44). Several miscellaneous factors have been related to atherosclerosis. Microvascular disease of the vaso vasorum may promote i n f i l t r a t i o n of lipids into the arterial wall and promote atherosclerosis. Diabetes also raises plasma VLDL and, to a lesser extent, LDL. Increased con-centrations of lipoproteins are mainly the result of their overproduc-tion. Both high levels and overproduction of these lipoproteins could enhance atherogenesis (45). Hyperglycemia may cause glycosylation of key proteins in the endothelium or subintimal spaces. Glycosylation may thus increase endothelial damage or enhance deposition of lipopro-teins in the arterial wall (46). Smoking is now accepted as a risk factor for atherosclerosis in the non-diabetic, but its effect in the 11 diabetic is not known. Under et al (47) have observed a progression of atherosclerosis in uremic patients undergoing dialysis and the presence of renal failure could accelerate diabetic CAD as well. Finally, the concept of accelerated aging has been reiterated. A recent study by Hamlin et al (48) showed that the determined age of diabetics as judged by collagen content of vessel walls was in excess of that of controls. Certainly, vascular disease increases with the duration of diabetes, as i t does with aging. 1.7 Intramural Vessels Obi iterative disease of the small coronary arteries has been thought to be important in the pathogenesis of cardiac disease in dia-betes. Thickened intramural arteries and occasional bridges of endo-skeletal cells have been attributed to diabetes. However, such les-ions have also been observed in non-diabetics without evident effects on cardiac muscle (49). In another disease with small vessel involve-ment, endomyocardial fib r o s i s , the process appears to be independent of tissue necrosis or fibrosis, presumably because the arterial dis-ease is patchy and its progression sufficiently slow to permit colla-teral development (50). The failure to find demonstrable obstructive lesions of intramural vessels in the free wall of the left ventricle in recent autopsy studies of diabetics suggests that small vessel lesions in this disease state may have l i t t l e or no relation to car-diac pathology (51). In vivo myocardial biopsies were recently reported in 12 adult diabetics with cardiac symptoms (52). Structural changes in the 12 microcirculation were not observed, a finding that was confirmed at autopsy in two of the patients and was also observed recently in a small series of diabetics (52,53). Although continuing controversy exists, the majority of recent studies support the view that occlusive disease of the intramural arteries and arterioles is not usually suf-ficient to account for heart muscle pathology. It should be added that a postmortem study of small vessels revealed saccular micro-aneurysms of arteriolar and capillary vessels in 3 of 6 patients (54). No specific abnormalities were observed in the tissue around these vessels. Although fibrosis or myocardial degeneration was seen, these abnormalities also occurred in other areas of myocardium where no aneurysms were observed. 1.8 Subclinical Diabetic Cardiomyopathy Requisite for a specific diabetic cardiomyopathy must be the demonstration of : 1) coronary arteriolar or capillary lesions unique to the diabetic state, 2) an identifiable microscopical myocardial lesion or 3) a myocardial contractile metabolic energy alterarions unique for the disease state. The generalized vascular changes throughout the body in long-term diabetes mellitus are of three types : 1) atherosclerosis of medium and large arteries. The changes begin as fatty streaks and raised lesions or plaques of the endothelium and are not structurally d i f f e r -ent from those encountered in the non-diabetic state, 2) arteriolar disease of the type seen in hypertension and 3) diabetic microangio-pathy-an accumulation of capillary basement membrane-type material 13 considered to be specific for this vascular lesion. To what extent these changes are uniquely specific for either the coronary arteries or the myocardium is not known. To test the hypothesis that a portion of the diabetic population may have asymptomatic myocardial abnormali-t i e s , diabetic patients aged 20-56 years who had no evidence of myo-cardial ischemia or other cardiovascular disease, underwent non-inva-sive measurement of the systolic time intervals for comparison with age-matched controls. Heart rate and arterial pressure were normal but the diabetic subjects had a shortened left ventricular ejection time, a longer pre-ejection period and a higher ratio of preejection period to left ventricular ejection time. The findings were apparent-ly unrelated to the types of treatment (55). The abnormality was thought to be related to either increased wall stiffness or reduced contractility. It is relevant that patients with classic angina pec-toris without cardiac decompensation usually have normal systolic time intervals at rest, so that myocardial ischemia is unlikely to be the basis of the preclinical abnormality. An additional report supports the hypothesis that some patients with diabetes mellitus have myocardial alterations without c l i n i c a l manifestations which may ultimately result in heart failure i f the process develops f u l l y . This study is of particular interest because the authors indicate that most normotensive persons with long-standing diabetes, but without c l i n i c a l evidence of heart disease, may have a preclinical abnormality of the left ventricle without overt manifesta-tions (56,57). 14 Evaluating the coronary arteries of nine juvenile diabetic sub-jects, Crall and Roberts (51) observed significantly more extramural coronary luminal narrowing by atherosclerotic plaques in diabetics than in the so-called normal individuals. These changes were seen both in the degree and the linear extent of narrowing throughout the coronary tree. Only minor degrees of intimal fibrous proliferation, considered of no functional consequence, were observed in the intra-mural coronary arteries in the ventricles of six of the nine diabetic subjects. Periodic-acid Schiff-positive (PAS) material, although never present in great degree, was always more frequent and of greater intensity in the diabetic subjects (58). The conclusions of the above studies were : 1) accelerated coron-ary atherosclerosis can be demonstrated in diabetic patients, but dia-betes does not per se always presuppose the presence of coronary art-ery disease, 2) intimal proliferation and PAS accumulation in the coronary circulation of maturity-onset diabetes was of minor or non-functional c l i n i c a l significance, negating the possibility of specif-ic microangiopathy. 1.9 Histopathology in Diabetic Cardiomyopathy The histopathologic changes in diabetic cardiomyopathy do not con-stitute pathognomonic histologic manifestations of the disease. The pathologic changes resemble those of ischemic heart disease (59). There are characteristic changes in the small vessels such as the arterioles and large coronary arteries. The arterioles are markedly narrowed and in some instances occluded (60). The impairment of blood 15 flow to the contracting myocardium results in degeneration of the myo-cardium to a variable extent, depending upon the degree of ischemia. Some areas show changes detectable only by electron microscopy, where-as other areas of the same heart display variable degrees of degenera-tion or even necrosis. There may be small areas of infarction or necrosis, focal scars, large fresh infarcts or large scars (61). These changes are similar to the well-known coronary arteriosclerosis of ischemic heart disease of non-diabetics. It is not possible by his-tology or electron microscopy alone to diagnose diabetic cardiomyo-pathy with certainty. Even though diabetic cardiomyopathy is a pre-valent disease, no one has characterised the nature of the cardiac lesion. The vascular changes tend to occur more rapidly and more extensively in diabetics than in non-diabetics (61). With the degenerative changes in the myocardium, functional and other physiologic changes of the myocardium develop (62,63). The hemodynamic changes are those of a fai l i n g heart or of congestive heart failure in the later stage of the disease. Disturbances in rhythm and order of depolarization and repolarization occur (63). The disturbances in conduction and other disorders of the heart beat are similar to those reported extensively for arteriosclerotic heart dis-ease. Almost any imaginable change in the heart beat or cardiac rhythm and abnormalities in the time-course of the order of depolariz-ation and repolarization can occur in ischemic heart disease and, therefore, in diabetic cardiomyopathy. The possible electrophysiolog-ic disturbances are so numerous that they are beyond the scope of this 16 thesis (64,65). 1.10 Hemodynamics in Diabetic Cardiomyopathy Non-invasive studies do not indicate the basis for the observed abnormalities and the relative role of impaired compliance or contrac-t i l i t y in diabetic cardiomyopathy. To explore the question of myo-pathy after the development of typical or atypical angina or dyspnea, left ventricular function was examined in adults with uncomplicated diabetes and a familial history of myocardial infarction to determine whether symptoms depended on the presence of significant atherosclero-s i s . These patients were well-controlled by diet, insulin or oral hypoglycemic agents and were without other cardiac risk factors (66). There was no significant obstructive disease by coronary arterio-graphy and ventriculograms showed no evidence of regional contractile abnormalities or mitral valve lesions in the non-coronary group. The latter were also without hypertrophy but had a significant reduction of stroke volume and elevation of end-diastolic pressure at rest com-pared with control subjects of similar age. End-diastolic volume was significantly less than in controls, so that the reduced stroke volume appeared to be secondary to abnormal f i l l i n g of the ventricle. Ejec-tion fraction did not differ significantly from controls, but this may have been due to enhanced end-diastolic wall stiffness (66). In diabetics with prior episodes of cardiac failure and normal coronary arteriograms, stroke volume was reduced more than in diabet-ics without f a i l u r e . This was not solely related to diminished dia-stolic f i l l i n g of the ventricle; decreased contractile function also 17 contributed since the ejection fraction was significantly reduced. Although the compliance abnormality was also evident, the sequence of diminished diastolic compliance followed by impaired contractility may have pathological significance (56,58). Since myocardial ischemia on a microvascular basis could not be excluded in these patients, atrial pacing was induced to determine whether or not lactate accumulation (as evidence of inadequate coron-ary blood flow) could be demonstrated, but no decrease in blood flow was observed. Consequently, i f abnormalities of small arteries or capillaries were present in these diabetic patients with altered ven-tricular function, they were apparently insufficient to restrict myo-cardial perfusion (58). The change in cardiac function is suggestive of enhanced wall stiffness and may be related to altered muscle composition in the form of i n t e r s t i t i a l collagen accumulation observed in morphologic studies. A diffuse distribution of this process throughout the left ventricle, as well as accumulation of triglyceride and cholesterol, supports the view that a cardiomyopathic process can exist in diabetes (56). The amount and distribution of glycoprotein in the interstitium may con-tribute to progression of the process. It is not yet known whether or not the onset of cardiac decompensation in diabetes involves a primary effect on contractile proteins or on calcium transport (66). 1.11 Catecholamines and Diabetes The preoccupation with diabetes as a coronary risk factor is so great that there is a tendency to forget that atherosclerosis is not 18 the only link between diabetes and its cardiovascular complications. The neurogenic and humoral factors that may adversely affect cardio-vascular hemodynamics in diabetes are often overlooked, particularly when the disease is complicated by autonomic neuropathy. Altered responses of diabetic patients to physiological, psychological and pharmacological stimuli may present serious c l i n i c a l problems that do not lend themselves to simple solutions. Among the humoral factors which have received l i t t l e attention in diabetes are the catechol-amines. The sympathetic nervous system is of major importance in the regu-lation of several physiological functions such as cardiovascular and metabolic homeostasis. The potential role of catecholamines in a num-ber of human diseases has been largely overlooked until recently. The metabolic changes observed in untreated diabetics are, in many respects, similar to those produced by infusion of catecholamines and include hyperglycemia, decreased glucose tolerance, elevated free fat-ty acids and ketone bodies in plasma and decreased insulin response to glucose (67). A number of studies indicate that emotional factors may aggravate the metabolic alterations in diabetic patients (68). Another aspect of diabetes that may well be relevant to the grave c l i n i c a l course of such patients with coronary artery disease is the occurence of defective autonomic control of the heart. Such defects of both sympathetic and parasympathetic nerves to the heart are common in patients with other evidence of neuropathy and are similar to the defective innervation noted in congestive heart failure (69,70). This 19 kind of autonomic impairment of the heart arising from diabetic neuro-pathy can predispose the patient to serious arrhythmias (71). It has recently been shown that cardiovascular responses to physi-cal exercise are impaired in diabetics. Resting heart rate was higher and the increased heart rate at low workload was diminished in diabet-ics compared to non-diabetics. Maximal heart rate, maximal systolic blood pressure, uptake and the greatest tolerable work load were reduced in diabetic patients. All the above observations are consist-ent with a form of autonomic neuropathy. The relationship between heart rate or systolic blood pressure and relative workload in patients with autonomic neuropathy suggests altered sympathetic ner-vous activity (72). Long-term diabetics (both type 1 and 11) are supersensitive to the intravenous infusion of catecholamines (73). This supersensitivity probably arises by several possible mechanisms. F i r s t , axonal uptake, a function which is important for the termination of catecholamine action decreases when the adrenergic nerves degenerate and the effect-ive concentration of amines at the receptor site thus increases. Secondly, the chronic absence of the neurotransmitter probably in-creases sensitivity by changing the availability or number of recep-tors analogous to denervation supersensitivity. Third, the impairment in blood pressure-controlling reflexes due to autonomic neuropathy may also be of importance (74,75). The potential for the role of elevated catecholamines to induce ischemic heart disease in the diabetic is not known. However, cate-20 cholamine-administration produces changes in the ECG pattern, especi-ally T-wave inversion, and displacement of the ST-segment, which are indicative of myocardial hypoxia. Multiple necrotic foci and myocar-dial alterations similar to those seen in myocardial ischemia have also been observed in experimental animals under these conditions (76). Since catecholamines are known to be elevated in diabetics (77), i t may therefore be postulated that such elevated catecholamine levels in the diabetic may play a role in determining the susceptibility of dia-betic hearts to myocardial ischemic injury. 1.12 Experimental Diabetes and Pathogenesis Abnormalities of the myocardium, apparently independent of coron-ary atherosclerosis, have been described in animals with spontaneous or experimental diabetes as well as in human diabetics (78,79). In the dog, one of the features of the disease is the accumulation of collagen in the myocardial interstitium. Under certain conditions, such as chronic ketoacidosis, such collagen deposition may not be ob-served because of an impairment in cardiac protein synthesis (80). Distinctly different cardiac abnormalities have been described in a rat model during streptozotocin-induced diabetes. Depressed con-t r a c t i l i t y , slowed rate of relaxation, abnormal molecular properties of myosin, and impaired calcium transport have all been observed (78, 79). In addition, various biochemical changes, including accumulation of long-chain acyl carnitines and l i p i d s , have been described in the dia- betic rat heart (81).These changes are normalized after 6 weeks of 21 insulin replacment, and may at least partially be dependent on the correction of acidosis (82). The latter can impair developed tension and calcium exchange in non-diabetic heart muscle (83). It should be noted that the incidence of heart failure in human diabetics after prolonged untreated ketoacidosis is unknown. 1.13 Metabolic Alterations in Diabetic Myocardium Myocardial carbohydrate metabolism in diabetes has been extensive-ly studied (84,85). The failure of the acute-diabetic animal hearts to u t i l i z e carbohydrates, the reversal of this defect by insulin, the bias towards l i p i d combustion, and the accumulation of intracardiac triglycerides all indicate profound myocardial metabolic abnormalities in the acute diabetic state (86). Contemporary studies with isolated Langendorff heart preparations have shown a series of acute metabolic defects which underlie these early observations (86). Impaired glu-cose transport across the cell membrane, decreased glucose phospho-rylation and impaired glycolysis by inhibiton of the rate limiting phosphofructokinase reaction have all been demonstrated (86). Lipid metabolism is both obligatory and preferential, and phosphofructokin-ase inhibition probably results from increased cellular citrate con-centrations, consequent to this increased l i p i d u t i l i z a t i o n . More c r i t i c a l l y , pyruvate dehydrogenase inhibition limits acetyl CoA entry into the c i t r i c acid cycle, this being reflected by an increased cyto-solic acetyl CoA: CoA ratio (86). The myocardium in diabetes, as in the fasting state, shows in-creased concentrations of intermediates of li p i d metabolism, the in-22 creased fatty acyl CoA, glyceride, and long-chain acyl CoA content a l l being demonstrable in the alloxan-diabetic perfused rat heart (86). Recent evidence indicates that accumulation of long-chain acyl CoA and carnitine can inhibit several enzymatic functions including mitochon-drial oxidative phosphorylation and adenine nucleotide translocase (87). Mitochondrial function could, thus, be impaired in diabetic hearts, as the synthesis of high energy phosphate compounds and their translocation across the mitochondrial membrane could be disrupted (86). The deleterious c l i n i c a l association of ischemic heart disease and diabetes mellitus has previously been mentioned. The obligatory pro-motion of fatty acid metabolism in the diabetic state is likely to be deleterious to the outcome of myocardial ischemia, where the u t i l i z a -tion of glucose promotes cellular survival and the combustion of free fatty acids has the reverse effect (86). The correct balance between the utilization of glucose and of free fatty acids by the ischemic heart seems c r i t i c a l , especially in the border zone between viable and necrotic tissue as visualized in the models of acute experimental myo-cardial infarction (86). Since diabetic ketoacidosis is known to worsen the outcome of dia-betic patients suffering from acute ischemic heart disease, i t seems reasonable to suppose that the acutely-diabetic myocardium might be more susceptible to coronary occlusion. Sinclair-Smith and Opie (87), therefore, perfused hearts from acutely-diabetic rats with glucose, beta-hydroxybutyrate or free fatty acids. After coronary occlusion , 23 hearts from severely diabetic rats with ketosis released considerably more creatine phosphokinase than did hearts from non-ketotic diabetic rats or controls; highest release rates were achieved in ketotic hearts perfused with ketone bodies or free fatty acids. Support was, therefore, given to the concept that promotion of the l i p i d uptake by the heart with regional ischemia exaggerates the extent of ischemic injury. Diabetes mellitus also alters the mechanical performance of the heart in the experimental situation (86). The glucose uptake of the insulin-deficient heart is decreased by about half when compared with the non-diabetic heart, but is accelerated by increased heart work. However, even with added insulin, the diabetic heart can not achieve normal rates of glucose uptake or glucose oxidation as seen by in-creased lactate production (86). Measuring aortic mean flow as an index of mechanical performance, Hearse et al (88) studied the abili t y of isolated perfused working hearts from diabetic rats to survive during, and recover from, 30 min-utes of anoxia. Compared with normal, diabetic hearts exhibited an early transient fai l u r e , subsequently recovering during a secondary phase, but not to the same extent as the hearts of normal animals. Increased vulnerability of diabetic hearts to anoxic damage was thus demonstrated. Using metabolic inhibitors, the biochemical basis of some of these reactions was investigated. Endogenous supplies of high energy phosphates and energy derived from the glycogenolysis and glycolysis was available in the immediate post-anoxic period, with 24 l i t t l e dependence on the oxidative metabolism of either endogenous or exogenous substrates. In normal hearts, the role of the major energy substrate provider was rapidly assumed by oxidative processes u t i l i z -ing exogenous substrates. In the insulin-deficient diabetic hearts, impaired glucose transport into the cells (with consequent energy shortage) led to an i n i t i a l cardiac failure and a delayed secondary recovery. Reversal of this effect was achieved by exogenous insulin or by the addition of pyruvate, acetate, citrate or beta-hydroxybu-tyrate as energy sources whose utilization was not impaired by insulin deficiency (88). In contrast to anoxic challenge, the effects of ischemia on rat myocardial function and metabolism in diabetes u t i l i z i n g the working heart preparation of Neely (89) was reported by Feuvray et al (90). When compared with control hearts, diabetic hearts perfused under aerobic conditions had higher tissue levels of total CoA than long-chain acyl CoA, lower levels of total carnitine but higher levels of long-chain acyl carnitine esters. Glucose utilization was lower than in normal hearts. Mild ischemia (50%reduction in coronary flow) re-sulted in : 1) greater glucose utilization in normal hearts compared with diabetics, 2) increased tissue levels of. acyl esters of CoA and carnitine in both groups but greater in diabetics. Severe ischemia resulted in a more rapid rise in tissue long-chain acyl CoA and acyl carnitine esters in diabetic hearts. Mechanical function of the heart did not differ in normal controls and diabetics at low work levels, but at higher work levels and more 25 severe ischemia, a faster rate of ventricular failure occured in the diabetic hearts (90). In hearts not working at maximum loads, energy production from oxidation of lipids compensates for reduced glucose u t i l i z a t i o n , which allows the diabetic heart to maintain normal function. However, under maximal stress an intrinsic defect of the diabetic heart to produce ATP may be present, which may be consequent to increased tissue levels of metabolites associated with free fatty acid oxidation. Tissue lev-els of triglycerides, free fatty acids, long-chain acyl CoA, acetyl CoA and citrate are a l l elevated. Ischemia greatly elevates tissue levels of both acyl CoA and acyl carnitine especially when exogenous fatty acids are available. It is to be anticipated that in the diabe-ti c ischemic heart these metabolites may be present at markedly ele-vated levels (86). 1.14 Carnitine Metabolism in Myocardial Ischemia In the carnitine carrier system, free fatty acids are taken up from the circulation and converted into acyl CoA within the cell and then transported across the inner mitochondrial membrane. Intramito-chondrial acyl CoA undergoes oxidative phosphorylation and produces ATP which is transported outward by means of the ATP translocase sys-tem for utilization by cellular systems including contractile mechan-isms (91,92). Ischemia inhibits oxidative phosphorylation and is also associated with increases in intramitochondrial levels of acyl CoA, thereby blocking the ATP translocase system. For such postulated mechanisms, the intramitochondrial localization of carnitine is obli-26 gatory-a finding recently confirmed by Idell-Wenger et al (93). Liedtke et al (94) tested the hypothesis that the replacement of carnitine, an intracellular carrier of free fatty acids and an agent which is lost to the heart during ischemia, might restore ventricular function when given to swine hearts perfused with high free fatty acids. Ischemia per se in normal hearts significantly decreased sev-eral parameters of global and regional mechanical function including left ventricular and mean aortic pressues, epicardial motion and left ventricular work, together with myocardial oxygen consumption. A fur-ther 25% reduction in the above indices was recorded when the perfu-sion solution contained elevated free fatty acids. DL-carnitine treat-ments in nine perfused hearts, not supplemented with extra free faty acids were without apparent effect in improving contractility. How-ever, DL-carnitine caused considerable improvement in high free fatty acid perfused ischemic hearts, suggesting that carnitine in ischemic hearts is capable of preserving mechanical function under conditions of excess free fatty acid challenge presumably through modification of intermediate free fatty metabolites (94). 1.15 Prevention of Ischemic Heart Disease in Diabetics The problem of an increased incidence and unfavorable prognosis of CAD in the diabetic is abundantly clear, but its solution is not. The prevention of myocardial ischemia is the goal to be attained, and in terms of its atherogenic potential, diabetes should never be treat-ed l i g h t l y . Obviously, control of numerous and as yet ill-defined factors involved in the pathogenesis of atherosclerosis must be sought. 27 Probably the two most important factors are the presence of diabetes it s e l f and the associated presence of hypertension, which appears to increase the incidence of arteriosclerotic heart disease by one and one-half times (5,6,8,9). Although hypertension certainly increases the severity of atherosclerosis in diabetics, the suggestion by Holmes that, in the absence of hypertension, atherosclerosis is no more sev-ere than in non-diabetics does not agree with the Joslin Clinic ex-perience (95). The influence of chronic treatment with insulin on the development and progression of the tissue complications of diabetes has been a matter of much controversy. Long-term use of insulin in diabetic ani-mals has apparently reduced the incidence of retinal microvascular disease but the influence of the hormone on the myocardium is less clear (96). In a chronic canine model of diabetes, collagen accumula-tion and diminished myocardial compliance were unaffected by insulin control of postprandial hyperglycemia (97). Similarly the lipi d ab-normalities of diabetic rat nerves are reportedly not corrected by insulin therapy (98). Treatment, of adult-onset diabetes is frequently approached by cal-oric restriction, perhaps combined with a modest exercise program that usually results in improved glucose tolerance. Although infrequent episodes of ketoacidosis or marked hyperglycemia may require intermit-tent use of insulin,only a minority of adult-onset diabetics do re-quire long-term insulin therapy for metabolic control (99). In view of the uncertain status of tolbutamide, other oral hypoglycemic agents 28 should only be used i f indicated. Treatment for hypertension and hyperlipidemia is at least as important as in non-diabetics i f not more so (100). Patients who develop heart failure requiring thiazide diuretics for treatment of edema or those with angina requiring beta-blocking agents may run the risk of impaired insulin secretion (101) . The latter problem is minimized by cardioselective agents with beta-1 action. Pre-load and after-load reducing agents should be used cautiously in diabetics with ischemic heart disease, since impaired arterial reflex responses due to autonomic dysfunction may be present (102) . Digitalis is particularly useful in diabetic patients with atrial f i b r i l l a t i o n and heart failure (103). The diabetic experience with CAD is sufficiently foreboding to make i t desirable not only to ut i l i z e every possible means to prevent the premature development of CAD, but also to consider the variety of techniques by which the diabetic patient threatened with serious cor-onary artery problems might undergo myocardial revascularization. Bilateral internal mammary artery revascularization has been performed in diabetic patients because of the simplicity and the low risk asso-ciated with the procedure. To date, however, the results have not been impressive (104). Through the studies of Sones, ut i l i z i n g selec-tive coronary arteriogaraphy, a reawakening of interest in the possi-ble benefits of transplantation of the internal mammary artery into the myocardium has given renewed hope of genuine benefit in appropri-ately selected patients (105). t With the widespread involvement of the coronary circulation in 29 diabetic patients, an additional problem is that of diffuse myocardial fibrosis and limited functional reserve which may further compromise coronary blood flow. At present, diabetic patients are being reevalu-ated as possible candidates for myocardial revascularization, using saphenous vein autografts. The benefits to be expected are as yet unknown, but i t appears almost certain that some patients will be helped. The primary objective will be to lengthen the l i f e of the diabetic patient with CAD and also to delay or lessen disability due to angina. 1.16 Problems with current data All of the c l i n i c a l and experimental literature leads to the con-clusion that diabetes causes a deterioration of cardiovascular func-tion, and makes the heart especially vulnerable to ischemic injury. At present, very few details regarding the elucidation of early and late molecular events in myocardial ischemic injury occuring in ex-perimental diabetes are available. Most studies to-date have focussed their attention on overall cardiovascular function in diabetes (106). Whether the increased susceptibility of diabetic myocardium to ischem-ic injury is associated with alterations in membrane integrity of the various subcellular organelles is not known. It is not even known to what extent the biochemical alteration in diabetic-ischemic injury are reversible, since no time-course studies have been done. In order to fully comprehend the nature of ischemic injury in the diabetic and to devise some form of rational pharmacological intervention, i t is necessary to look at several levels of cellular organization ranging - 30 -from whole organ function to the integrity of the subcellular organ-elles using a multilevel-analytical approach. 1.17 Model of Diabetes From the above cl i n i c a l and experimental data, i t is clear that cardiovascular disease represents one of the major causes of morbidity and mortality in the diabetic patient, yet a serious lack of informa-tion is available regarding the mechanisms by which diabetes exerts its effects. The use of animal models for studies of the cardiovascular compli-cations of diabetes has been complicated either by problems in availa-b i l i t y or by the fact that the most widely available models have ex-hibited only minimal cardiovascular alterations. The small size of some animal models does not permit extensive investigations of the cardiovascular system. Nevertheless, important information can be obtained from studies of these models, particularly i f they are selected carefully to examine specific research problems. The model of diabetes we have utilized is the rabbit. Although a colony of dia-betic rabbits with spontaneous diseases has been described, no studies regarding their cardiovascular system have been performed (107). Alloxan treatment has been widely used to induce diabetes in rab-bits because of d i f f i c u l t i e s experienced with strepotozotocin in this model (108). Rabbits tend to tolerate diabetes well as long as severe ketosis and dehydration are avoided during the f i r s t week. A stable, non-ketotic form of diabetes can be achieved and the growth rate of these animals is near normal over an extended period of time, thus 31 making the animals potentiality useful for chronic studies. In addi-tion, the size and longevity also permit a wide range of metabolic, physiologic and pathologic studies relating to the cardiovascular sys-tem. Finally, the ready availability and relatively low cost of these animals are obvious advantages in using rabbits as a model to study the diabetic cardiovascular complications. 1.18 Experimental Approach to the Study of Myocardial Ischemic  Injury in Diabetes One of the major problems faced in attempting to study the nature of myocardial damage produced by ischemia is the multiplicity of lev-els of structural and functional organization at which alterations may be investigated. One may focus on changes which are chemical (e.g. ATP, lactate, inorganic cations, etc), ultrastructural, biochemical (at the level of various subcellular organelle systems), electrophy-siological (patterns of abnormal electrical activity) or functional (abnormalities in the contractility of the myocardium). It is clear that a l l of these manifestations of myocardial ischemia are very clo-sely interrelated and the subdivisions somewhat a r t i f i c i a l . Nonethe-less, most experimental studies focus on one or at best a few, of these diverse aspects of the overall problem usually because of tech-nical limitations and/or expertise on part of the investigator. As a result, uncertainties are introduced when observations obtained with one experimental model of ischemia are rather freely extrapolated to those made in other systems by other investigators using different analytical approaches. 32 Species differ widely in their susceptibility to myocardial i s -chemia injury following coronary artery ligation, with factors such as animal size, metabolic rate and particularly the degree of collateral circulation being c r i t i c a l considerations (109). In experiments in-volving coronary ligation, the particular vessel occluded is an impor-tant determinant of the nature of the injury produced and its response to drugs. Thus, ligation of the circumflex branch of the cornary artery yields predominantly posterior infarcts while occlusion of the left anterior descending coronary artery yields mainly anterior in-farcts (110). In cases of experiments ut i l i z i n g anaesthetized ani-mals, the type and dose of the agent employed may markedly influence the extent of damage either directly (by modifying properties of cel-lular membranes) or indirectly, as the result of hemodynamic altera-tions producing changes in oxygen supply and demand (111). It is only recently that the subcellular basis of cardiac contrac-t i l e failure involving different membrane systems is beginning to re-ceive the attention of various investigators, and the results thus far have already led to the formulation of a new concept (112). Accord-ingly, i t is believed that irrespective of etiological factors, heart damage is ultimately referable to defects in membrane handling of cal-cium, leading to intracellular calcium overload (113). A survey of the existing literature shows that myocardial ischemic injury is asso-ciated with alterations in one or more membrane systems such as those of the sarcolemma, sarcoplasmic reticulum and mitochondria (114, 115). In most experimental models of myocardial ischemic injury, 33 alterations in calcium accumulation and membrane-bound enzyme activit-ies have been identified (116). Therefore, not only should various experimental models of myocardial ischemic injury be used in investi-gating membrane defects, but i t is also essential that different mem-brane systems from the same experimental model be examined concomit-antly. A close examination of the literature reveals that very few at-tempts have been made to test the reversibility of the membrane de-fects in myocardial ischemic injury in diabetes (117). There are also genuine gaps in our knowledge concerning the changes in the membrane systems in experimental models of ischemic injury which simulate com-monly encountered human disease. Therefore, in our study we propose to study the time-course of changes in membrane functions in myocar-dial ischemic injury in diabetic rabbits and correlate these with chemical, ultrastructural and functional changes. It is hoped the information gained from such a multi-disciplinary approach to analyz-ing myocardial ischemic injury will help in earlier diagnosis, allow more accurate indication of prognosis, will suggest approaches to arresting i f not reversing the course of the disease and open up new avenues for the treatment of myocardial ischemic injury not only in diabetic but also in non-diabetic patients. 1.19 Objectives of the Study With all these limitations and uncertainties in mind, we have un-dertaken a study of myocardial ischemic injury in the diabetic rabbit. The overall objective of this thesis was to understand the nature and 34 extent of myocardial ischemic injury (induced by catecholamine admini-stration or coronary artery ligation) in diabetic rabbits and compare it to that in control animals. The following were the major objec-tives of the investigation: a) To study alloxan-induced diabetes in the rabbit as a model of insulin-dependent diabetes. b) To determine the biochemical, functional and ultrastructural alterations in the myocardium of diabetic rabbits. c) To develop a model of isoproterenol (ISO)-induced myocardial i s -chemic injury in the rabbit and determine the extent of injury using biochemical, functional and ultrastructural parameters. d) To determine the effects of isoproterenol (ISO)-induced myocardial ischemic injury in the diabetic rabbit. e) To determine the incidence of arrhythmias and ischemic injury in the diabetic rabbit following coronary artery ligation and reper-fusion. f) To determine the extent of ischemic injury in conscious diabetic rats following ligation of the left anterior descending coronary artery. 35 2 . MATERIALS AND METHODS 2.1 Animal Studies 2.1.1 Induction of diabetes in the rabbit Adult male New Zealand white rabbits (1.5-2.0 kg) were fasted overnight before injection with alloxan. Alloxan monohydrate (Sigma Chemical Company, St. Louis, Missouri) was dissolved in sterile saline (0.85%) immediately before use and injected (100 mg/kg) intravenously into the caudal ear vein of lightly anesthetized rabbits (sodium pen-tobarbital, 20 mg/kg, intravenously). Control rabbits received intra-venous saline (0.85%) alone. Rabbits which failed to show an increase in blood glucose were given another injection of alloxan 24-48 hours later. Because alloxan injection is i r r i t a t i n g and painful, 0.5 ml of 2%xylocaine was injected subcutaneously into the ear just before the injection of alloxan. Because alloxan is capable of producing fatal hypoglycemia as the result of massive insulin release from the pan-creas, animals were treated with 20% glucose (15-20 ml) subcutaneously every 4-6 hours following alloxan administration for the f i r s t 24 hours. To prevent dehydration from the severe polyuria, intravenous saline (0.85%, 10 ml/kg) was also administered every 12 hours for the f i r s t 36-48 hours. 2.1.2 Induction of diabetes in the rat Male Wistar rats weighing 150-200 g were fasted overnight and made diabetic by injecting streptozotocin (Sigma Chemical Company, 55 mg/kg, dissolved in 0.05 M citrate buffer, pH 4.5) into the t a i l vein with the animals under light ether anaesthesia. Control animals re-36 ceived citrate buffer only. All the animals were housed under identi-cal conditions and allowed free access to Purina Rat Chow and water. Four days after the injection with streptozotocin, 24 hour urine sam-ples were collected and tested with L i l l y Tes-tape to determine urine glucose levels as an indication of the presence or absence of diabetes. Ketosis was monitored using ketostix. Animals were sacrificed at 6 and 12 weeks after injection with streptozotocin and the blood col-lected was analyzed for serum glucose, insulin, lipids and thyroid hormone status. 2.1.3 Myocardial ischemia produced by coronary artery ligation Male New Zealand white rabbits (2.0-2.5 kg) were anaesthetized by injecting 30 mg/kg of sodium pentobarbital into the ear vein. A tracheotomy was subsequently performed to permit positive pressure ventilation using a Palmer pump. Blood gas measurements were done periodically and were in the normal range. A polyethylene cannula was inserted into the carotid artery and connected to a Bell and Howell pressure transducer (Model 4-327-0010) in order to monitor blood pres-sure. The chest was opened by a sternotomy, the rib cage retracted and the heart suspended in a pericardial cradle. A 20-gauge Jelco catheter was inserted into the left ventricle through the apex and connected via a polyethylene tubing to a Statham P231D pressure trans-ducer. The output of this left ventricular pressure recording was fed into a calibrated differentiator (Grass Instruments, Model 7P20 C) to obtain the differentiated waveform - an indirect measure of cardiac contractility (dP/dt) max. Maximal rates of rise of left ventricular 37 pressure were well within the response range of the pen recorder. Electrocardiograms were recorded from both limb (Leads 1, 11, 111, aVr, aVf, aVl) and chest (V1-V5) leads . Elevation of the ST-segment and R-wave enlargement were monitored in order to assess whether or not the left circumflex coronary artery had been successfully ligated. In addition, the electrocardiogram was used to determine the incidence of arrhythmias during the ligation and reperfusion periods. Ligation was performed by inserting a 4-0 silk suture around the left circumflex coronary artery at its origin. The two free ends of the ligature were passed through a short length of polyethylene tubing and ischemia was produced by pulling up and securing the ends with a haemostat. Clamping the suture in this way made the occlusion readily reversible when reperfusion was part of the protocol. Tightening of the ligature resulted in an occluded zone of approximately 60 ± 5% (mean ± SD, n = 30) of the left ventricle. The percent occluded zone was measured as: wet weight of occluded zone x 100 total left ventricular wet weight If f i b r i l l a t i o n occurred during the ligation or reperfusion per-iods, brass electrodes were placed directly on the heart and a 0.5 Watt-second D.C. - countershock was applied from a defibrillator (American Optical Company). This low level of stored energy was usually effective in restoring sinus rhythm and did so at a level well below the threshold shown by Koning et al (118) to produce tissue dam-age in isolated rabbit hearts. ST-segment elevation was measured above the T-P segment after the 38 end of the QRS complex. The amplitude of the R-wave was measured in mm to the nearest 0.5 mm, using the same isoelectric l i n e . The R wave amplitude at each site was calculated throughout the experiment, and the difference between each R wave at the start of each experiment and at the end of the experiment was determined. If the R wave in the remote epicardial positions or the limb leads showed progressive chan-ges in axis, the recordings were rejected. Chest ECG recorded during the 20 minute control period showed no significant change in the ST-segment or the R-wave. The ECG recordings were recorded at 3 minute intervals before and after occlusion of the left circumflex coronary artery. The effects of respiration were taken into consideration by measuring the mean ST-segment elevation and R wave amplitude from 5-ECG complexes with each observation. When the experiment was complete, the heart was quickly removed by cutting the aorta and was immediately dropped into ice-cold sucrose (0.25M). After removal of the heart, a l l procedures were carried out a 4'C. I n i t i a l l y the occluded zone as identified by perfusing the heart retrogradely via the aortic root with indocyanin green (2.5 mg/ml at 100 mmHg pressure). The ligature was reclamped i f the heart was removed during reperfusion. Thus, the ligated zone remains unper-fused, whereas the non-occluded zone turned green after the infusion of the dye. This method of quantitation of the occluded zone provided us an estimate of the size of the occluded zone. However, since the dye may affect the biochemical assays that were to be performed on the ischemic tissue, the hearts were perfused with sucrose (0.25 M). 39 Again, the ligated zone remained unperfused and could be distinguished from the surrounding perfused tissue which was blanched. No major difference was observed when the occluded zone was identified by per-fusing the tissue with either sucrose or the dye. An excess of tissue was trimmed from the border of the ischemic zone to ensure that the isolated area contained only ischemic tissue. Sample for ATP analysis were obtained directly from the heart in situ and were rapidly frozen in liquid nitrogen at -70° C. Similarly, for hearts that were to be analyzed by electron microscopy, tissues were again obtained directly from the heart in s i t u . The procedures for preparing tissues for electron microscopy are described below. The remainder of the tissue from the occluded zone was subjected to subcellular fractionation as outlined below. Control tissue (taken from the same area of the ventricle) was always obtained from sham-operated animals subjected to a l l surgical procedures except tighten-ing of the ligature. The same area of the left ventricle was used for tissue sampling after the appropriate time lag between removal and freezing of the tissue. 2.1.4 Coronary ligation in rats The conscious rat coronary artery ligation preparation is an ex-tension of methods used in anaesthetized rats (119,120). Approximate-ly one week before ligation, rats were anaesthetized with halothane (1.%) and the chest opened. Under positive pressure respiration, a 5-0 polypropylene suture was passed around the left anterior descend-ing coronary artery and arranged so that the ligature could be tight-40 ened from outside the chest. Permanent stainless steel ECG leads were placed subcutaneously in each limb (using a long subdermal trocar) and exteriorised near the occluder. A chest lead was also used and this was placed in the pectoralis muscle overlying the chest incision (4-5th intercostal space). After 7 days of recovery from the surgery, the animals were left in the cage for 1 hr prior to ligation. To ob-tain ligation, sufficient traction was exerted between the polypro-pylene suture and the polyethene tubing guide to close the ligature. Ligation was easily completed within seconds in the unrestrained ani-mal. Ligation was performed during daylight hours on rats in their cages and animals tended to sleep during the various procedures. Rats responded to ligation with few apparent outward signs of discomfort (restlessness and mild piloerection). Generally, no other marked changes in behaviour were seen and many animals assumed a sleeping position shortly after ligation. If ligation produced ventricular f i b r i l l a t i o n which did not spontaneously revert within 10 seconds, i t was usually successfully terminated by tapping the chest. After 24 hours of ligation, the animals were sacrificed and the heart removed. The heart was perfused (Langendorff technique) with Krebs solution at 22° C and 100 mmHg pressure for 5 minutes to remove a l l blood. A bolus of 2.0 ml cardio-green dye (Indocyanine Green, Hynson, Westcott and Dunning Inc., Baltimore, MD) (1.0 mg/ml in Krebs solution) was then used to differentiate the perfused (green) from the unperfused or occluded zone (pink). The unperfused region was immedi-ately cut out and weighed. The entire ventricle was also weighed and 41 the occluded zone was expressed as the percent of total left ventricu-lar weight. Immediately thereafter, the heart tissue was sliced long-itudinally into 1.0 mm thick sections and incubated in tetrazolium dye (10 mg/ml, 2-3-5 triphenyl tetrazolium chloride in 70 mM sodium phos-phate buffer, pH 8.5) at 37° C for 30 minutes. At the end of the in-cubation period all sections were placed in 10%formalin (in normal saline) for 2-3. days before the undyed (white) infarcted tissue was dissected from viable tissue (purple). Infarcted tissue was expressed as percent (by weight) of total ventricular tissue. 2.1.5 Induction of myocardial ischemic injury by isoproterenol Male rabbits (2.0-2.5 kg) were treated with isoproterenol hydro-chloride (ISO) administered subcutaneously in aqueous solution at an i n i t i a l dose of 0.5 mg/kg on the f i r s t day, with an increase of 1 mg/kg on each subsequent day for 15 days. Control animals were in-jected subcutaneously with water only. Because rabbits are highly sensitive to catecholamine-induced arrhythmias, ISO was administered in 4-divided doses given over a period of 12 hours. ISO solutions were freshly prepared daily using sterile water. 2.2 Isolation and Characterization of subcellular organelles 2.2.1 Preparation of mitochondrial and sarcolemmal membrane-en- riched fractions from myocardial tissue 1 g of normal or infarcted rabbit left ventricle was minced with scissors and homogenized in 10 ml' buffer (1.25 KC1, 2 mM dithio-t h r e i t o l , 0.5 mM CaC^, 10 mM T r i s , pH 7.4). The tissue was homo-genized on ice with a Polytron PT-10 homogenizer (Brinkman Instru-42 ments) for 5 seconds at one-quarter maximal speed. The homogenate was filtered through a 0.5 mm nylon mesh and centrifuged at 1200 x g for 10 minutes. The supernatant was discarded and the pellet resuspended in 10 ml homogenization buffer and homogenized in a 40-ml glass homo-genization tube with a tight f i t t i n g teflon pestle using 3 up and down strokes with a Potter Elvejhem homogenizer. The homogenate was then centrifuged at 3000 x g for 10 minutes. The supernatant was discarded and the pellet resuspended in 3.0 ml of 10%w/v sucrose buffer con-taining DTT-10 mM T r i s , pH 8.2. The suspension was homogenized to homogeneity with a glass hand homogenizer.- 1.0 ml of the homogenate was layered on top of each of 6 gradient tubes which contained discon-tinuous sucrose gradients with 2.5 ml 60% sucrose as bottom layer and on which was layered 2.5 ml portions of 55%', 52.5% and 50%w/v. The sucrose for the preparation of the gradients was dissolved in 2 mM DTT-10 mM Tris pH 8.2. The gradient was centrifuged at 40,000 x g for 1 hour in a Beckman ultracentrifuge (Rotor SW 21). The sarcolemmal fraction equilibrated at the 55-60%sucrose interface while the mito-chondrial and sarcoplasmic reticulum fraction was equilibrated on top of the 50%sucrose fraction. The sarcolemmal and mitochondrial frac-tions were removed with a Pasteur pipette, washed in 5 volumes of 10 mM Tris pH 7.4, and centrifuged at 30,000 x g for 15 minutes. The pellet was resuspended in double d i s t i l l e d water to yield a protein concentration of approximately 3-4 mg/ml. The membranes were quickly frozen in vials by immersion in acetone/dry ice and stored at -20° C. 43 2.2.2 Characterization of sarcolemmal and mitochondrial membrane  fractions The activity of the mitochondrial marker enzyme cytochrome c oxi-dase was measured in both the sarcolemmal and mitochondrial fractions in order to assess the mitochondrial contamination of the sarcolemmal fraction. Cytochrome c oxidase activity was assayed in membrane fractions by the procedure outlined by Cooperstein and Lazarow (121). A stock solu-tion of 0.03 M phosphate buffer pH 7.4 was prepared by mixing 805 ml 0.03 M Na2HP04 with 195 ml 0.03 M KH2P04. A solution of re-duced cytochrome c was freshly prepared by adding 100 yl of freshly prepared 1.2' M sodium hydrosulfite to a 17 M solution of cytochrome c (Sigma Type III) dissolved in 0.03 M phosphate buffer. The cytochrome c solution was shaken vigorously for several minutes to remove excess hydrosulfite. Three ml of this reduced cytochrome c solution was transferred to a spectrophotometric cuvette and to this an aliquot of 50 yl of sample was added. The absorbance at 550 nm was measured at 30 second intervals for 3 minutes. The cytochrome c in the cuvette was then f u l l y oxidized by the addition of a few crystals of potassium ferricyanide. Absorbance of this f u l l y oxidized solution of cyto-chrome c was subtracted from each of the absorbances obtained at the 30 second time intervals following sample addition. The logarithm of these incremental absorbance values when plotted against time gave a straight line with a negative slope from which could be determined the amount of cytochrome c (in ymoles) oxidized per minute per mg sample 44 protein. 2.2.3 Triton X-100 addition For all experiments, 0.05% and 0.001% Triton X-100 was added to the assay medium for the mitochondrial and sarcolemmal fractions, respectively, in order to eliminate the effects of vesiculation on measured enzyme activities (122). The effects of detergents on enzyme activity followed a bell-shaped curve, i.e., activation at low concen-trations followed by inhibition at concentrations higher than those listed above. 2.2.4 Isolation of mitochondria Mitochondria were isolated according to the method of Peng et al (123). Samples of left ventricular tissue were weighed and homogen-ized as a 10% suspension (w/v) in 0.25 M sucrose using a PT-10 Poly-tron instrument (one quarter maximal speed for five seconds). The resulting mixture was centrifuged (1200 x g for 10 minutes) and the pellet discarded. The supernatant was strained through several layers of gauze and centrifuged at 10,000 x g for 15 minutes. The resulting pellet was resuspended in sucrose and recentrifuged at 10,000 x g for 15 minutes. The second washing procedure was repeated to remove cel-lular contaminants adhering to the mitochondria. Mitochondria were fin a l l y resuspended in sucrose (0.25 M) to yield a suspension contain-ing 2-3 mg mitochondrial protein/ml. 2.2.5 Isolation of lysosomes Samples (1 g) of normal or infarcted tissue were homogenized as a 10%suspension (w/v) in buffer (0.25 M KC1, 1 mM EDTA and 50 mM Tris 45 pH 7.4) using a Polytron (PT-10) instrument (two bursts of 15 seconds at one quarter maximal speed). The resulting mixture was centrifuged' (500 x g f o r 5 minutes) and the pellet discarded. The supernatant was centrifuged at 40,000 x g for 30 minutes. The pellet was resuspended in 7.5 ml homogenization buffer containing 0.1% Triton and homogenized (PT-10, one burst of 35 seconds at a setting of 4.5) to achieve total disruption of the lyspsomes. Lysosomal latency was measured by e s t i -mating the activities of the lysosomal hydrolases (cathepsin D, acid phosphatase and N-acetyl-B-glucosaminidase) in both non-sedimentable and the sedimentable pellet fractions. The percent latency of the enzymes is expressed as follows: Activity in lysosomal supernatant x 100 Activity in pellet supernatant + lysosomal supernatant 2.3 Biochemical Assays 2.3.1 Nitrophenylphosphatase activity Nitrophenylphosphatase activity was measured according to the method of Godin et al (124) by measuring the hydrolysis of p-nitro-phenylphosphate in the presence of magnesium and potassium at 37° C and pH 7.4. The reaction mixture consisted of 1.0 ml imidazole buffer (0.15 M, pH 7.4) 0.1 ml 0.09 M p-nitrophenylphosphate, 0.1 ml 0.09 M MgC^, 0.1 ml 0.9 M KC1, 0.2 ml membrane suspension and double dis-t i l l e d water in a final reaction volume of 3.0 ml. The reaction was stopped by the addition of 1.0 ml cold 10% trichloroacetic acid and the protein precipitated by centrifugation at 2000 x g for 10 minutes. A 3.0 ml aliquot of the supernatant was taken and alkalinized with 1.0 ml of 1.5 M Tris solution. The absorbance of the resulting solution 46 was measured at 412 nm. The K-stimulated (ouabain-sensitive) compon-ent of the enzyme was determined by subtracting the activity of the enzyme assayed in the presence of magnesium (basal activity) from the activity of the enzyme assayed in presence of both magnesium and potassium (total a c t i v i t y ) . 2.3.2 Sarcolemmal Na+K+-ATPase Sarcolemmal ATPase activity was measured according to the method of Godin et aT (124). Na K -ATPase activity was assayed in the heart membrane preparation by incubating for 15 minutes at 37° C a reaction mixture that consisted of 1.0 ml 156 mM Tris-HCl buffer, pH 7.4, 0.1 ml 0.09 M MgCl2, 0.1 ml 3 mM EGTA, 0.3 ml 30 mM ATP, 0.1 ml 0.6 M KC1, 0.1 ml 2.4 M NaCl, 0.2 ml membrane suspension and double d i s t i l l e d water to make the volume 3.0 ml. The protein concentration of the heart membrane suspensions was about 0.7-0.8 mg/ml. The reac-tion was stopped by the addition of 1.0 ml cold 10%,trichloroacetic acid and the mixture centrifuged at 2000 x g for 10 minutes. A 3.0 ml aliquot of the supernatant was combined with 1.8 ml molybdate solution (1.4 ml d i s t i l l e d water plus 0.4 ml 5% ammonium molybdate). Color development for inorganic phosphorous was initiated by adding 0.2 ml Fiske-Subbarow reagent (see below for preparation) to the mixture and the absorbance at 660 nm was determined spectrophotometrically after 15 minutes. These absorbance readings were compared to the readings obtained with standards containing known quantities of inorganic phos-phorous. The Na+K+-stimulated component of the enzyme was deter-mined by subtracting the activity observed in the presence of magne-47 sium alone from the activity seen when magnesium, sodium and potassium were all present in the reaction mixture. Similar results were obtain-ed with the addition of ouabain (30mM). The Fiske-Subbarow reagent was prepared weekly by the addition of 0.25 g l-amino-2-napthol-4-sulfonic acid (ANS), followed by the addi-tion of 15% sodium bisulfite and 0.5 g of anhydrous sodium sulfite with the volume made up to 100 ml. The solution was stored in a dark bottle for no longer than 1 week. 2.3.3 Mitochondrial azide-sensitive ATPase activity Mitochondrial ATPase activity was assayed in the presence and ab-sence of sodium azide (5 mM) as described by Godin et al (124) except that the reaction time was 15 minutes and each tube contained 15-30 yg of protein. Otherwise, the assay medium had the same composition as that for the Na+K+-ATPase assay except that NaCl and KC1 were omitted. 2.3.4 Sarcoplasmic reticulum ATPase activity Sarcoplasmic reticulum ATPase activity was measured according to the method of Jones et al (122). To 1.0 ml of imidazole buffer (0.15M pH 7.4) was added 0.1 ml 1.5 mM calcium chloride, 0.1 ml magnesium chloride (92 mM), 0.1 ml 10 mM Tris-EGTA, 0.1 ml 0.15 M sodium azide, 0.1 ml 30mM ATP, 0.1 ml 3 M KC1 and 0.2 ml of the membrane preparation. After incubation at 371C for 15 minutes, the reaction was stopped with 1.0 ml 10% trichloroacetic acid and the phosphate released was measured as described previously. The azide-insensitive, EGTA inhibitable sarcoplasmic reticulum ATPase activity is expressed as ymoles phosphate/mg protein/hour . 48 2.3.5 Mitochondrial ATP generation Mitochondrial ATP generation was measured by the method of Nayler (125). The mitochondria for these studies were extracted under condi-tions which favored retention of their endogenous calcium as described previously. ATP generating capacity was measured with the use of a reaction medium containing 250 mM sucrose, 12.5 mM Tris-HEPES (N-2-hy-droxyethylpiperazine-N'-2-ethanesulphonic acid) pH 7.2, 3.0 mM Tris-glutamate and 3.0 mM potassium dihydrogen phosphate. Mitochondria were added to provide a final concentration of 2.5 mg/ml protein, and the reactions, which were performed at 37° C, were started by the add-ition of ADP to provide a final concentration of 2.5 mM. Fifteen, 30, 45, 60 and 120 seconds later, 50 yl samples were removed from the re-action tubes and 50 yl of 6% perchloric acid added. The tubes were centrifuged at 5000 rpm for 10 minutes and to 200 yl of the superna-tant was added 100 yl of 1.5 mM ammonium bicarbonate. The tubes were centrifuged at 5000 rpm for 5 minutes and the resulting supernatant was assayed for ATP as described below. 2.3.6 Serum creatine phosphokinase Creatine phosphokinase activity in the serum was assayed according to the Sigma Technical Bulletin (no. 661). To 0.3 ml of serum was added 1.0 ml of creatine solution (0.06 M) and 1.0 ml water. The re-action was initiated by adding 0.1 ml ATP-glutathione solution (300 mg ATP and 200 mg glutathione in 5 ml of Tris-HCl buffer, pH 9.0) and incubated at 37° C for 30 minutes. To terminate the reaction, 1.6 ml of cold trichloroacetic acid (20%) was added and the tubes centrifuged 49 at 3000 rpm for 5 minutes. To 1.0 ml of clear supernatant was added 4.0 ml water, 1.0 ml acid molybdate solution and 0.25 ml Fiske-Subba-row solution. The mixture was allowed to stand at room temperature for 30 minutes and the color developed was read at 660 nm. Standard phosphorous calibration curves were run according to the procedures outlined in the Bulletin and the results expressed as Sigma units/ml. 2.3.7 Myocardial creatine phosphokinase activity Ischemic or normal ventricular tissue was minced with scissors and homogenised as a 25% (w/v) suspension in 0.25 M sucrose, 0.001 M EDTA, and 0.1 ml mercaptoethanol in a Polytron homogeniser (PT-10) at one quarter maximal speed for 5 seconds. The homogenate was then cen-trifuged at 16,000 x g for 10 minutes and the supernatant fraction was assayed for creatine phosphokinase activity as described above. 2.3.8 Measurement of Myocardial ATP ATP was measured according to the method of Jaworek et al (126). Samples of control or ischemic myocardial tissue (50 mg) were quick-frozen in liquid nitrogen and stored at -70° C for periods not exceed-ing 1 week. Prior to analysis, samples were ground up in liquid ni-trogen with 6% perchloric acid (4 pi per mg tissue) and thawed on ice. Following centrifugation at 3000 rpm for 5 minutes, an aliquot (80 pi) was neutralized with 60 pi of 1.4 M ammonium bicarbonate, left standing on ice for 15 minutes and centrifuged at 3000 rpm for 5 min-utes. An aliquot of the resulting mixture was used to estimate ATP content by a coupled enzymatic assay procedure, wherein 3-phosphogly-ceric acid is phosphorylated to 1,3-diphosphoglyceric acid in the 50 presence of ATP and phosphoglycerate kinase. The reduction of 1,3-diphosphoglyceric acid to glyceraldehyde-3-phosphate by glycer-aldehyde-3-phosphate dehydrogenase and NADH was monitored by measuring the conversion of NADH to NAD at 340 nm. Because water content may be increased in ischemic tissue, ATP content is expressed as nmoles/mg tissue dry weight. 2.3.9 Acid phosphatase Acid phosphatase activity was determined as described in Sigma technical bulletin number 104. The reaction mixture consisted of 0.5 ml 2.09 M citrate buffer (pH 4.8), 0.5 ml p-nitrophenylphosphate solu-tion (4 mg/ml) and 0.2 ml of sample. Samples were appropriately d i l -uted so that absorbance values did not exceed 0.900. The reaction was carried out at 37° C for 30 minutes and stopped with 5.0 ml 0.1 N NaOH. .The absorbance of the resulting solution was measured at 410 nm and divided by a factor of 0.3 to convert the results into Sigma units of acid phosphatase activity determined from a standard curve as des-cribed in Sigma technical bulletin number 104. 2.3.10 N-acetyl-g-glucosaminidase N-acetyl-e-glucosaminidase activity was determined by measuring the extent of hydrolysis of p-nitrophenyl N-acetyl-e-glucosamine sub-strate to p-nitrophenol under acidic conditions. The reaction mixture consisted of 0.5 ml of 0.3 M citrate buffer (pH 4.3), 0.5 ml p-nitro-phenyl-N-acetyl-e-glucosamine (3 mg/ml), 0.3 ml of d i s t i l l e d water and 0.2 ml sample. Samples were appropriately diluted in order that ab-sorbance values of the reaction did not exceed 0.300, the point after 51 which the relationship between absorbance and enzymatic quantity becomes non-linear. Plasma samples were usually diluted 1:3 with sal-ine. The reaction was carried out at 37° C for 30 minutes and then terminated with 1.5 ml cold 3%trichloroacetic acid. The solution was then centrifuged at 10,000 x g for 5 minutes and a 2.0 ml aliquot of the supernatant was taken. To this aliquot 1.0 ml bicarbonate buffer consisting of 0.5 M NaHC03 and 0.5 M Na2C03 was added to develop the color. The absorbance was measured at 420 nm. The results were expressed as absorbance/mg protein or absorbance/ml. 2.3.11 Cathepsin D Assays for cathepsin D activity were performed as described by Barett (127). The total volume of the reaction mixture was 0.4 ml and consisted of 0.1 ml 1 M formate buffer (pH 3.0) 0.1 ml 8 (w/v) hemo-globin substrate and 0.2 ml sample. The reaction was started with the hemoglobin substrate and incubated at 45° C for 60 minutes, after which i t was stopped by the addition of 2.5 ml cold 3%trichloroacetic acid. The reaction mixture was allowed to stand on ice for 30 minutes and then centrifuged at 3000 x g for 10 minutes. An aliquot of the supernatant (0.5 ml) was then assayed for small molecular weight pep-tides using the Lowry protein assay. Blanks for the cathepsin D assay contained water instead of 1 M formate buffer. Results are expressed as mg/protein liberated /hour. 2.4 Chemical Assays 2.4.1 Intracellular sodium The measurement of intracellular sodium content of myocardial t i s -52 sue involved washing out the sodium of the extracellular space (128) by transferring the tissue through a series of tubes containing 8 ml of oxygenated Na-free, K-free Ringer solution (composition 98 mM MgCl2, 1.4 mM MgS04, 1.6 mM MgHP04 and 1 mM ouabain) for 22 min-utes at 4° C. After washout, the tissues were blotted on f i l t e r pap-er, weighed and dried at 110° C overnight and reweighed to determine wet and dry weights. Tissues were then prepared for atomic absorption spectroscopy as described below. 2.4.2 Intracellular potassium, calcium and magnesium Intracellular magnesium, calcium and potassium were measured using the procedure of James and Roufogalis (129). Samples of each tissue (25 mg) were incubated in tubes containing 5 ml of oxygenated buffer (50 mM Tris HC1, pH 7.4 and 5 mM LaCl3) for 30 minutes on ice. The tissues were blotted on f i l t e r paper, weighed and dried at 110° C overnight and reweighed to determine the wet and dry weights. The dry tissues were then processed for atomic absorption spectroscopy as described below. 2.4.3 Red blood cell magnesium content Magnesium content of the red blood cells was measured by washing the cells three times in cold saline. It is essential to wash the cells in cold saline to prevent the leakage of intracellular ions. The packed red blood cells were lysed with water and an aliquot of the lysate was assayed for magnesium content. To 0.2 ml of the aliquot was added 0.2 ml of 100%trichloroacetic acid : 17 M acetic acid (v/v, 1:1)). After boiling for 30 minutes, 0.2 ml of ImM LaClo was added 53 and the supernatant analyzed for magnesium as described below. 2.4.4 Plasma electrolytes To 0.2 ml of the plasma was added 0.2 ml of 100%trichloroacetic acid : 17 M glacial acetic acid (v/v, 1:1) and 0.2 ml ImM LaCl3. The mixture was made up to 2 ml with water, an aliquot was assayed for electrolytes as described below. 2.4.5 Atomic absorption spectroscopy Tissue levels of sodium, potassium, magnesium and calcium were measured by atomic absorption spectroscopy using a modification of the technique of James and Roufogalis (129). The dried samples from the occluded zone or the comparable area of control hearts (approximately 10 mg) were dissolved in boiling 100%trichloroacetic acid: 17 M gla-cial acetic acid (v/v, 1:1) and 1 mM LaCL^ was added to precipitate phosphate. After centrifugation (5000 x g for 5 minutes) the superna-tant was analyzed using a Varian Techtron atomic absorption spectro-scope (Model AA 5) calibrated with freshly prepared standard solutions. Standard solutions contained the same proportion of acetic acid, lan-thanum chloride, potassium chloride and cesium chloride as did the sample solutions as well as the appropriate volume of atomic absorp-tion stock solution for each element (Fischer Chemical Company, stock solution concentration 1000 ug/ml). All solutions were prepared using freshly de-ionized water. Air-acetylene fuel was used for the meas-urement of sodium, potassium and magnesium. Calcium absorbance was measured in a nitrous oxide-acetylene flame. Suppression of ioniza-tion was effected by the addition of CsCl? (20 mg/ml) to the potas-54 sium samples and KCl (2.5 mg/ml) to the calcium and magnesium samples. The amount of the element in each tissue sample was determined by in-terpolation from the standard curve and all the results are expressed as nanogram atoms/mg dry weight. Results for plasma and red blood cells are given as mmol/1. 2.4.6 Mitochondrial calcium content Mitochondria were isolated as described above. To one ml of the isolated mitochondria (approximately 2-3 mg/ml), lanthanum chloride (0.2 ml, ImM) and 0.2 ml trichloroacetic acid (100.9$: glacial acetic acid (17 M) (1:1 v/v) was added. The volume was made up to 2.0 ml with de-ionized water and centrifuged at 5000 x g for 5 minutes. One ml of the supernatant was then analyzed for calcium as described pre-viously. 2.4.7 Hydroxyproline assay The method used to assay hydroxyproline was that described by Woessner (130). Tissue samples (50-100 mg) were sealed in small Pyrex test tubes and hydrolyzed with 2 ml HC1 (6N) for 3 hours at 130° C. The tubes were then opened and the contents decanted into a graduated cylinder. The tubes were thoroughly washed with water and the wash-ings combined with the hydrolyzate. Several drops of 0.025 methyl red indicator were added, followed by sufficient drops of 2.5 N NaOH to neutralize the acidic hyrolyzate. After neutralization, the volume of hydrolyzate was made up to 10 ml with water. To determine hydroxypro-line in the hydrolyzate, 1 ml of chloramine T (0.05 M in water) was added to 2 ml of the hydrolyzate. The tube contents were mixed and 55 allowed to stand at room temperature for 20 minutes. This was follow-ed by the addition of 1 ml perchloric acid (3.15 M) and the mixture allowed to stand for a further 5 minutes at room temperature. Final-ly, 1 ml of p-dimethylaminobenzoate (20%solution in methyl cello-solve) was added, the tubes were incubated at 60° C for 20 minutes and the absorbance was read at 557 nm. To prepare the standard curve, a stock solution of 25 mg hydroxyproline was dissolved in 250 ml 0.001 N HC1 and the stock diluted to obtain concentrations of 1, 2, 3, 4, 5, 10, 25 and 50 ng/ml and processed as described above. Results are expressed as pg/mg. 2.4.8 Glycosylated hemoglobin assay Glycosylation of hemoglobin was determined by the method of Subramanian et al (131). Approximately 2 ml of blood was collected in heparinized tubes. The red blood cells were separated from serum by centrifugation (2000 x g for 10 minutes) and washed 3 times with sal-ine (0.85%) at 4° C. The packed red blood cells were then lysed with 2 ml water and 0.5 ml carbon tetrachloride was added. The suspension was centrifuged at 3000 g x 10 minutes at 4° C and the pellet discard-ed. To 1 ml of clear hemolysate was added acidified acetone (1 ml 12 N HC1 and 99 ml acetone) to separate heme and iron from globulin. The suspension was centrifuged at 5000 x g for 10 minutes and the supernatant discarded. The pellet (globin) was washed once with acidified acetone and 4 times with cold acetone. The final pellet was suspended in diethyl ether and dried under nitrogen. A 75 mg sample of the globin was digested with 4.0 ml of 10 M acetic acid for 16 56 hours at 105° C. Following incubation, 2 ml of 10%trichloroacetic acid was added and the mixture allowed to sit for 5 minutes. To meas-ure the formation of hydroxymethylfurfural (HMF), 2 ml of thiobarbi-turic acid (0.35 g in 100 ml 0.25 M sodium sulfate) was added. The reaction mixture was incubated for 50 minutes at 40'C and the absorb-ance read at 443 nm. Standard solutions of varying concentrations of HMF (20 to 150 viM) were prepared in 2 M acetic acid and also read at 443 nm. Results are expressed as ymol of hydroxymethylfurfural per g of globin. 2.4.9 Tissue glycogen Glycogen was measured according to the method of Seifter and Dayton (132). Immediately after cardiac excision, approximately 50 mg of tissue from the ischemic zone was dropped into a tube containing 30% KOH. After digesting the tissue for 20 minutes in a boiling water bath and cooling, 1.25 ml of 95% ethanol was added and the contents mixed. The contents of the tubes were gently brought to a boil in a water bath and centrifuged for 15 minutes at 3000 rpm. The superna-tant was decanted and the precipitate was redissolved in 1 ml water and reacted with 2 ml 0.2%anthrone reagent (0.2 g anthrone in 100 ml of 95% sulfuric acid). The mixture was allowed to stand for 30-45 minutes at room temperature and the optical density read at 565 nm. The amount of glycogen was calculated using the following equation =: v9 of glycogen x 100 x A 1.211 x s where A is the optical density of unknown test solution, s is the op-57 ti c a l density of 100 pg glucose standard solution, and 1.211 is the multiplication factor for the conversion of glucose to glycogen. 2.4.10 Tissue l i p i d extraction One g of heart tissue was weighed and homogenized as a 10%suspen-sion (w/v) in a chloroform: methanol (2:1 v/v) mixture at one quarter maximal speed for 5 seconds using a Polytron homogenizer. The result-ing mixture was kept at 4° C for 30 minutes and centrifuged at 5000 rpm for 15 minutes and the extract stored in a glass v i a l . The pellet was again re-extracted with the chloroform-methanol mixture and the procedure repeated as above. The volume of the combined extracts was recorded and aliquots were removed to determine cholesterol, phospho-l i p i d , triglycerides and free fatty acids. 2.4.11 Serum triglycerides Triglycerides were according to the method of Kaplan and Lee (133). To 0.5 ml of undiluted serum sample was added 2.0 ml heptane, 3.5 ml isopropanol and 1 ml 0.08 N ^SO^. The mixture was vortexed and left standing at room temperature for 5 minutes. 0.2 ml of the upper heptane layer was transferred to a tube and 2 ml of isopropanol and one drop of 6.25 M KOH was added. The tubes were warmed up to 70° C for 10 minutes and after cooling, 0.2 ml of periodate reagent (0.6 g sodium metaperiodate in 5 ml glacial acetic acid and 95 ml water) and 1 ml of acetylacetone reagent (1.5 ml of acetylacetone diluted to 200 ml with 200 ml of 2 M ammonium acetate). The samples were again warm-ed up to 70° C for 10 minutes and the absorbance read at 425 nm. To obtain a standard curve, the following amounts of stock stand-58 ard (1 g triolein in 100 ml isopropanol) were diluted to 50 ml with isopropanol: 2.5, 5.0, 10, 15 and 17.5 ml. The concentrations of these standards were 500, 1000, 2000, 3000 and 3500 yg/ml, respecti-vely. These standards were analysed in the same manner as the test samples. 2.4.12 Cholesterol The method used for cholesterol analysis was based on the proce-dure of Levine and Zak (134). A stock reagent was prepared by dissol-ving 2.5 g FeCl^ in 50 ml glacial acetic acid. This stock reagent was stored at -20° C. A working Zak solution was prepared by diluting the stock iron solution 1:100 with concentrated sulfuric acid. Samples of serum (50 ul) were mixed with 2.0 ml glacial acetic acid and let stand at room temperature for 30-45 minutes. Then 1.3 ml of the dilute iron reagent was carefully added so as to underlay the glacial acetic acid layer. The layers were then mixed thoroughly and the color was allowed to develop for 30 minutes and the absorbance read at 565 nm. Sample absorbances were compared to a standard absor-bance where a known quantity of cholesterol (50 yg) was assayed. 2.4.13 Free fatty acids Free fatty acids were measured according to the method of Kelly (135). To 50 yl of serum in a centrifuge tube was added 1 ml phos-phate buffer (33 mmol/1, pH 6.4) and 6 ml extraction solvent (CHC1^ ^H^CH^OH, 5:5:1). The tubes were vortexed for 90 seconds, kept at room temperature for 15 minutes and centrifuged at 2000 rpm x 10 minutes. The buffer was carefully removed by suction and 5 ml of the extract was removed. To the extract was added 2 ml copper reagent 59 (10 ml of 500 mmol/1 Cu(N03)2, 6 ml of 1 mmol/1 NaOH and 33 g NaCl made to 100 ml with water) and shaken vigorously for 50 seconds. The tubes were centrifuged at 4000 rpm x 5 minutes and 3 ml of the upper layer was transferred to a tube containing 0.5 ml phenylcarbazide sol-ution (4 g/1 methanol) and mixed. The optical density was measured after 15 minutes at 550 nm. To prepare the standard curve, 5 ml of stock standard (palmitic acid, 2 mmol/1) was diluted to 20 ml with extraction solvent to give a solution containing 500 ymoles/1. For a standard curve, 2, 4, 6 and 8 ml stock standards were diluted to 20 ml with the extraction solvent. These are equal to 200, 400, 600, 800 and 1000 ymoles/1, respectively. 2.4.14 Phospholipid analysis Phospholipid content was determined by assaying for phospholipid phosphorous as described by Bartlett (136), except that the samples were digested with perchloric acid instead of sulfuric acid. Stock solution A (Fiske-Subbarow reagent) was prepared by adding 0.25 g 1-amino-2-napthol-4-sulfonic acid (ANS) to 100 ml freshly prepared 15_%(w/v) sodium bisulfite and then adding 0.5 g anhydrous sodium sul-f i t e . The solution was filtered and then stored in a dark bottle for a week. Stock solution B consisted of a 5%(w/v) ammonium molybdate solution in water. To a 20-50 yl serum sample, 1.5 ml of 70%per-chloric acid was added and the mixture digested at 230° C in a sand bath for 30 minutes. After the samples had cooled, 7.6 ml d i s t i l l e d water was added and the solution mixed. A 4.5 ml aliquot was taken and color was developed by adding 0.5 ml d i s t i l l e d water, 0.2 ml of 60 stock solution B, 0.2 ml of stock solution A, mixing and placing the mixture in a boiling water bath for 7 minutes. After cooling, the absorbance of the sample was determined at 830 nm. Sample absorbance was compared to absorbance that was obtained from a standard solution containing a known quantity of inorganic phosphate (20 pg). To con-vert pgrams phosphorous to pmoles phospholipid the conversion factor of 25 and an average molecular weight of 700 were used. 2.4.15 Serum glucose determination Glucose content of the serum was determined by an enzymatic method using a glucose-oxidase kit obtained from Sigma. The assay involves an enzymatic reaction in which glucose is converted to gluconolactone in the presence of ^ © 2 . Hydrogen peroxide, in the presence of the enzyme peroxidase, oxidises the colorless reagent 0-dianisidine to a reddish-brown dye and the color developed is read at 450 nm using a spectrophotometer. A 100 mg/100 ml glucose standard solution was used as a reference stock solution. Serum glucose levels were expressed as mg/dl. 2.4.16 Serum T^  determination The T^ content of the serum was determined by a radioimmunoassay D method using the Tetrabead -125 T^ diagnostic kit (Amersham). The assay involves competition between the radioactive and non-radioactive T^ in the standard or serum sample for the binding sites present in a limited quantity of^ the T^ antibody immobilized in beads. Separa-tion of the bound and free radioactive T^ was achieved by centrifu-gation (3600 rpm x 15 minutes) of the reaction liquid from the beads. 61 The radioactivity bound to the beads was estimated using a gamma coun-ter. Serum T^ content was expressed as yg/dl. 2.4.17 Serum "^determination Serum T^ content was determined by a charcoal bead uptake method R using the Triobead -125 T^ uptake kit (Amersham). The assay in-volves the partition of the radiolabel led T^ between the primary binding sites (serum proteins such as albumin, globulin, etc.) in the serum and secondary binding sites such as the charcoal-coated beads. The radioactivity taken up by the beads gives an indication of the thyroid status. In hypothyroidism, the primary binding sites are less saturated and they take up more radioactivity and the beads less. Conversely in hyperthyroidism, the primary binding sites are supersat-urated and so the beads take up more radioactivity. The radioactive T^ bound to the charcoal-coated beads was separated by centrifuga-tion (3600 rpm x 15 minutes) the reaction liquid from the beads and was counted in a gamma counter. The charcoal bead uptake was express-es ed as a percent of the T^ uptake value for the reference control serum provided with the k i t . 2.4.18 Serum insulin Insulin content of the serum was measured by a radioimmunoassay using the Becton Dickinson insulin radioimmunoassay k i t . The assay 125 involved a competitive-binding reaction between I-labeled insulin (porcine insulin) and the non-radioactive insulin in the standard (human insulin) or serum sample (rabbit insulin). Bound and unbound radioactive insulin were separated using dextran-coated charcoal. 62 Dextran-coated charcoal is known to readily adsorb free insulin but not the antibody-bound insulin. After incubation, a suspension of dextran-coated charcoal was added to the incubation mixture, vortexed, centrifuged and the antibody-bound radioactivity in the supernatant was measured using a gamma counter. Serum insulin is expressed as p Units/ml. 2.4.19 Water content Transmural tissue samples were blotted lightly with f i l t e r paper and weighed in porcelain crucibles. The tissues were dried at 110° C overnight and the dry weight was determined by subtraction. Water content is expressed as a percent of left ventricular wet weight. 2.4.20 Protein assay Protein concentration was measured by the method of Lowry et al (137) using bovine serum albumin as a standard. 2.5 Ultrastructural analysis 2.5.1 Preparation of samples for electron microscopy The tissues were fixed by immersion in 2%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and immediately cut into 1 mm cubes to fa-c i l i t a t e penetration of the fixative into the tissue. The cubes were fixed for 4 hours at 4° C followed by two rinses in 0.1 M cacodylate buffer (pH 7.4). The cubes were then postfixed in 1% osmium tetroxide for 60 minutes followed by two washes in d i s t i l l e d water. This was followed by dehydration for 10 minutes (twice) in a series of methanol concentrations (25, 50, 70, 95 and 100%). Following propylene oxide dehydration (twice for 20 minutes each), the cubes were embedded with EP0N 812. The blocks were incubated in plastic vials for 24 63 hours at 60° C and were then ready for sectioning. Prior to section-ing, the block surface was trimmed into a 0.5 mm square. All section-ing was done using glass knives on an ultramicrotome (model MT-1). The sections were stained with uranyl acetate (30 minutes) and lead citrate (5 minutes). To remove a l l areas which might show evidence of mechanical damage, the periphery of each block was trimmed away and sections cut only from the middle of the block. Tissues for electron microscopy were obtained from a minimum of 3 or 4 animals in each group and 7 blocks were prepared and blindly analyzed. In addition, 10 electron micrographs were obtained from each section. In instances where severe and normal ultrastructure alterations were both observed in samples from one segment, the data were excluded. Therefore, con-clusions regarding cellular alterations were based only on sections where uniform changes were present. Various features of the micro-graphs were each scored according to a pre-determined scoring system in which 0 = normal, 1 = mild abnormality, 2 = moderate abnormality and 3 = severe abnormality. 2.6 Microsphere Study to Determine Regional Blood Flow 2.6.1 Blood flow studies In order to measure the extent of blood flow reduction after cor-onary artery ligation and, more importantly, the level of reperfusion when the ligature is released, radioactively-labeled microspheres were injected before and after ligation/reperfusion. Two isotopes were used, "^Sn and ^Co. In order for each animal to serve as i t s own control, the study was divided into two sections. The f i r s t study 64 involved the reduction of blood flow as a result of ligation and the second measured the extent of reperfusion after 40 minutes of ligation. 2.6.2 Microsphere preparation ^Co and ^ S n - l a b e l l e d microspheres (15 ± 3y) were obtained from New England Nuclear and suspended in physiological saline. They were vortexed for 20 minutes before adding them to a 10% F i c o l l solu-tion (with the addition of 0.05%chlorobutanol as a preservative) con-taining a drop of Tween 80% to prevent aggregation of the microspheres'. 113 Immediately before use, the Sn microspheres were diluted again with the same vehicle so that each injection contained approximately 57 126,000 microspheres. Because of the higher activity, the Co-labeled microspheres were diluted to a greater extent resulting in a total of 25,000 microspheres per injection. When the hemodynamic variables had stabilized after surgery, 113 Sn-labelled microspheres, prepared as described above, were in-jected directly into the left ventricle of the animal. Immediately prior to injection, a reference blood sample from the femoral artery was withdrawn at a constant rate of 1.1 ml/minute with a Harvard pump and this was maintained for at least 2 minutes after completion of the microsphere injection. This procedure provided an estimate of control blood flow. After 3-5 minutes, the left circumflex coronary artery was occluded. Microsphere injection was done prior to ligation, at 5 minutes after occlusion and 60 minutes after reperfusion. After the hearts were removed, radioactivity in the occluded zone, the non-occluded left ventricle, right ventricle and kidneys was 65 counted on a Beckmann Gamma 8000 radiation counter. 3 STATISTICAL ANALYSIS When two samples were compared, stat i s t i c a l analysis was performed using the unpaired Student's t-test. For multiple comparison, one way analysis of variance, followed by the Newman-Keuls test was used. A probability of P< 0.05 was used as the level of significance. 4 MATERIALS Pentobarbitone sodium, osmium tetroxide and propylene oxide were obtained from BDH. Acids, solvents and standard ion solutions for atomic absorption were all reagent grade. Enzyme-grade sucrose (Ultrapure) was purchased from Schwarz-Mann. EM grade glutaraldehyde was obtained from Merck (Darmstadt). Radioactive insulin, T3 and T^ kits were purchased from Amersham and New England Nuclear. Alloxan, l-amino-2-napthol-4-sulfonic acid, streptozotocin and all the other necessary chemicals were obtained from Sigma Chemical Company. 66 5 RESULTS 5.1 Model of Alloxan-Induced Diabetes in Rabbit 5.1.1 General features Among the doses of alloxan studied, 100 mg/kg was found to be op-timal in consistently producing a diabetic state which was sustained with minimal mortality for the 10-12 week experimental period. Doses less than 100 mg/kg ( i . e . 50 or 70 mg/kg) did not produce a long-term diabetic state and doses higher than 100 mg/kg resulted in an in-creased rate of mortality. The onset of alloxan diabetes in the rab-bit is characterized by a triphasic pattern of changes of blood glu-cose levels (Figure 1). A few minutes after injection of alloxan, there is a transient hyperglycemia which lasts for 2-4 hours. From 4-16 hours after alloxan administration, a period of hypoglycemia is observed, which is particularly severe in the rabbit. After 16-24 hours, there is sustained hyperglycemia after 2-3 days. All animals injected with alloxan also showed a significant serum hyperlipidemia which in some rabbits disappeared after 6 weeks. The diabetic state in the rabbits was manifested by increased food and water uptake and polyuria associated with glycosuria throughout the 10-week period (Table 1). The availability, of some functional insulin in the diabet-ic rabbits eliminated the necessity for insulin therapy in these rab-bits for survival. 5.1.2 Body weight and mortality The mean body weight of control and diabetic animals is shown in Table 2. It was observed that there was a considerable fluctuation in 67 BLOOD G L U C O S E A F T E R A L L O X A N I N J E C T I O N FIGURE 1 Blood glucose levels following alloxan injection. Transient hypergly-cemia (2-4 hours) followed by a prolonged hypoglycemia (4-17 hours) followed by sustained hyperglycemia is observed. Values are expressed as mean ± SEM (n = 6 animals). 68 Table 1. General features of diabetic animals after 10 weeks Control Diabetic Food uptake (g/ 24 h) 150 ± 25 400 ± 50* Water uptake (ml/24 h) 150 ± 50 650 ± 200* Urine output (ml/24 h) 200 ± 50 700 ± 300* Urine glucose (mg/dl) 5640 ± 650 Ketosis^- mild (3/12 animals) P < 0.05. Significantly different from control. Values are expressed as mean ± SE (n = 6 animals). * Ketosis was only evident during the f i r s t 7-10 days following a l -loxan injection. 69 Table 2. Effect of 10 weeks of alloxan-induced diabetes on rabbit body and heart weight Control Diabetic Body weight (kg) Heart weight (g) Left ventricular weight (g) Heart weight g/kg 3.8 * 0.3 5.5 ± 0.3 3.9 ± 0.3 1.4 ± 0.2 Body weight Dry heart weight (mg/g) 227 * 18 Water content 77.6 ± 0.3 (%'left ventricular wet weight) 3.3 ± 0.5 3.8 ± 0.6* 2.4 ± 0.6* 1.1 ± 0.1 242 * 12 78.2 ± 0.7 P < 0.05. Significantly different from control. Values expressed as mean ± SE (n = 8 animals). 70 the body weight of individual diabetic rabbits but the mean body weight of diabetic rabbits was not significantly different from control. All diabetic animals which showed hyperglycemia without hyperl ipidemia did not show a decrease in body weight (60%' of the animals), whereas those diabetic animals which developed both hyper-glycemia and hyperlipidemia showed a significant decrease in body weight (35%rof the animals). The mortality rate of the diabetic animals not administered 10% glucose was 100% after injection with alloxan. All rabbits in-jected with alloxan died within 24 hours, mainly from severe hypogly-cemia, which occurs due to the massive insulin release from the pan-creas. To prevent the fatal hypoglycemi-a, a l l animals were given 10%glucose subcutaneously. In addition, those diabetic animals show-ing severe dehydration (as evidenced by a decrease in body weight) were treated with intravenous saline. This precautionary measure dro-pped the mortality rate to less than 10 . Despite the above precau-tionary measures 5-10^of the diabetic animals died within the f i r s t 10 days after alloxan administration. All of the rabbits that died had significantly elevated serum l i p i d s . Whenever hyperlipidemia was observed within the f i r s t 5 days after alloxan administration, insulin treatment was initiated (8 units/kg subcutaneously) for 2-3 days and this considerably improved survival. Ketosis, as evidenced by the excretion of acetone bodies in the urine, occured in approximately 25 .'of the animals and was usually f i r s t detected on the third day after alloxan injection, the maximum levels being reached towards the end of the f i r s t week. Some animals 71 died at this stage in acidosis and coma. In the rabbits which sur-vived this c r i t i c a l period, the excretion of acetone bodies gradually decreased and towards the end of the second week following alloxan administration, urinary ketone bodies were no longer detectable. 5.1.3 Left ventricular weight and water content The alloxan-treated rabbits had a decrease in both the heart and left ventricular weight as compared to control rabbits after 10 weeks (Table 2). More recent observations have indicated that there appear to be two subtypes of diabetic states produced by alloxan in the rab-b i t , i.e., one with only hyperglycemia and one with hyperglycemia and hyperlipidemia. All rabbits with hyperglycemia and hyperlipidemia showed not only significant decreases in body weight, but also de-creases in their heart and left ventricular weights. Since we were not aware of this phenomenon e a r l i e r , the present study has included data from both groups of animals. The heart weight to body weight ratio was not significantly altered in diabetic animals as compared to controls. No changes were observed in either the mean dry weight or total water content of hearts of diabetic animals com-pared to control animals. 5.1.4 Serum chemical properties The results of serum chemical analyses in control and diabetic rabbits are summarized in Table 3. Time-course studies revealed that a significant increase in blood glucose occurred 3 days after alloxan administration and these levels remained high for the duration of the 72 Table 3. Effects of 10 weeks of diabetes on serum chemical components Control animals Alloxaq-tr^ated Glucose (mg/dl) 79 14 467 ± 39* Insulin (yU/ml) 23.2 2.3 17.9 * 1.8* HbAlc (ymol HMF/g globin) 1.2 0.4 4.7 ± 0.9* Cholesterol (mg/dl) 79 11 176 ± 22* Phospholipid (mmol/1) 0.14 0.04 0.12 ± 0.04 NEFA (mmol/1) 1.1 0.2 1.7 ± 0.3 Triglycerides (mmol/1) 0.8 0.04 1.5 * 0.1* Results expressed as mean ± SEM (n = 8 animals). P < 0.05, Significantly different from control. 73 study. A few animals did start to show a decline in blood glucose 6-8 weeks after the administration of alloxan. To these rabbits, a smal-ler dose of alloxan (50 mg/kg intravenously) was administered, result-ing in an elevation of the blood glucose levels. Increases in serum cholesterol and triglycerides were observed 4 weeks after the injec-tion of alloxan. These levels remained high for the duration of the study, but showed considerable fluctuations. No change was observed in serum phospholipid and free fatty acids (NEFA) in the diabetic ani-mals. Hemoglobin glycosylation, an index of the severity and duration of hyperglycemia, was elevated 2-4 weeks after the induction of diabe-tes and remained elevated for the remainder of the study. In con-trast, there was a decrease in serum insulin after 8-10 weeks of dia-betes (Table 3). No change in either the circulating serum T^ or T^ levels were observed in the diabetic rabbits after 10-12 weeks. Both blood urea nitrogen and creatine phosphokinase activity were significantly ele-vated in the diabetic animals compared to controls (Table 4). Meas-urements of the serum electrolytes, Na, K, Mg and Ca, revealed no significant differences between control and diabetic animals. 5.1.5 Status of atherosclerosis Prior to analyzing the myocardium in control and diabetic animals, the extent of atherosclerosis in these animals was assessed. Aortae from the diabetic animals showed no difference in cholesterol or t r i -glyceride content in comparison to controls (Table 5). Sudanophi1ic-ity (as assessed with Sudan Black Dye) also revealed no significant fatty streaks in diabetic aortae as compared to controls. 74 Table 4. Effects of 10 weeks of diabetes on serum T^, T^, blood urea nitrogen, electrolytes and creatine phosphokinase activity Control animals Alloxan-treated animals T3 (ng/dl) 7.1 ± 0.3 6.4 ± 1.1 T4 (pg/dl) 4.0 * 0.2 3.6 ± 0.5 Blood urea nitrogen (mg/dl) 1.2 ± 0.3 2.7 * 0.9* Creatine phosphokinase (Sigma units) 17 ± 2.0 25 ± 3.8* Na (mmol/1) 144 * 2.50 139 * 3.90 K (mmol/1) 4.1 ± 0.2 4.7 * 0.6 Mg (mmol/1) 1.2 ± 0.3 1.4 ± 0.4 Ca (mmol/1) 2.6 * 0.2 2.2 ± 0.5 Results expressed as mean ± SEM (n = 8 animals). P < 0.05, Significantly different from control. 75 Table 5. Extent of li p i d accumulation in aortae from 10-week alloxan-diabetic rabbits Control animals Alloxan-treated animals Cholesterol (mg/g) 3.4 ± 0.1 3.6 * 0.4 Triglycerides (mg/g) 15.8 ± 0.9 16.7 ± 1.9 Sudanophilicityl Aortic Arch + + Thoracic aorta — Abdominal aorta — Values are expressed as mean ± SE (n = 6 animals). Sudanophi1icity was measured by staining the aortae with Sudan Black dye (2.5 mg/ml in ethanol). In the thoracic and abdominal aorta, no significant staining of the aorta was observed by Sudan Black in either control or diabetic animals. 76 5.1.6 Glucose tolerance test Diabetic animals subjected to an oral glucose tolerance test showed an abnormal glucose disposal test (Figures 2 and 3). In con-trol animals, there was a significant increase in blood glucose 5-15 minutes after oral administration of glucose. The peak glucose levels occurred between 15-30 minutes and thereafter declined, reaching nor-mal blood glucose levels within 2 hours. Diabetic animals showed rises in blood glucose levels at about 5-30 minutes after oral glucose administration but the levels failed to decline even 2 hours after the administration. Similarly, insulin levels in the control animals peaked with the rise in glucose and declined thereafter. In the dia-betic animals, a slight increase in serum insulin was observed during the glucose test, but the levels did not reach levels comparable to those observed in the control animals. 5.1.7 Hemodynamic measurements The effects of alloxan-induced diabetes on hemodynamic properties are shown in Table 6. After the development of steady-state pentobar-bital anaesthesia, hemodynamic studies revealed that left ventricular pressure, heart rate and the positive rate of rise of left ventricular developed pressure were significantly decreased in the diabetic ani-mals. No changes were observed in either the left ventricular end-diastolic pressure or systemic blood pressure in diabetic compared to control animals'. Electrocardiogram recordings from chest and limb electrodes did not reveal any significant cardiac rhythm abnormalities in diabetic animals (data not shown). 77 G L U C O S E T O L E R A N C E T E S T — G L U C O S E L E V E L S o-j 1 1 1 1 ; — i 1 o 20 40 60 80 100 120 T I M E CMINS> FIGURE 2 Blood glucose levels in oral glucose tolerance testing (1 g/kg, p.o.) of control { ) and 10-week diabetic ( ) animals. Blood samples were obtained every 15 minutes for 2 hours and analyzed for glucose. Values are expressed as mean ± SEM (n = 6 animals) P < 0.05. Significantly different from control. 78 ioH 1 1 1 1 1 1 D ZD 4 0 GO S O l O O 1 2 0 T I M E <MINS> FIGURE 3 Serum insulin levels in oral glucose tolerance testing (1 g/kg, p.o.) of control ( ) and 10-week diabetic ( ) animals. Blood samples were obtained every 15 minutes for 2 hours and analyzed for serum insulin. Values are expressed as mean ± SEM (n = 6 animals). *P < 0.05. Significantly different from control. 79 Table 6. Effects of 10 weeks of diabetes on various cardiovascular parameters Control animals Alloxan-treated animals Left ventricular pressure (mmHg) 125 ± 10 100 ± 5* Positive rate of left ventricular pressure development (mmHg/s) 4000 ± 360 3200 ± 220* Blood pressure (mmHg) Systolic: Diastolic: 120 * 10 85 ± 5 115 ± 10 85 * 5 Heart rate (beats/min) 300 ± 10 260 ± 10* Left ventricular end diastolic pressure (mmHg) 5 ± 2 8 ± 4 Results expressed as mean ± SEM (n = 8 animals) P < 0.05. Significantly different from control. 80 5.1.8 Myocardial composition Analyses of left ventricles from control and diabetic animals are shown in Table 7. After 10 weeks of diabetes, tissue levels of Na and K in the left ventricles of diabetic- animals showed no differences relative to control. However, a significant reduction in tissue Mg and a slight but insignificant accumulation of Ca was observed in the left ventricles of diabetic animals. Further analyses of left ventri-cles from diabetic animals revealed significant increases in choles-t e r o l , triglyceride, hydroxyproline and glycogen with no change in phospholipid levels. Of the various subcellular organelle ATPases studied in the left ventricles from diabetic animals, both myofibril-lar and sarcoplasmic reticulum K ,Ca -stimulated (EGTA-inhibit-able) ATPases showed significant reductions (Figure 4). Mitochondrial azide-sensitive ATPase, sarcolemmal Na+,K+-stimulated (ouabain-sensitive) ATPase and K+-stimulated p-nitrophenylphosphatase showed smaller but non-significant decreases in the left ventricles of diabe-tic animals compared to controls. 5.1.9 Magnesium metabolism Measurement of magnesium in the red cells of diabetic animals revealed a significant decrease in comparison to control red c e l l s . Time-course studies revealed that significant magnesium depletion in red cells from diabetic animals occurred 6-8 weeks after the onset of diabetes and the depletion appeared to increase with the duration of diabetes (Figure 5 and 6). Measurements of urinary magnesium levels revealed a significant increase in excretion 81 Table 7 The effect of 10 weeks of diabetes on left ventricular content of ionsl and biochemical substances Control animals Diabetic animals Na 29.2 ± 11.1 37.6 ± 6.6 K 292 ± 13 268 ± 21 Mg 28.7 ± 2.3 22.6 ± 1.7* Ca 5.3 ± 0.9 6.7 ± 0.6 Cholesterol (mg/g) 3.1 ± 0.8 5.9 ± 0.6* Triglyceride (mg/g) 12.7 ± 1.9 36.8 ± 3.9* Hydroxyproline (yg/mg dry weight) 3.2 ± 0.4 5.0 0.3* Glycogen (mg/g wet weight) 5.2 ± 0.8 8.7 0.9* Phospholipid (mg/g wet weight) 6.6 ± 1 .1 6.9 ± 0.9 Results expressed as mean ± SE (n = 8 animals). P < 0.05. Significantly different from control. '''Ion values are expressed as ng atoms/mg dry weight. 82 Q C O N T R O L [0 D IABETIC o 250 w a 01 ^ 200 o 150 E 3. 100 o < 50|-III 10 2 -P<0.05 I 1.0 0.8 -0.6 0 .4 0.2 -20r P<0.05 16 i < A Z I D E - S E N S I T I V E N a * , K * - A T P a s e K*. C a " - A T P a s e K * - N P P a s e M g " - A T P a s e ( m i t o c h o n d r i a ) ( s a r c o l e m m a ) ( s a r c o p l a s m i c ( s a r c o l e m m a ) (myof ib r i l l a r ) re t i cu lum) FIGURE 4 Mitochondrial, sarcolemmal, sarcoplasmic reticulum and myofibrillar ATPase activities in the left ventricles of control and diabetic rabbits. Results expressed as mean ± SEM (n = 8 animals). -83-WEEKS FIGURE 5 Red cell magnesium content at various times in control and diabetic animals . Values are expressed as mean ± SEM (n = 6 animals) *P < 0.05. Significantly different from control. 84 FIGURE 6 Relationship between increasing blood glucose concentration and plasma or red blood cell magnesium concentrations. Each point represents the mean of 2-3 animals. 85 Table 8 Plasma and urinary magnesium concentration in control and 10-week alloxan-diabetic rabbit Control animals Alloxan-treated animals Plasma Mg (mM/1) 1.3 ± 0.5 1.4 0.09 Red cell Mg (mM/1) 4.2 ± 0.2 2.4 ± 0.7* Urine volume (ml/day) 126 17 471 54* Magnesium (excretion) in urine (yg/ml) 466.7 ± 14 160.7 ± 29* Total urinary magnesium excretion (mg/day) 58.8 5.9 75.7 ± 7.1* P < 0.05. Significantly different from control. Values are expressed as mean ± SE (n = 6 animals). No changes were observed in the plasma and urine levels of Na and K in control versus diabetic animals. 86 Table 9 Magnesium content in tissues of 10-week alloxan-induced diabetic animals Control animals Alloxan-treated animals Liver 16.6 ± 1.3 17.5 ± 0.4 Kidney 24.5 * 2.2 20.8 ± 1.1 Spleen 23.0 ± 3.6 10.8 ± 4.6* Aorta 7.8 ± 1.2 5.3 * 1.2* Right ventricle 14.9 * 3.3 17.2 ± 1.3 Left ventricle 21.7 ± 2.2 15.2 ± 2.2* Lung 17.7 ± 1.3 14.1 ± 0.4* Smooth muscle 11.0 * 2.4 15.0 * 3.5 Skeletal muscle 16.4 ± 2.5 15.0 ± 4.7 Right atrium 13.0 * 1.7 15.8 3.9 Left atrium 17.9 * 1.4 17.4 * 3.8 Pancreas 11.8 ± 2.1 6.9 * 1.7* * P < 0.05. Significantly different from control. Values are ex-pressed as mean ± SE (n = 6 animals). Magnesium concentration expressed as ng atoms/mg dry weight. 87 of this electrolyte after 6-10 weeks of diabetes (Table 8). Tissue magnesium measurements after 10 weeks of diabetes revealed significant decreases in the left ventricle, aorta, spleen, lung and pancreas. No changes were observed in the magnesium .content in the smooth and skeletal muscle, right ventricle, l i v e r , left and right atrium (Table 9). It should be noted that only rabbits showing blood glucose levels above 400mg/dl (9 out of 12 rabbits) showed significant depletion of tissue magnesium content. Rabbits with blood glucose levels less than 300 mg/dl did show some decreases in the red cell magnesium content, but the levels were not significantly different from control animals. 5.1.10 Lysosomal enzymes The effects of diabetes on left ventricular lysosomal enzymes are shown in Figure 7. Analyses of myocardial homogenates from diabetic and control animals revealed a small, but significant increase in the non-sedimentable fraction of N-acetyl-e-glucosaminidase activity. In contrast, acid phosphatase, a less specific marker for myocardial ly-sosomal structures than N-acetyl-e-glucosaminidase, did. not show any significant changes in sedimentation characteristics following alloxan treatment. Cathepsin D, another specific lysosomal enzyme, did not show any significant difference in activity compared with control. 5.1.11 Myocardial ultrastructural alterations Control. Myocardial tissue from control animals showed normal ultrastructural features of myocardial cells (Figure 8). Myocardial cells had parallel arrays of myofibrils which were in register 88 r— 100-i > i— 90-O < 80-1X1 70-_ i CD < 60-f-Z 50-LU 40-D 30-LU CO 20-NO 10-z 0-C D N - A C E T Y L - 3 - P H O S P H A T A S E G L U C O S A M I N I D A S E ACID C D C A T H E P S I N D. FIGURE 7 Release of the lysosomal enzymes, N-acetyl e-glucosaminidase, cathe-psin-D and acid phosphatase, in the left ventricles of control ( C ) and diabetic ( D ) rabbits. Lysosomal enzyme activity is expressed as percent non-sedimentable activity relative to the total (sedimentable + non-sedimentable) activity. Results are expressed as mean ± SEM (n = 8 animals). *P < 0.05. Significantly different from control. 89 with the mitochondria. The mitochondrial membranes were intact and contained tightly packed cristae. The ultrastructure of left ventri-cular myocardium observed in control animals conformed with previous descriptions of normal rabbit myocardium (115). Diabetic. Using electron microscopy to evaluate the ultrastruc-ture of hearts from 4 and 10-week diabetic rabbits, i t was observed that abnormalities ranging from mild to severe, were present in 9 of the 12 animals. Prior to the 4-week period, no significant altera-tions in the diabetic rabbit myocardium were apparent (Figure 9) At the end of 10 weeks, considerable structural alterations in the diabetic hearts were quite evident (Figure 10). Most myofibrils in diabetic left ventricles were intact. However, some myofibrils did appear damaged and showed tearing of the Z-bands and disruption of the intercalated disk. An irregular appearance of Z-bands along with varying degrees of contraction of myofibrils were also observed. Of a l l the subcellular organelles studied, the ultrastructural alterations of diabetic mito-chondria were the most conspicuous (Figure 10). Both normal and dam-aged mitochondria were seen, with the former being more prominent. The distribution of damaged mitochondria was generalized throughout the myocardium. The damaged mitochondria contained large amorphous electron-dense bodies, some mitochondria showed a decrease in the num-ber of cristae and some contained a less-electron dense matrix. Spread throughout the left ventricle were some severely damaged mitochondria exhibiting disruption of the cristae network, and loss of 90 FIGURE 8 Electron micrograph showing ultrastructure of control rabbit myocar-dium. Note the well preserved features of myocyte ultrastructure. Myofibrils (My) form a regular array and glycogen (g) f i l l s a ll avail-able space . Mitochondria (M) are moderately dense and cristae appear intact. Nuclear chromatin (N) is evenly distributed. Glutaraldehyde-osmiun fixation. X 16,000. 91 FIGURE 9 Ultrastructure of 4-week diabetic myocardium. No significant visible alterations are present. Some mitochondria (M) have their cristae network broken. Myofibrils (My) appear intact with normal Z-lines. Nuclear chromatin (N) is evenly distributed. Glycogen (arrows) is also present in and around mitochondria. Glutaraldehyde-osmium fixa-tion. X 12,500. 92 FIGURE 10 This section shows ultrastructure of 10-week diabetic rabbit myocar-dium. Mitochondria show pallor, dilatation and disruption of their cristae network. Myofibrils (My) appear damaged and do not show characteristic Z-lines. Lipid droplets and dilatation of sarcoplasmic reticulum (arrow) are also evident. X 16,000. 93 FIGURE 11 Swollen mitochondria (M) with disruption of cristae network in 10-week old diabetic rabbit myocardium are apparent. Few electron-dense bod-ies are also visible in the mitochondria (arrows). Myofibrils (My) appear intact and glycogen deposits seen surrounding the mitochondria. Dilatation of sarcoplasmic reticulum (SR) is most evident. X 18,000. 94 structural integrity (Figure 11). In addition, granules of glycogen between myofilaments and beneath the sarcolemma were evident. The glycogen granules were also preval-ent, both around and inside the nucleus and outside the mitochondria. One other prominent finding in the ultrastructure of diabetic rabbit ventricles was the presence of l i p i d droplets. These droplets varied in size and were distributed unevenly throughout the ventricle. The normally fl a t and rough endoplasmic reticulum cisternae were dilated and contained varying degrees of electron-dense material. Objective ultrastructural assessment of diabetic hearts in comparison to controls is shown in Table 21. 5.2 ISO-Induced Myocardial Ischemic Injury 5.2.1 General features All animals exhibited ventricular tachycardia and became prostrate within 5-15 minutes of each ISO injection and some animals also devel-oped deep abdominal breathing. The deep abdominal breathing persisted for about 45-90 minutes. The ventricular tachycardias subsided within 45 minutes of ISO-injection. Despite evidence from other workers (138), our animals could only tolerate a maximum dose of only 0.5 mg/kg, s.c. on the f i r s t day. When doses greater than 0.5 mg/kg s.c. were administered on the f i r s t day, severe ventricular arrhythmias occurred within 10 minutes, culminating consistently in ventricular f i b r i l l a t i o n . The mortality from ventricular f i b r i l l a t i o n was 100%, with death occurring within 5-10 minutes after ISO-administration. 95 Because rabbits are prone to developing fatal ventricular arrhythmias, it was necessary to administer ISO in 4-divided doses over a period of 12 hours. This precautionary, measure s t i l l did not invariably abolish mortality, the aeath rate being about 7-10% during the f i r s t 2 days and less than 5%for the remainder of the study period. 5.2.2 Electrocardiograms The normal rabbit electrocardiogram showed P, Q, R, S and T-waves as in man. All 3 standard limb leads and the precordial lead were used to assess ischemic changes. In control animals, Q-waves when they could be distinguished, were always small. No ST-segment elevation or deflection between QRS and T-wave was observed in the control animals. Precordial leads showed upright P-waves followed by ventricular complexes with upright T-waves. There was l i t t l e change in the ECG as the chest lead position was moved across the pericar-dium, the main deflection being the R-wave., which was closely followed by T, with a shallow trough of S-T segment between. The heart rate of the control animals was about 300 ± 10 beats/min and abnormal rhythms were not seen. Electrocardiograms in the unanaesthetized control animals were obtained soon after the injection of saline and every 3 days until the end of the treatment schedule. Serial electrocardiograms in the ISO-treated animals showed a num-ber of distinct changes as compared to controls. Occasionally, nega-tive T-waves were seen in the ECG recordings 2-10 hours after ISO treatment or on days 4-7. 96 Table 10 Heart rate, P-R interval, QRS complex, QT interval and ST-segments in control and 15-day isoproterenol (ISO)-treated animals Control animals ISO-treated animals Heart rate (beats/min) 300 ± 10 350 ± 10* P-R interval (msec) 37 ± 4.0 32 ± 6.0 QRS complex (msec) 16 ± 3.0 18 ± 2.0 QT interval (msec) 137 * 3.0 143 ± 4.0 ST-segment elevation^ (mm) 2 ± 1 6 * 2* * P < 0.05. Significantly different from control. Values are expressed as mean ± SE (n = 6 animals). ^"ST-segment elevation was observed in leads I, II, III, aVr and epicardial lead V,-. Q-waves were seen in 33%(2 out of 6 animals). 97 All of the treated animals which died within 10 minutes after ISO-administration showed severe irreversible ventricular f i b r i l l a t i o n . The incidence of ventricular f i b r i l l a t i o n was observ-ed for up to 4-5 days, but after this period, the occurrence of ventricular f i b r i l l a t i o n decreased despite the increase in dose of ISO. The ECG evidence of myocardial ischemic injury in our animals was based on the appearance of Q-waves, reduction in amplitude of the R-wave and ventricular arrhythmias. We found that the presence of the Q-wave in both the standard and precordial leads was the most characteristic ECG abnormality in ISO-induced myocardial damage. However, the appearance of the Q-wave was not found to be consistent and was present in only 33%of the ISO-treated animals. Peak Q-wave amplitude was seen 8-9 days following the initiation of ISO treatment. In addition, ISO treatment revealed significant ST-segment elevations in leads, I, III, aVr and precordial lead V^. The amplitude of ST-segment elevation is shown in Table 10. In 3 animals, T-inversion was complete, negative T-waves following R with l i t t l e to indicate an S-T segment between them. Heart rate was significantly increased in a l l the ISO-treated ani-mals. Maximum heart rate increases were seen soon after ISO admini-stration but declined with time, yet were higher than those of control animals. Accurate measurements of P-R and Q-T intervals were obtained by running the ECG tracings at a maximum speed of 100 mm/second. When they coulo be measured, the ranges for P-R, QRS complex and Q-T inter-val in both control and ISO-treated animals were similar (Table 10). 98 5.2.3 Body weight and serum lipids Despite fluctuations in the body weight of individual rabbits, there was no significant difference in the mean body weight of rabbits during or at termination of the ISO treatment (Table 11). However, a l l ISO-treated rabbits showed significant increases in heart and left ventricular weight. The most pronounced increases in the heart and left ventricular weights were observed after 12-15 days of ISO-treat-ment. All ISO-treated animals also showed a significant increase in total left ventricular water content 15 days after ISO injection. This increase in water content was only evident in tissue sections obtained from the apex. Tissue sections obtained from other regions of the heart, such as the septum, showed an increase in the mean dry weight and a lower water content, perhaps indicating hypertrophy of the heart. Unlike the increases in heart weight, the most pronounced increases in water content were observed at around day 12. Table 12 summarizes serum abnormalities during ISO-induced myocar-dial ischemic injury. Total cholesterol, free fatty acids and glucose were all significantly elevated in the ISO-treated animals by day 3 and they remained elevated for the remainder of the study. In con-trast, a significant decrease in serum triglycerides was observed in the ISO-treated animals. An unexpected result was that ISO-treated animals also had lower serum T^ levels compared to controls after 15 days. Total serum creatine phosphokinase activity, an indicator of 99 Table 11 Body, heart and left ventricular weights after 5 and 15 days following isoproterenol (ISO) treatment Control animals ISO-5 days ISO-15 days Body weight (kg) 2.8 * 0.4 2.9 * 0.3 2.9 * 0.4 Heart Weight (g) 5.2 ± 0.2 5.8 ± 0.6 6.9 ± 0.3* Left ventricular weight (g) 3.3 ± 0.3 3.6 ± 0.3 4.8 * 0.6* Heart weight n/kn Body weight g K g 1.9 ± 0.3 1.9 ± 0.3 2.4 * 0.2* Water content ( % l e f t ventricular wet weight) 77.5 ± 0.3 77.8 ± 0.2 81.1 * 0.4* * P < 0.05. Significantly different from control. Values expressed as mean ± SE (n = 8 animals). 100 Table 12 Changes in blood components 15 days after isoproterenol (ISO) treatment Control animals ISO-treated animals Blood glucose (mg/dl) 98 ± 13 178 ± 16* Triglycerides (mg/dl) 120 ± 16 76 ± 9* Free fatty acids (mg/dl) 33 ± 9 69 ± 11* Total cholesterol (mg/dl) 94 ± 7 168 ± 17* Creatine phosphokinase^ (Sigma units) 32 ± 11 114 ± 24* T4 (yg/100 ml) 4.0 ± 0. .2 3.3 ± 0.3 Values expressed as mean ± SE (n = 8 animals). P < 0.05. Significantly different from control. ''"Peak creatine phosphokinase activity was seen after 5 days of treatment with isoproterenol (812 ± 79 S.I. units). 101 myocardial ischemic injury, was significantly elevated following ISO administration. Peak creatine phosphokinase levels were observed 5-7 days after the initiation of ISO treatment. These elevated levels, indicating severe myocardial damage, persisted until day 10 and then progressively decreased. 5.2.4 Myocardial chemical and biochemical alterations Analyses of left ventricles from control and ISO-treated animals are shown in Tables 13 and 14. Ventricles obtained from rabbits pre-treated with the above regimen were significantly enlarged and exhi-bited pale apical regions upon visual examination. The hearts appear-ed slightly hemorrhagic with diffuse nodular spreads. Most of these visual observations on the hearts of ISO-treated animals were conspi-cuous at about days 12-15. Rabbits which died from ventricular f i b r i l l a t i o n did not show any of the above gross abnormalities. Of the various subcellular organelle ATPase activities chosen as markers for myocardial ischemic injury on the basis of earlier studies involv-ing coronary artery ligation (115), we observed significant decreases by day 15 (but not day 5) in mitochondrial (azide-sensitive) ATPase, sarcolemmal (ouabain-sensitive) ATPase, K+-stimulated-p-nitrophenyl-phosphatase as well as K ,Ca -stimulated sarcoplasmic reticulum (ouabain-insensitive, EGTA inhibitable) ATPase activities in ISO-treated animals (Table 13). Measurement of myocardial tissue ions indicated a significant accumulation of Na and Ca in the left ventricles of ISO-treated animals. Further analysis revealed Ca accumulation by the 102 Table 13 Effects of 5 and 15 day isoproterenol (ISO) treatment on subcellular organelle ATPase a c t i v i t i e s ! Control IS0-5 days IS0-15 days Mitochondrial ATPase (azide-sensitive) 220 10 205 ± 12 148 11* Na+,K+-ATPase (ouabai n-sensitive) 6.7 ± 0.4 7.1 0.2 3.8 ± 0.8* P-nitrophenyphos-phatase (ouabain-sensitive) 1.2 0.1 0.9 ± 0.2 0.6 0.2* K+-Ca2+-sarco-plasmic reticulum ATPase 2.9 0.2 2.5 ± 0.2 2.0 0.3* *P < 0.05. Significantly different from control. Values expressed as mean ± SE (n = 8 animals). "'•ATPase activities expressed as pmol Pi/mg protein/h. 103 Table 14 Changes in intracellular ions and biochemical substances 15 days after isoproterenol (ISO) treatment Control ISO-treated Mg 25.2 * 1.2 23.0 * 1.1 Ca 3.7 ± 0.5 6.9 ± 0.6* Na 85.4 ± 4.9 112.9 ± 11.1* K 294.7 * 11.7 288.9 * 15.4 Mitochondrial Ca (nmol/mg protein) 5.4 ± 1.4 9.3 ± 1.5* ATP (nmoles/mg dry weight) 13.2 * 0.3 8.6 ± 0.4* Glycogen (mg/g wet weight) 5.7 ± 0.4 2.7 ± 0.8* Hydroxyproline (wg/mg dry weight) 3.7 ± 0.4 5.8 * 0.4* Values expressed as mean ± SE (n = 8 animals). P < 0.05. Significantly different from control. *Ion values are expressed as ng atoms/nig dry weight. 104 mitochondria (Table 14). Measurement of myocardial ATP and glycogen contents showed signi-ficant decreases in the ISO-treated animals. Time-course studies revealed that significant ATP depletion was present at about day 10, whereas significant glycogen depletion was observed much earlier (at around days 5-7). ISO-induced myocardial ischemic injury is associ-ated with scarring and significantly elevated levels of hydroxypro-line, indicative of increased collagen synthesis. Measurement of myo-cardial creatine phosphokinase revealed a significant decrease in the ISO-treated animals compared to controls (Control 22 ± 4, ISO-treated 13 ± 4, Sigma units/mg protein). 5.2.5 Lysosomal enzymes The effects of ISO treatment on left ventricular lysosomal enzyme latency are shown in Figure 12. Analyses of left ventricular homogen-ates revealed a significant increase in the non-sedimentable fraction of N-acetyl-B-glucosaminidase and cathepsin D activity in the ISO-treated animals. In contrast, acid phosphatase, a less specific marker for myocardial lysosomal structures, did not show any s i g n i f i -cant changes in sedimentable characteristics following ISO treatment. 5.2.6 Hemodynamics Prior to sacrifice, 24 hours after the last injection of ISO, rab-bits were anaesthetized with sodium pentobarbital and hemodynamic parameters were measured. The effect of the ISO treatment protocol on hemodynamic properties are shown in Table 15. After the development 105 80- P>0.05 70-u < Ul _J 0 < 60-§ 30H 20-p>ao5 i o J C T C A T H E P S I N 0 C T A C I D P H O S P H A T A S E C T N - A C E T Y L - B Q L U C O S A M I N I D A S E FIGURE 12 Lysosomal enzyme activity .in control and 15-day ISO-treated animal hearts. Lysosomal enzyme activity is expressed as percent non-sedi-mentable relative to total (sedimentable + non-sedimentable) activity. Values are expressed as mean ± SEM (n = 6 animals) 106 Table 15 Hemodynamic alterations in rabbits treated with isoproterenol (ISO) for 15 days Control ISO-treated Left ventricular pressure (mmHg) 105 10 125 10 Positive rate of rise of left 4250 100 4850 ± 300* ventricular pressure (mmHg) Blood pressure (mmHg) Systolic: 100 5 80 5* Diastolic: 80 ± 5 65 5* Left ventricular end 4 ± 2 8 ± 5 diastolic pressure (mmHg) Left ventricular blood flow 4 .9 * 0.4 5, .6 ± 0.7 (ml/min/g) P < 0.05. Significantly different from control. Values are expressed as mean ± SE (n = 6 animals). 107 of stable anaesthesia, hemodynamic studies revealed a significant decrease in systemic blood pressure in the ISO-treated animals. After each injection of ISO, blood pressure recordings made through the cen-tral ear artery revealed a decrease in systolic pressure which per-sisted for at least 2-3 hours. On the other hand, significant in-creases in heart rate, and in the rate of positive left ventricular pressure development were observed in the ISO-treated animals. The increases in heart rate (330-420 beats/minute) were evident after each injection of ISO and were s t i l l apparent 30-60 minutes after the in-jection of ISO. The left ventricular pressure was increased in the ISO treated animals, but. the increase was not significant in compari-son to controls. 5.2.7 Myocardial ultrastructural alterations Using electron microscopy to assess ultrastructural changes in the ISO-treated rabbit hearts, i t was observed that abnormalities ranging from mild to severe were seen in 5 of the 6 animals studied. Examina-tion of hearts following 4 days of ISO treatment showed some changes in lipids and glycogen as well as swelling of the mitochondria and slight disruption of the cristae network (Figure 13). Moderate mar-gination of nuclear chromatin was observed after 4 days of ISO treat-ment. After 10 days of ISO treatment, the abnormalities observed at 4 days had a more uniform distribution. In addition, the number of glycogen granules was less. The margination of the nuclear chromatin appeared to be slightly more than that observed at day 4. Other 108 FIGURE 13 Ultrastructure of rabbit heart after 4-days of ISO treatment. Nuclear chromatin (N) is finely granular and evenly dispersed. Myofibrils (My) are intact with the characteristic Z-lines appearing dense. Some mitochondria (M) are swollen and the cristae network appear disrupted. Large l i p i d droplets (L) and glycogen (g) are also v i s i b l e . X18,000. 109 FIGURE 14 This section shows ultrastructure of 7-day ISO-treated rabbit myocar-dium. Mitochondria (M) appear swollen, pale and appear to have dis-rupted cristae network. Small amorphous dense bodies are also visible in the mitochondria. Very few glycogen (g) granules are present sur-rounding the mitochondria . X18.000, 110 -mm N FIGURE 15 This section shows mitochondria (M) from 15-day ISO-treated animal hearts. The mitochondria appear swollen and show a loss in the matrix density. There is also disorganization of the cristae. The nuclear chromatin (N) appears slightly marginated. Not many glycogen granules are v i s i b l e . Cross section of the myofibrils (My) indicates lack of prominent Z-bands. The sarcoplasmic reticulum (SR) is dilated and some li p i d droplets (L) are also v i s i b l e . X 16,000. I l l FIGURE 16 This section shows ultrastructural alterations in the mitochondria (M) of 15-day ISO-treated rabbit heart. The mitochondria are severely damaged and some appear to have been fragmented. The cristae are in the process of disruption. Few glycogen (g) granules are visib l e . The sarcoplasmic reticulum (SR) and lipid (L) are v i s i b l e . Some elec-tron dense bodies are visible in the mitochondria (arrows). X18,000. 112 changes included an increased number of irregular l i p i d droplets and dilatation of the sarcoplasmic reticulum throughout the myocardium (Figure 14). Following 15 days of ISO treatment, most of the ultrastructural changes appeared to worsen. Myofibrillar Z-bands in many of the affected cells were dense and thickened. Some Z-bands did lose their electron density and were poorly defined (Figure 15). The myofibrils also appeared contracted, with some fibers appearing pale and poorly defined. The most marked effects of ISO treatment were on the mito-chondria. Mitochondrial alterations appeared in nearly every myocyte. They consisted of increases in mitochondrial swelling, the presence of numerous electron dense bodies, pale matrices and disruption of the cristae network. A few mitochondria were fragmented. Intramitochon-drial granules were irregular in shape and were often seen in the cen-ter of the mitochondria. Also striking was the abundance of li p i d droplets and l i t t l e or no glycogen was v i s i b l e . The sarcoplasmic reticulum was significantly dilated in animals treated with ISO (Figure 16). Objective ultrastructural assessment of control and ISO-treated hearts are shown in Table 21. 5.3 ISO-Induced Myocardial Damage in Diabetes 5.3.1 General considerations All animals exhibited ventricular tachycardia and f i b r i l l a t i o n after injection with ISO. The diabetic animals, like the controls, became prostrate and developed deep abdominal breathing after ISO administration. The onset of ventricular tachycardia and f i b r i l l a t i o n occurred within 10-15 minutes after ISO injection to diabetic 113 rabbits. Like controls, diabetic animals had a 100%mortality from ventricular f i b r i l l a t i o n i f the dose of ISO was not divided in 4 equal doses. Despite the precaution of administering ISO in 4 divided doses, the overall death rate of the diabetic animals was 25%. No differences in the incidence and frequency of ventricular arrhythmias were observed between control and diabetic animals injected with ISO. 5.3.2 Serum chemical properties The effects of ISO treatment on serum chemical changes in control and diabetic animals are shown in Table 16. ISO treatment in diabetic animals resulted in significantly higher values of blood glucose, total cholesterol and free fatty acids when compared either with ISO-treated controls or diabetic animals not receiving ISO. On the other hand', a significant decrease in serum triglycerides was seen in the ISO-treated diabetic animals. The activity of serum creatine phosphokinase was also significantly greater in the ISO-treated diabe-ti c animals compared to control and diabetic animals. Serum insulin was decreased in the diabetic animals prior to ISO treatment and re-mained low despite ISO treatment. In contrast, hemoglobin glycosyla-tion which was elevated prior to ISO treatment in the diabetic animals was not altered by ISO treatment. The effect of ISO treatment on body and heart weight of control and diabetic animals is shown in Table 17. Despite fluctuations in body weight of individual diabetic ani-mals treated with ISO, no significant differences in mean body weight were found relative to control animals treated with ISO. All ISO-treated animals showed significant increases in heart and left ventri-cular weights as well as increases in total 114 Table 16. Effects of Isoproterenol (ISO) treatment on serum biochemical changes in control and diabetic rabbits Control Diabetic Control Diabetic animals animals animals animals + ISO + ISO Blood glucose (mg/dl) 126 13 420 ± 28* 190 ± 14* 570 i 13* ** t Urinary glucose (mg/dl) - 5600 604 6100 ± 790 Serum insulin (pU/ml) 24.3 ± 2.7 19.3 ± 1.8* 26.4 2.3** 18.7 ± 3.4* HbAlc (nmol HMF g globin) 1.7 ± 0.3 3.9 ± 0.4* 2.1 0.2** 4.4 ± 0.3* t ; Serum cholesterol (mg/dl) 78 ± 16 232 39* 179 ± 19* 261 21* t Serum triglycerides (mg/dl) 85 7 128 ± 17* 47 ± j* ** 17 ± Serum free fatty acids (mg/dl) 57 6 54 ± 4 74 •j* ** 104 Creatine phosphokinase (Sigma Units) 28 ± 7 39 5 89 113 15* ** t Values are expressed as mean ± SE (n = 5 animals). P < 0.05. Significantly different from control. P < 0.05. Significantly different from diabetic. f P < 0.05. Significantly different from ISO-treated control. 115 Table. 17. Body, heart, and left ventricular weights in control, diabetic and isoproterenol (ISO)-treated animals Control Diabetic animals animals Control amma is + ISO Diabetic animals + ISO Body weight (kg) Heart weight (g) Left ventricular weight (g) Water content (%) Scarring (visual scoring) 3.3 ± 0.3 2.9 ± 0.2 4.9 ± 0.2 4.1 ± 0.3 3.3 ± 0.2 2.9 ± 0.2 77.1 ± 0.2 77.7 ± 0.3 3.2 ± 0.3 6.9 ± 0.3* ** 4.8 ± 0.4* ** 3.1 ± 0.2 6.7 ± 0.4 4.9 ± 0.4 * ** * ** 81.1 ± 0.3 * ** 81.7 ± 0.6 * ** ++ ++ Values are expressed as mean ^ SE (n = 5 animals) *P < 0.05. Significantly different from control. **P < 0.05. Significantly different from diabetic. Objective scoring was based on a scale where + = mild scarring and ++ = moderate scarring of the hearts. 116 The heart weight to body weight ratio was increased in both groups of ISO-treated animals, suggesting the presence of cardiac hypertrophy. Visual observation of control and diabetic hearts treated with ISO indicated the same amount of hemorrhage and diffuse nodules. 5.3.3 Myocardial enzymatic and compositional properties Of the various subcellular organelle ATPase activities studied + p+ only the K , Ca -stimulated (ouabain-insensitive, EGTA inhibit-able) ATPase activity was significantly decreased in the left ventri-cles of diabetic animals (Table 18). ISO-treatment reduced this en-zyme activity in both controls and diabetics, the decrease in diabetic animals being somewhat greater (67% versus 5 9 % ) . No significant dif-ferences were observed between ISO-treated control and ISO-treated diabetic animals with regard to the activities of mitochondrial ATPase (azide-sensitive) or sarcolemmal (ouabain-sensitive) Na+,K+-ATPase and K+-p-nitrophenylphosphatase, although a l l showed significant decreases relative to their appropriate controls not treated with ISO. Measurement of tissue ions indicated significant depletion of Mg in non-ISO-treated diabetic animals with no further increases or de-creases in the non-treated diabetic animal being observed. ISO-treat-ment resulted in a significant accumulation in Na in control and dia-betic animals. Similarly, an increased Ca accumulation was observed in control and diabetic left ventricles after ISO treatment, with the increase being much more significant in the diabetic animals (83%ver-sus 123%) (Table 19). ISO treatment resulted in similar degrees of ATP depletion in dia-betic (60%) and control (52%) animals. The extent of glycogen 117 Table 18. Subcellular organelle ATPase activities* in left ventricles of control and diabetic animals treated with isoproterenol (ISO). Control Diabetic Control Diabetic animals animals animals animals + ISO + ISO Mitochondrial ATPase (azide-sensitive) 220 ± 10 190 i 18 133 13* ** 130 -j* ** l\la+,K+-ATPase (ouabain-sensitive) 6.2 ± 0.8 5.8 ± 0.4 2.3 ± 0.4* ** 2.1 ± 0.7* ** p-Nitrophenyl phosphatase (ouabai n-sensitive) 1.1 ± 0.1 1.0 ± 0.2 0.5 ± 0.1* ** 0.3 ± 0.2* ** Sarcoplasmic reticulum Ca++-K+-ATPase (EGTA inhibitable) 2.9 ± 0.2 1.8 ± 0.2* 1.7 ± 0.2* 1.2 ± 0.3* ** Values expressed as mean ± SE (n = 4 animals). *P < 0.05. Significantly different from control. * P < 0.05. Significantly different from diabetic. + P < 0.05. Significantly different from ISO-treated control. ATPase activity expressed as pmol Pi/mg protein/hr. 118 Table 19. Myocardial ATP, glycogen, hydroxyproline and intracellular ions in control and diabetic animals treated with isoproterenol (ISO) Control Diabetic Control Diabetic animals animals animals animals + ISO + ISO Na 24.1 4.8 31.3 5.5 44.9 ± 6.7* 47.3 ± 4.6* K 296.0 ± 14.0 302.0 11.0 274.0 ± 7.0 289.0 ± 10.0 Mg 23.1 1.1 19.4 1.0* 20.6 * 1.3 20.4 1.0* Ca 3.6 ± 0.3 3.9 ± 0.4 6.6 ± 0.4* ** 8.7 ± 0.4* ** ATP (nmol/mg dry wt) 13.4 ± 1.2 10.2 1.1* 6.4 ± 1.8* ** 4.1 1.2* ** Glycogen (ug/mg) 4.1 ± 0.2 5.7 ± 0.3* 3.1 ± 0.2* ** 2.9 ± 0.4* ** Hydroxyproline (ug/mg) 3.2 ± 0.4 5.0 ± 0.3* 5.1 ± 0.2* 6.1 ± 0.4* ** Values expressed as mean ± SE (n = 4 animals). P < 0.05. Significantly different from control. **P < 0.05. Significantly different from diabetic. tP < 0.05. Significantly different from ISO-treated control. Ion values expressed as ng atoms/mg dry weight. 119 depletion following ISO administration was significantly greater in diabetics as compared with controls (40%versus 241%). Finally, the increase in myocardial hydroxyproline content ISO-treated diabetics was significantly less than that for ISO-treated controls (22%versus 59%) (Table 19). 5.3.4 Lysosomal enzymes The effects of ISO treatment on diabetic left ventricular lysoso-mal enzyme latency are shown in Figure 17. No differences in the la-tency of cathepsin D, N-acetyl-e-glucosaminidase or acid phosphatase were observed in ISO-treated diabetic animals as compared with ISO-treated controls. However, the non-sedimentable activity of N-ace-tyl-e-glucosaminidase was significantly increased ( i . e . latency was decreased) in the diabetic, ISO-treated controls and diabetics as com-pared to non-treated control animals. 5.3.5 Hemodynamics ISO administration caused similar decreases in both systolic and diastolic pressures in control and-diabetic animals (Table 20). The diabetic animals had a significantly lower left ventricular pressure prior to ISO administration. ISO administration caused a significant increase in the left ventricular pressure of control and diabetic ani-mals (approximately 10%in both cases). Similarly, comparable increa-ses in the positive rate of rise of left ventricular pressure and heart rate were observed after ISO administration to both control and diabetic animals. ISO-treated non-diabetics and diabetic animals did not show any difference in the left ventricular end diastolic pressure. 120 [~| contro l §53 isoproterenol PJ d iabet ic £ rjjjj isoproterenol & diabet ic > O 1 0 0 r < FIGURE 17 Effect of ISO-treatment on left ventricular lysosomal enzyme latency in control, diabetic, ISO-treated control and ISO-treated diabetic animals. Lysosomal enzyme activity is expressed as percent non-sedimentable relative to total (sedimentable + non-sedimentable) activity. Non-sedimentable activity is an index of enzyme release from lysosome. Values are expressed as mean ± SEM (n = 4 animals). *P < 0.05. Significantly different from control. 121 Table 20 Hemodynamic alterations in control and diabetic animals injected with isoproterenol (ISO). Control Diabetic Control Diabetic animals animals animals animals + ISO + ISO Blood pressure: (systolic) 115 * 10 100 ± 5 80 * 10* 85 * 5* (mmHg) (diastolic) 80 * 10 75 * 10 60 * 5 60 * 5 Left ventricular 115 ± 5 95 * 5* 125 * 5** 110 * 5** pressure (mmHg) Left ventricular end 5 * 2 5 * 2 7 * 3 8 * 4 diastolic pressure (mmHg) (+)Rate of rise of 4600 * 150 3900 * 100* 4900 * 100* ** 4400 * 150**t left ventricular pressure (mmHg/s) Heart rate 300 * 5 260 * 10* 345 * 10* ** 290 * 10* * * t (beats/min) Values expressed as mean * SE (n = 4). P < 0.05. Significantly different from control. P < 0.05. Significantly different from diabetic. l-f-'P < 0.05. Significantly different from ISO-treated control. 122 5.3.6 Ultrastructural alterations Electron micrographs showing control, diabetic and isoproterenol-treated rabbit hearts have been presented earlier (Figures 8,9,10,11, 12,13,14,15). The effects of isoproterenol on ultrastructure of left ventricles from diabetic hearts are shown in Figures 18, 19 and 20. Hypercontraction bands were apparent in most cells treated with ISO. Mitochondria showed^enlargement and pallor due to increases in inter-cristal space. A number of mitochondria showed disruption of their cristae network. The myofilaments appeared dense and did not show clear Z-lines. In some c e l l s , no details were visible in the myofilaments and they appeared only as a homogeneous sheet of material. Glycogen was sparse and the sarcoplasmic reticulum was dilated. A number of amorphous electron-dense bodies were visible in and around the mitochondria. These electron-dense bodies became increasingly prominent in areas of severe mitochondrial damage. A comparison of ultrastructural features of the left ventricles in ISO-treated controls and diabetic animals is shown in Table 21. 5.4 Coronary. Artery Ligation and Reperfusion in Control and  Diabetic Rabbits The model of coronary artery ligation used in this study has been adequately described in detail by Godin et al (124) and Moore and Godin (115). The c r i t e r i a used to assess myocardial ischemic injury used in the study of Godin et al (124) have been used in the present study. 123 FIGURE 18 This electron micrograph shows the ultrastructure of a 10-week diabet-ic myocardium treated with ISO for 5 days. The mitochondria are fragmented and the cristae network is disorganized. The sarcoplasmic reticulum is swollen and the myofibrils appear as a homogeneous mass, showing no distinct Z-bands. xl3,500 124 FIGURE 19 This electron micrograph shows the effects of 15-day ISO-treatment on 10-week diabetic rabbit left ventricle. The mitochondria are swollen, appear pale and fragmented. Lipid accumulation is seen but no glycogen is evident. X15,000. 125 FIGURE 20 Mitochondria from diabetic animals after 15-day ISO-treatment. No Z-lines are seen and the myofibrils are damaged. Extensive damage to the mitochondria is seen. X15,000. 126 Table 21 Objective assessment of ultrastructural integrity of control, diabetic and isoproterenol (ISO)-treated hearts Mitochondri a fragmentation Mitochondrial amorphous bodies Myofibrillar integrity Nuclear chromatin clumping Glycogen depletion Sarcoplasmic reticulum dilatation Control hearts 0-1 0 0 0 0 0 10-week alloxan diabetic hearts 0-1 0-1 0-1 1 1 1-2 15-day ISO treated heart 2-3 1-2 2-3 1-2 2-3 1-2 ISO-treated diabetic hearts 3 1-2 2-3 1-2 2-3 2-3 *Scoring: 0 = normal, 1 = mild abnormality, 2 = moderate abnormality, 3 = severe abnormality. 6 blocks from each sample and 7 electron micrographs obtained from-each section. 6 control and 6 ISO-treated hearts were used for the electron microscopy study. 127 5.4.1 Occluded zone and ECG alterations The occluded zone in both control and diabetic animals following coronary artery ligation and reperfusion, as assessed by the dye indo-cyanin green, was similar (C 60 ± 4.0, D 66 * 3.0, expressed as .'. left ventricular weight, mean ± SD). Measurement of the occluded zone with the dye does not provide any estimate of the infarct size but does give some indication of the area at risk . Since the time of occlusion was only 40 minutes, cell death or infarct size could not be determin-ed. In order to get minimal detection of cell death/infarct, the ani-mal heart must be ligated for at least 6 hours and then quantified with nitroblue tetrazolium (139). Immediately following coronary artery ligation in control and dia-betic animals, the segment of the myocardium supplied by the ligated artery ceased to contract, appeared cyanotic, dilated and exhibited bulging. The incidence of arrhythmias and cardiogenic shock in control and diabetic animals subjected to 40 minutes of ischemia induced by liga-tion of the coronary artery followed by 60 minutes of reperfusion are shown in Table 22. During the non-1 igated period in a l l animals and during the experiment in the sham-operated animals, there were no sig-nificant ST-segment changes or alterations in R-wave amplitude. Dia-betic animals showed a significantly higher frequency of spontaneous ventricular f i b r i l l a t i o n following occlusion and reperfusion as com-pared to control animals. In both groups of animals, the highest incidence of this irreversible ventricular f i b r i l l a t i o n occurred 128 between 0-12 minutes following occlusion and 0-9 minutes after reper-fusion. If ventricular f i b r i l l a t i o n occurred during the occlusion period, administration of a D. C. shock usually restored sinus rhythm in both groups of animals. However, i f the ventricular f i b r i l l a t i o n occurred during the reperfusion period, reversal with D. C. shock was much less effective in the diabetic animals as compared to controls {50%versus 85%). Similarly, the incidence of cardiogenic shock (as evidenced by zero left ventricular and systemic blood pressure) was also s i g n i f i -cantly higher in the diabetic animals. In particular, diabetic ani-mals with high serum l i p i d levels fared considerably worse than diabe-tic animals with normal serum l i p i d s . In both control and diabetic animals, cardiogenic shock resulting mainly from left ventricular f a i -lure was almost always fatal as evidenced by a drastic drop (0-10 mm Hg) in left ventricular pressure. The appearance of Q-waves and ST-segment elevation was seen in both groups of animals after ligation. In the sham-operated animals there were no signficant ST-segment changes or alterations in R-wave amplitude. ST-segment elevations in both control and diabetic animals were observed within 30-45 seconds after occlusion. The elevation in ST-segments reached a maximum of 45 ± 10 mm within 20-30 minutes in both groups of animals. The most pronounced ST-segment elevation was observed in leads I, III, aVr, aVr and chest lead Vc. Q-waves (which are diagnostic of myocardial infarction) were also evident in both control and diabetic animals following reperfusion . These Q-waves were observed in leads II, III and chest lead 129 Table 22. Incidence of ventricular f i b r i l l a t i o n , cardiogenic shock and appearance of Q-waves in control and diabetic animals following 40 min ligation (40 min 0) of the left circumflex coronary artery and 60 min reperfusion (60 min R). Control animals Di abetic animals 40 min 0 60 min R 40 min 0 60 min R Ventricular f i b r i l l a t i o n9 1/9 (11%) 4/9 (44%) 4/8 (50%) 6/8 (75%) Cardiogenic shock^ - 2/11 (18%) 4/10 (40%) 6/9 (67%) Appearance of Q-waves 3/9 (33$ 7/9 (78%) 4/8 (50%) 6/8 (75%) Premature ventricular^ contractions 2.1 * 0.6 1.1 * 0.3 2.2 * 0.3 1.6 * 0.2 In both groups of animals, the highest incidence of ventricular f i b r i l l a -tion was observed shortly after occlusion (0-12 min) and immediately after reperfusion (0-9 min). During cardiogenic shock, the mean arterial pressure dropped to zero, and resuscitation in both groups of animals was impossible. Q-waves were most evident in leads I, III and epicardial lead Vs. ST-seg-ment elevation was observed in a l l leads in both groups of animals soon after occlusion. Premature ventricular contractions are expressed as logio Mean * SEM (n = 9 animals). 130 Vg. There was no difference in the amplitude of Q-waves in control and diabetic groups, and both groups showed a higher incidence of Q-waves during reperfusion as compared with ischemia. The number of premature ventricular contractions in control and diabetic animals following occlusion and reperfusion was similar. The highest incidence of premature ventricular contractions occurred 20 minutes after occlusion and shortly after the init i a t i o n of reperfu-sion. 5.4.2 Effect of coronary ligation on ATP The effects of coronary ligation on ATP levels are shown in Figure 20. Prior to ligation, ATP levels in the diabetic animal hearts were significantly lower than control (13.8 ± 1.2 versus 10.4 ± 1.6 nmoles/mg dry wt). After 40 minutes of occlusion of the coronary art-ery, ATP levels in both control and diabetic animals dropped s i g n i f i -cantly compared to their preligated levels. The percentage decrease of ATP levels in control and diabetic animals was 42%and 56%, respectively. Following 60 minutes of reperfusion, there was a fur-ther decrease in the myocardial ATP content in both groups of animals. The percentage decreases compared to the preligated levels in control and diabetic animals were 74%and 82;%, respectively. 5.4.3 Ions and water content The effects of coronary artery ligation and reperfusion on ion distribution in control and diabetic animals are shown in Table 23. Prior to ligation, no changes were observed in the ion contents of K, Ca and Na in control versus diabetic animals. However, a significant 131 FIGURE 21 Myocardial tissue ATP levels in in control (L~Z3) and diabetic (£==)) animal hearts after 40 min coronary artery occlusion 60 followed by 60 min of reperfusion. Values are expressed as mean ± SE (n = 6 animals). * P < 0.05. Significantly different from respective pre-ligated value. P < 0.05.. Significantly different from corresponding control. 132 Table 23. Ionic alterations in hearts of control and diabetic animals after 40 min occlusion and 60 min reperfusion of the left circumflex coronary artery Control animals Diabetic animals Preligation 40 0 + 60 R Preligation 40 0 + 60 R Na 29.2 * 5 . 4 66.4 * 5.9 37.6 6.6 78.7 * 7.1* K 294.1 * 13.3 254.1 ± * 10.1 268.4 11.4 241.3 * 7.7* Mg 28.7 ± 2.3 23.1 3.10* 22.6 ** 1.7 * ** 16.4 * 3.1 Ca 5.6 * 0.6 10.4 2.0* 5.9 0.4 15.9 * 2.1* ** Values are expressed as mean ± SE (n = 6 animals). P < 0.05. Significantly different from respective preligated value. P < 0.05. Significantly different from corresponding control. Ion values are expressed as ng atoms/mg dry weight. 133 reduction in the myocardial Mg level was apparent in the diabetic ani-mals. No changes in the ion levels were observed following 40 minutes of coronary artery occlusion (data not shown). Upon reperfusion, a significant increase in Na was observed in both control and diabetic animals compared to their preligated values. In contrast, a s i g n i f i -cant decrease in the K content of the myocardium was observed in both these groups of animals. In control animals, a significant decrease in Mg levels was observed. This trend was also observed in the diabe-t i c animals, but the decrease was significantly greater than that ob-served in control animals. With respect to Ca, a significant increase was observed in both control and diabetic animals following reperfu-sion, the magnitude of the Ca accumulation being significantly greater in the diabetic animals. No difference in the total water content was observed between con-trol and diabetic animals prior to ligation. After 40 minutes of oc-clusion of the left circumflex coronary artery, no increase in the water content was seen in either group. However, following reperfu-sion, a significant increase in total water was observed in both con-trol and diabetic animals (C 80.1 ± 1.0, D 81.3 ± 1.3 % ) in compari-son to their preligated values (C 77.2 ± 0.4, D 78.1 ± 0.1 % ) . 5.4.4 Subcellular organelle ATPases The effects of coronary ligation and reperfusion on subcellular organelle enzyme markers in control and diabetic animals is shown in Figures 22, 23 and 24. The effects of 40 minutes of coronary ligation on mitochondrial (azide-sensitive) ATPase activity are shown in Figure 134 FIGURE 22 Mitochondrial (azide-sensitive) ATPase activity in control (CZJ) and diabetic (JEEEJ) animal hearts after 40 min occlusion and 60 min reper-fusion. Values are expressed as mean ± SE (n = 6 animals). P < 0.05. Significantly different from respective pre-ligated value. ** P < 0.05. Significantly different from corresponding control value. .135 FIGURE 23 Sarcolemmal Na+,K+-ATPase activity (ouabain-sensitive) in control (I |) and diabetic animal hearts after 40 min occlusion and 60 min reperfusion. Values are expressed as mean * SE (n = 6 animals). P < 0.05. Significantly different from respective preligated value. ** P < 0.05. Significantly different from corresponding control value. 136 4-n PRE-LIQATION 40-MINS 60-MINS OCCLUSION REPERFUSION FIGURE 24 Sarcoplasmic reticulum ATPase activity (EGTA inhibitable, ouabain-insensitive) control ([" |) and diabetic (|=!) hearts after 40 min occlusion and 60 min reperfusion. Values are expressed as mgan ^SE (n = 6 animals). P < 0.05. Significantly different from respective pre- ligated value. P < 0.05. Significantly different from corresponding control value. 137 22. Forty minutes of ligation decreased the mitochondrial ATPase act-ivity in both control and diabetic animals (59% versus 61%). Follow-ing 60 minutes of reperfusion, a significant recovery in the mitochon-drial ATPase activity was observed in control animals and while that in diabetic animals did show a tendency to recover, i t was s i g n i f i -cantly less than that observed in controls (61%versus 83%). The sarcolemmal Na+,K+ (ouabain-sensitive) ATPase activity following 40 minutes occlusion and 60 minutes reperfusion is shown in Figure 23. The decreases in the sarcolemmal ATPase activity after 40 minutes occlusion were 33%and 34% in control and diabetic animals, respectively. Following reperfusion, no recovery of this enzyme was observed in either group of animals but rather a further decrease was observed. The decreases compared to preligated levels were 49% and 61% in control and diabetic animals, respectively, with the decrease being significantly greater in the diabetic animals. Prior to ligation, the sarcoplasmic reticulum ATPase activity was significantly less than in the diabetic animals compared to control (Figure 24). After 40 minutes of occlusion, both groups of animals showed a decrease in this enzymatic activity, with the depression in diabetic animals being significantly greater (33% versus 45%). With reperfusion, the sarcoplasmic reticulum ATPase in control animals showed no further change while that in the diabetic animals showed a further decrease. 5.4.5 Hemodynamics The hemodynamic parameters in control and diabetic rabbits 138 following ligation and reperfusion are shown in Table 24. The mean heart rate in the diabetic animals was significantly lower than that in controls prior to ligation. Following ligation both control and diabetic animals had similar heart rates which changed minimally with reperfusion. The left ventricular end diastolic pressure in both con-trol and diabetic animals did show a tendency to increase with ischem-ia and reperfusion, but the increases were not significant when com-pared to preligated values. The systemic arterial pressure after coronary ligation and reperfusion was significantly decreased in con-trol (40%) and diabetic animals (52%) compared to their respective preligated values. In addition, a similar decrease in the left ven-tricular pressure after coronary ligation and reperfusion was observed in the control (40%) and diabetic animals ( 5 0 % ) . The rate of rise of positive left ventricular pressure, like the heart rate, was signi-ficantly lower in the diabetic animals prior to ligation. Following ligation and reperfusion, however, no significant changes were ob-served in this parameter in either group. 5.4.6 Mitochondrial ATP generation The ability of the mitochondria to generate ATP after ligation and reperfusion in control and diabetic animals is shown in Table 25. Mitochondria obtained from diabetic animals prior to ligation had a significantly lower ability to generate ATP in comparison to con-t r o l s . Following ligation, mitochondria obtained from control and diabetic animals both showed a decrease in their abi l i t y to generate ATP (15%versus 27%). After 60 minutes of reperfusion, a further 139 Table 24. Hemodynamics in control and diabetic animals following occlusion and reperfusion of the left circumflex coronary artery Control animals Diabetic animals Pre 40 min 0 60 min R Pre 40 min 0 60 min R Left ventricular pressure (mmHg) 115 * 5 105 * 10 70 * 10* 100 * 10 80 * 10 50 * 5* ** Left ventricular end diastolic pressure (mmHg) 4 * 2 6 * 4 10 * 5 5 * 2 5 * 2 10 * 4 Heart rate (beats/min) 290 * 5 300 * 10 315 * 5* 265 * 10** 290 * 10* 290 * 10* Mean arterial pressure (mmHg) 115 * 5 105 * 10 70 * 10* 95 * 10 85 * 10 50 ±10* Rate of rise of ventricular pressure (mmHg/s) 4000 * 300 3600 * 250 3850 * 300 3300*300** 3450 * 250 3300 * 250 Values are expressed as mean * SE (n = 6 animals) P < 0.05. Significantly different from preocclusion value. P < 0.05. Significantly different from corresponding control. 140 Table 25 Mitochondrial ATP generation in control and diabetic animals following coronary artery occlusion (40 min 0) and reperfusion (60 min R) Control Diabetic Preligation 40 min Occlusion 40 min Occlusion + 60 min Reperfusion 0.55 ± 0.03 0.47 ± 0.04' 0.17 ± 0.04' 0.41 ± 0.09 0.30 ± 0.06 ** * ** 0.06 ± 0.07' P < 0.05. Significantly different from respective preligated value. P < 0.05. Significantly different from corresponding control. Values are expressed as mean ^ SE (n = 6 animals). ATP generation is expressed as ymoles/mg/min. ** 141 decrease in the ability of mitochondria to generate ATP in both con-trol and diabetic animals was observed, with the decrease in the dia-betic animals being significantly greater than that in controls (69%versus 85%). 5.4.7 Ultrastructural alterations Time-course myocardial ultrastructural alterations in control and diabetic hearts after coronary ligation and reperfusion are shown in Figures 25, 26, 27, 28, 29 and 30. Ultrastructural features of con-trol and diabetic hearts prior to ligation have already been described (Figures 8, 9, 10, 11,). After 20 minutes of occlusion, no major ultrastructural alterations were seen in either group of animal. Mod-erate margination of nuclear chromatin was evident in both groups of animals. Most myofibrils appeared intact and glycogen depletion was not evident in either group. The mitochondria appeared less granular and the matrix less electron-dense than in the preligated tissues (Figures 25, 28). After 40 minutes of occlusion, fine structural changes were ob-served in both groups of animals (Figures 26, 29). The severity of ischemic injury varied from cell to cel l in both groups of animals. However, both control and diabetic animals showed similarities in the ultrastructural alterations but the magnitude of the changes appeared to be significantly more marked in the diabetic animals. The charac-t e r i s t i c feature of the ischemic injury in both groups of animals was lack of glycogen, relaxation of myofibrils with prominent I-bands. Of the subcellular organelles examined, the mitochondria 142 FIGURE 25 Ultrastructure of control rabbit myocardium after 20 minutes of coron-ary artery ligation. Mitochondria appear to be swollen with very few intramitochondrial dense bodies. Glycogen is sparse and the myofib-r i l l a r integrity is maintained. X16.000. 143 FIGURE 26 This section shows ultrastructure of control rabbit myocardium after 40 minutes of coronary artery ligation. The mitochondria appear mark-edly swollen and contain amorphous dense bodies. Glycogen is not vis-ible. The myofibrils are dispersed and appear contracted. X16.000. 144 FIGURE 27 Ultrastructure of control rabbit myocardiun after 40 minutes occlusion and 60 minutes reperfusion. The mitochondria are severely damaged and fragmented. Numerous electron-dense bodies appear within the mitoc-hondria. The myofibrils appear dispersed with prominent I-bands. No glycogen is v i s i b l e . X16,000. FIGURE 28 Ischemic myocardium from diabetic animal after 20 min of coronary artery occlusion. Mitochondria (M) appear swollen with some disrup-tion of their cristae network. Myofibrils appear dispersed (X13,000). 146 FIGURE 29 Ischemic myocardium from a diabetic animal after 40 min of coronary artery occlusion. Most mitochondria (M) are swollen with disrupted cristae and contain amorphous dense bodies. Glycogen is absent. Myofibrils (My) appear relaxed with prominent I-bands (X16,000). 147 FIGURE 30 This electron micrograph shows severe irreversible ischemic injury in hearts of diabetic animals following 60 min reperfusion of the left circumflex coronary artery. Most mitochondria (M) appear severely damaged, showing clearing of the matrix, small dense granules and fragmentation. Large amorphous dense bodies are more evident in the mitochondria. No glycogen is present and clumping of nuclear chromatin is evident (X16,000). 148 appeared to be more damaged in the diabetic myocardium. Mitochondria in both control and diabetic myocardium were swollen, fragmented and contained large amorphous dense bodies, consisting presumably of either calcium-lipid deposits or denatured protein. These amorphous dense bodies had a different appearance from those in hearts of sham-operated animals and they were clearly distinguishable from the floc-culent matrix by their high electron-density. Mitochondria from the diabetic animals, in particular, showed more disruption of their c r i s -tae network. The nucleus in both groups of animals appeared coarse, densely aggregated and margination of the chromatin was quite evident. Occasionally, disarrangement of whole sarcomeres or of myofibrils was evident. Sarcoplasmic reticulum appeared slightly dilated. The i n t e r s t i t i a l space in both groups of animals was considerably widened and exhibited an accumulation of f l u i d , protein-like material. A few hypercontractions bands were observed in both groups of animals. Following reperfusion, the ultrastructural changes in the myocar-dium worsened in both control and diabetic groups (Figures 27,30) Again, i t appeared that the ultrastructural alterations following reperfusion were significantly greater in diabetic than those observed in the control animals. Both groups of animals showed severely dam-aged mitochondria with clearing of the matrix. Diabetic animals show-ed a greater number of damaged mitochondria, and in many cases these mitochondria were fragmented. Large amorphous electron-dense bodies were present in the mitochondria, with the frequency of the electron-dense bodies appearing to be much greater in the diabetic animals. 149 In both control and diabetic animals, the frequency of occurrence of these dense bodies was higher than that observed during the period of occlusion. The nuclear chromatin in both groups of animals appear-ed clumped, dense and was marginated towards the periphery of the nuclear membrane. No glycogen was evident during reperfusion. The disarrayed myofibrils exhibited numerous contraction bands. These hypercontraction bands were more frequently observed during the reper-fusion period than after the ischemic interval. Myofibrillar disper-sion and intermyofibrillar oedema was also observed in both groups of animals. Table 26 illustrates the results of an objective scoring of ultrastructural alterations in hearts of control and diabetic animals following occlusion and reperfusion. 5.4.8 Blood flow following occlusion and reperfusion The effects of occlusion of the left circumflex coronary artery and subsequent reperfusion on blood flow are shown in Table 27. Because of the d i f f i c u l t y in excising endocardium and epicardium in rabbit hearts, all data are based on total heart weights. Occlusion of the coronary artery had a significant effect on blood flow, de-creasing the flow in both control and diabetic hearts by about 95%. Reperfusion in both control and diabetic animals restored the blood flow back to the original value. The restoration of the blood flow following reperfusion in both groups of animals eliminates the possi-b i l i t y of a no-reflow phenomenon in our studies. 150 Table 26. Objective assessement of myocardial ultrastructure integrity of diabetic and control animals before and after 40 min occlusion (0) and 60 min reperfusion (R) Mitochondria Mitochondrial fragmentation amorphous bodies Myofibrillar i ntegrity Nuclear chromatin clumping Glycogen depletion Sarcoplasmic reticulum dilatation Preligati on 40 min 0 40 min + 60 min R 0- 1 (0) 0-1 (0) 0-1 (0) 0 (0) 0 (0) 0 (0) 1- 2 (0-1) 1-2 (0-1) 0-1 (0-1) 0-1 (0-1) 1-2 (1-2) 1-2 (1-2) 3 (2-3) 3 (2-3) 3 (3) 3 (2-3) 2-3 (2-3) 2-3 (2-3) Scoring: 0 = normal, 1 = mild abnormality, 2 = moderate abnormality, 3 = severe abnormality. Numerous sections and electron micrographs were obtained from each animals. Tissues were obtained from a minimum of 3 animals at each time point. Numbers in brackets reflect myocardial ultrastructural alterations in control hearts. 151 Table 27. Blood flow in ischemic (40 min 0) and reperfused (60 min R) left ventricles of control and diabetic animals Control Diabetic Preligation 5.90 * 0.7 4.65 * 1.85 40 min Occlusion 0.27 * 0.14* 0.35 ± 0.16* 40 min Occlusion 6.47 * 1.24 5.56 ± 0.87 + 60 min Reperfusion *p < 0.05. Significantly different from respective preligated value. Blood flow ml/min/g = cpm sample/cpm reference blood x withdraw! rate tissue weight in g Values are expressed as mean * SE (n = 5 animals). 152 5.5 Coronary Artery Ligation in Conscious Control and Diabetic Rats In our previous study, we examined the effects of coronary artery ligation and reperfusion in the diabetic rabbits. Since the rabbit model is not appropriate for the study of arrhythmias, the conscious rat model was studied to compare the incidence of post-1igation ar-rhythmias in control and diabetic rats. The general features of 6 and 12 week diabetic rats are given in Table 28. Injection of streptozotocin resulted in a profound state of hyperglycemia and hyperlipidemia within 3 days. Compared to controls, diabetic rats after 6 and 12 weeks had higher blood glucose, elevated hemoglobin glycosylation and serum lipids and depressed body weight and levels of serum insulin and thyroid hormones. The effects of ligation of the left anterior descending coronary artery in conscious control, 6 and 12 week diabetic rats are shown in Table 29. within 5 minutes after ligation, a l l animals began to show marked ventricular ectopic activ i t y . These dysrhythmias included isolated extrasystoles and ventricular tachycardias, including ventricular f i b r i l l a t i o n . In control animals, only 3% of the animals showed reversion of the ventricular f i b r i l l a t i o n to spontaneous sinus rhythm. In contrast, in a l l 6 week diabetic animals, reversion to spontaneous sinus rhythm did not occur. In controls and 6 week diabetic animals which did not show reversal of ventricular f i b r i l l a t i o n to sinus rhythm, tapping of the chest (as a means of defibrillation) was done. This procedure resulted in the reversion of 153 Table 28 General features of control, 6- and 12-week diabetic rats Control 6-week 12-week Body weight (g) 197 ± 6 148 ± 5* 102 ± 11* Blood glucose (mg/dl) 106 ± 9 596 * 28* 514 ± 17* Serum insulin (nil/ml) 19.2 * 2. .6 7.6 ± 3.4* 5.3 ± 3.1* HbAic (umol HMF/g globin) 1.3 * 0. ,2 4.5 ± 0.6* 5.9 ± 0.8* T3 (ng/dl) 4.3 ± 0. .4 2.4 ± 0.4* 1.8 ± 0.6* T4 (Jig/dl) 5.8 ± 0. ,3 3.1 * 0.3* 2.1 ± 0.7* P < 0.05. Significantly different from control. Values expressed as mean ± SE (n = 30 animals). 154 ventricular f i b r i l l a t i o n to sinus rhythm in a l l control and 6-week diabetic rats. Having passed the c r i t i c a l stage of early post-1igation arrhythm-ias, a l l animals survived. At about 2-4 hours post-1igation, a second phase of arrhythmias was observed in the animals. In most cases, the dysrhythmias spontaneously reverted to sinus rhythm and the mortality rate was about 20%in both groups of animals. Coronary artery liga-tion in 12 week diabetic rats was markedly different from the situa-tion in controls. Soon after ligation a l l diabetic animals developed ventricular f i b r i l l a t i o n which failed to spontaneously revert to sinus rhythm. Unlike the controls, or 6 week diabetic rats, tapping the chest was unsuccessful in defibri1lating. The animals were observed for the remainder of the time period and sacrificed 24 hours post-1igation. The occluded and infarcted zones in the control and 6 week diabetic rats were similar (Table 30). The mortality rate and the incidence of ventricular f i b r i l l a t i o n appeared greater in the 6 week diabetic animals, but did not differ significantly from control. Unlike the situation in the 6-week diabetic rats, the mortality rate in 12 week diabetic rats was 100.% within 1-7 minutes post-1igation. Examination of hearts from a l l three groups revealed that both the 6 and 12 week diabetic rats had a significant magnesium deficiency prior to ligation. 155 Table 29 Effect of coronary artery ligation in control and 6-week diabetic rats on mortality, infarct and occluded zones and incidence of arrhythmias Control Diabetic Occluded zone (%) 41 ± 4.0 39 ± 5.0 Infarcted zone (%) 25.5 ± 1.3 23.8 * 2.6 Mortality (%): 0-2 h 2-24 h 84 10 88 10 Incidence of f i b r i l l a t i o n (%) 92 100 Magnesi urn (ng atoms/mg dry wt) 33.2 ± 1.5 27.2 ± 2.9* Values expressed as mean ± SE (n = 30 animals). P < 0.05 Significantly different from control. Occluded and infarcted zones are expressed as percentage of total ventricular weight. 156 Table 30 Effect of coronary artery ligation in control and 12-week diabetic rats on mortality, infarct and occluded zones and incidence of arrhythmias Control Diabetic Occluded zone (%) 37 ± 4.0 40 ± 5.0 Infarcted zone (%) 21 ± 3.0 -Mortality (%): 0-2 h 2-24 h 75 18 100 Incidence of f i b r i l l a t i o n (%) 84 100 Magnesi urn (ng atoms/mg dry wt) 3.6.8 ±2.3 29.6 ± 1.7* Values expressed as mean ± SE (n = 30 animals). * P < 0.05 Significantly different from control. Occluded and infarcted zones are expressed as percentage of total ventricular weight. 157 6 DISCUSSION 6.1 Aim The purpose of the work in this thesis was to investigate the in-fluence of al loxan-induced diabetes in rabbits on myocardial ischemic injury produced either acutely by coronary artery ligation or by re-peated isoproterenol treatment. The work involved the characteriza-tion of the model of diabetes and the model of myocardial ischemic injury using isoproterenol in the rabbit. These experiments have at-tempted to measure the extent and nature of myocardial ischemic in-jury using biochemical markers (activity of subcellular organelle en-zymes), chemical markers (alterations in cellular ATP and ion levels), ultrastructural changes (electron microscopy) and functional altera-tions.- In addition, the susceptibility of diabetic rats to coronary artery ligation has also been studied using the conscious rat model developed by Johnston et al (120). 6.2 The Choice of the Animal Model In the study of myocardial ischemic, injury there is no ideal model which exactly simulates the typical c l i n i c a l situation. The dog is frequently used because of the presence of pre-existing coronary co-llateral vessels thought to resemble the situation in man. However, because of this extensive collateral network in the dog, a well-defined ischemic zone is d i f f i c u l t to obtain. The pig is essentially free of coronary collateral vessels but does not develop characteristic electrophysiological disturbances (arrhythmias) after coronary occlusion (140). The acute open-chest rabbit provides a sta-ble, physiological preparation which allows both functional and - 158 -biochemical measurements to be performed in the same animal. The yield of ischemic tissue is sufficient to allow sampling for ATP, ions, electron microscopy, as well as separation into mitochondrial and sarcolemmal fractions, without having to pool tissue from several animals. A further advantage of using the rabbit as the experimental animal is that left circumflex coronary artery occlusion produces a well-defined zone of ischemia without interdigitating normal tissue, thus making for more reproducible and accurate sampling. A disadvantage of the open-chest rabbit model is the failure to develop consistent electrical disturbances (arrhythmias) following ligation. In view of this limitation, we have also included experi-ments performed using a conscious rat model which we believe is ideal to study post-1igation arrhythmias. 6.3. The Diabetic Rabbit Model Alloxan treatment generally has been used to induce diabetes in rabbits because of the d i f f i c u l t i e s experienced with streptozotocin in this species. Unlike the reports of Lazarus and Shapiro (141), we were unable to induce diabetes in rabbits with streptozotocin (150-300 mg/kg, intravenously), suggesting that rabbits are resistant to strep-tozotocin and thus confirming the reports of Kushner et al (142). Rabbits treated with alloxan developed a diabetic state characterized by the classical symptoms of insulin-dependent diabetes mellitus, such as hyperglycemia, hypoinsulinemia, glycosuria, polyuria and hyper-1 ipidemia. - 159 -Alloxan diabetes is characterized by the specific necrosis of the beta cells of the islets of Langerhans with resulting insulin defi-ciency (143). Although morphometric analysis to determine the total number of islets and beta cells has not been accomplished in the rab-b i t , preliminary electron microscopic studies reveal that beta cells of diabetic rabbits show an increased number of beta granules (144). From the results obtained, i t appears that the absence of insulin alone is not responsible for all the above-mentioned symptoms of ex-perimental diabetes in the rabbit. Ketosis, particularly, is not a result of insulin deficiency alone, as can be seen from the fact that when the excretion of ketone bodies became normal, the urinary excre-tion of sugar was s t i l l high. The rapid rise in the urinary excretion of acetone bodies for the f i r s t few days after the injection of alloxan indicates that there is increased ketogenesis, possibly in the l i v e r . The f a l l in the urinary excretion of ketone bodies at the end of the second week may be due either to diminished formation of ketone bodies or to their increased utlization. Approximately one-third of the animals who originally developed diabetes following a single dose of alloxan appeared to es-cape from its effects within a 6-month period, and additional doses may be required to maintain the diabetic state. However, rabbits ge-nerally tolerate diabetes relatively well as long as severe ketosis and dehydration are avoided in the f i r s t week following alloxan ad-ministration. A stable non-ketotic form of diabetes can be achieved, and the growth rate of the animals is near normal over an - 160 -extended period of time, thus making the animal potentially useful for chronic studies. The alloxan-diabetic rabbit should be a useful ex-perimental model of human diabetes mellitus. The relatively late on-set of overt symptoms, lack of obesity, severe hyperglycemia and de-pressed insulin secretion in this animal model are some of the charac-ter i s t i c s observed in insulin-dependent diabetic humans. In addition, the animals may be particularly useful for the investigation of the macrovascular complications of diabetes, as the rabbit species is one of the best studied models of dietary cholesterol-induced athero-sclerosis. 6.4 Diabetic Cardiomyopathy One of the major problems in attempting to study the nature of cardiovascular complications in diabetes relates to the multiplicity of levels of structural and functional organization at which altera-tions may be investigated. Because i t is clear that chemical, bioche-mical and. functional abnormalities are closely interrelated, and be-cause of the d i f f i c u l t y in comparing results from different species, we have explored some of these diverse aspects of myocardial changes in a diabetic rabbit model in order to gain a better understanding of events leading to experimental diabetic cardiomyopathy. The bioche-mical and functional changes were studied after 10-12 weeks since our preliminary time-course studies revealed that no significant altera-tions were present prior to this time period (145). Because of the limited quantity of tissue available from the a t r i a , the studies were limited to the left ventricles. One of the most distinctive - 161 -differences between the diabetic rabbit and the diabetic rat was the lack of any thyroid hormone depression in the former model. Thyroid hormone depression in diabetic rats has been reported by numerous la-boratories (146). The reason for the absence of any thyroid hormone depression in the diabetic rabbit is not yet understood, but could perhaps be dependent upon the severity and duration of the diabetic state or could be a species-dependent phenomenon. In our study we observed that hyperglycemia alone did not s i g n i f i -cantly affect the body weight of the diabetic rabbits. However, hy-perglycemia in the presence of hyperlipidemia significantly decreased not only the body weight, but also the heart and left ventricular wei-ghts of the diabetic rabbits. Recent studies in our laboratory have shown that diabetic rats treated with hydralazine show a complete ab-sence of hyperlipidemia associated with improved myocardial function (147). * Of the various subcellular organelle-specific ATPases examined, we found a depression only in the myofibrillar and sarcoplasmic reticulum K ,Ca - stimulated ATPase a c t i v i t i e s . The decreased activity of the latter is consistent with the studies of Lopaschuk et al (81) who also showed an impairment in the abi l i t y of cardiac sarcoplasmic re-ticulum to accumulate calcium in streptozotocin-diabetic rats. The inhibition of the sarcoplasmic reticulum ATPase activity in diabetic rat hearts has been related to long-chain acylcarnitines (81). Membrane-bound sarcolemmal ATPase and p-nitrophenylphosphatase were not significantly altered in the diabetic rabbits and the normal - 162 -myocardial contents of water, sodium and potassium lend further sup-port to the idea that, unlike the situation in ischemic injury, sarco-lemmal integrity was largerly preserved. However, a recent study by Regan at al_ (97) in diabetic dogs demonstrated changes in tissue sodium and increases in water content. These alterations have been attributed to an elevation in i n t e r s t i t i a l glycoprotein anionic bind-ing sites rather than to a change in sarcolemmal membrane permeability. We also observed an increase in the hydroxyproline content of dia-betic animal hearts. Such an increase in myocardial hydroxyproline in diabetes presumably indicates increased collagen synthesis. The basis for the collagen increase in the absence of hypertrophy in diabetes has not been well studied. Regan et al (97) suggest that degradation of collagen is decreased, because insoluble collagen was increased in heart muscle, while the acid-soluble fraction declined. It could also be that collagen deposition has occured as the result of tissue in-jury. Whatever the reason, the increase in myocardial "stiffness" in diabetic patients has been ascribed to compositional changes in the fibrous protein (66). Elasticity of normal muscle has been attributed predominantly to extracellular structures, with collagen being of par-ticular significance (148). This fibrous protein is considered to increase prior to the development of ventricular hypertrophy in aortic banded animals and the early increase of myocardial stiffness has been ascribed to this change (149). A qualitatively similar increase of collagen content in the left ventricle of diabetic dogs appears to be - 163 -the basis of the changed compliance (79). Total ion measurements demonstrated calcium accumulation and mag-nesium depletion in left ventricles of diabetic animals. As yet the importance of an alteration in the calcium/magnesium ratio in the left ventricles of diabetic animals has not been definitely established. However, c l i n i c a l data indicate that diabetic patients are more prone to medial arterial calcification which increases with age and duration of diabetes (150). The etiology and pathogenesis of this type of cal-cification in diabetes is as yet unknown. Decreased levels of serum magnesium have until recently not been a well recognized feature of diabetes (151). In diabetic ketoacidosis, gross urinary losses of Mg occur and marked hypomagnesemia is known to develop during chronic insulin therapy (152). Previous studies of magnesium levels in plasma and erythrocytes of diabetic patients have shown either no changes or decreases (153). However, a recent study by McNair et al (152) has shown the presence of hypomagnesemia in insulin-dependent human dia-betics.. Experimental studies by Aikawa (154) on levels of tissue mag-nesium revealed no difference between diabetic and control rabbits. However, this study was a short term (11 day) investigation and our results in alloxan-diabetic rabbits have shown that magnesium deple-tion in red cells and other tissues develops only after several weeks. The cause of diabetic hypomagnesemia is unknown but several factors have been implicated. The osmotic action of hyperglycemia-re-lated glycosuria is known to depress the net tubular reabsorption of 164 Mg in man (155). Studies by Aikawa (154) indicate that renal excre-tion of is increased in diabetes and that the mean exchangeable magnesium content of tissues is decreased. In addition, an increased translocation of Mg to bone may be involved. Conversely, De Leeuw et al (156) have recently reported a significant decrease in trabecular bone magnesium content in i l i a c crest biopsy specimens from juvenile-onset diabetics suggesting that there,may be a decrease in transloca-tion of magnesium to bone. Unfortunately, there are no comparable data on soft tissue magnesium contents in diabetic patients. The relevance of the hypomagnesemia found in this study to tissue and/or total body magnesium status in diabetes is presently unclear. Plasma magnesium comprises only about 0.3%'of total body magnesium and may be an unreliable indicator of tissue magnesium content. Neverthe-less, in normal subjects plasma levels are maintained within narrow limits by sensitive homeostatic mechanisms and the decreased tissue levels found in our study suggest some disturbance of magnesium hand-ling accompanying the diabetic state. It has been proposed that hypo-magnesemia might be a risk factor in the development of diabetic r e t i -nopathy, and a case for magnesium supplements has been put forward as a possible means of reducing the vascular complications of diabetes (157). Studies by Altura et al (158) have indicated that magnesium defi-ciency in rats can produce graded elevations of arterial blood pressure. In agreement with the above study is our recent finding that - 165 -16-week alloxan diabetic rabbits show an elevation of blood pressure in association with hypomagnesemia (unpublished observations). Changes in total activity of lysosomal enzymes and the morphology of cardiac lysosomes have been found to accompany pathological changes in the heart. One of the earliest described biochemical manifesta-tions of tissue damage is a decreased sedimentabi1ity of lysosomal hydrolases (159). It is generally unclear, however, whether these are a cause or a consequence of injury. Such changes, which may be sug-gestive of decreased lysosomal stability either in vivo or in vitro during homogenization, have not been seen in our diabetic rabbits. Similar studies by Chua et al (160) have shown that alterations in cardiac lysosomal enzymes are associated with accelerated rates of proteolysis in working hearts of diabetic rats. These authors believe that the decreased sedimentabi1ity of the lysosomal enzymes may con-tribute to increased protein breakdown and membrane alterations in diabetic cardiomyopathy. Our findings indicating the presence of impaired contractile func-tion in the hearts of diabetic animals parallel the results of studies using isolated perfused hearts (106,146). Frequent abnormalities of left ventricular function attributed to a "preclinical cardiomyopathy" have been detected by hemodynamic and non-invasive methods in diabetic patients without c l i n i c a l evidence of heart disease (161). Histolo-gical and necropsy reports are, however, conflicting as to the presen-ce, extent, and significance of atherosclerosis and diabetic microvas-cular changes of the coronary circulation in the above patients (165). - 166 -The molecular basis of the depression of left ventricular func-tion in experimental diabetes has not yet been definitely established. Work by Lopaschuk et al (81) indicates that elevated levels of long-chain acylcarnitines in the sarcoplasmic reticulum of hearts of diabetic rats may play a role in this regard. The depression of myosin ATPase and the defective utilization of high energy phosphates may also contribute to the depression in contractile function in diabetic hearts (163). Barany (164) has observed a close relationship between ATPase activity and contractile function in skeletal and cardiac muscle, and numerous other studies in diabetic rat hearts suggest a similar parallel relationship between contractile function and myofibrillar ATPase (165,166). Examination of diabetic rabbit hearts using electron microscopy revealed varying degrees of ultrastructural changes. Although myocar-dial ultrastructural changes in diabetic rabbits have not been pre-viously investigated in d e t a i l , data have been reported for chemi-cally-induced diabetes in the rat (167). These latter studies in dia-betic rat hearts demonstrated progressive ultrastructural alterations involving the mitochondria and myofibrils which were only apparent after significant depression in myocardial function. The early phase of ultrastructural changes was characterized by swelling of the mito-chondria, disruption of the myofilaments and disorganization of the mitochondrial cristae network. Since the integrity of the coronary - 167 -vasculature in diabetic animals has not been f u l l y studied, one can-not rule out the fact that the changes may be secondary to coronary obstruction. In our studies, the ultrastructural changes in diabetic hearts were not very specific. Alloxan-induced diabetes was accompanied by mitochondrial alterations, including swelling and inclusions, forma-tion of contraction bands and an increased number of l i p i d droplets. Based on histological studies, Giacomelli and Wiener (168) reported the presence of a primary cardiomyopathy in genetically diabetic mice, even though degenerative changes in the intramural coronary vascula-ture as well as in the perivascular nerve endings were seen to accom-pany the pathological changes in the cardiac muscle c e l l s . Their con-clusion in favor of a primary cardiomyopathy was mainly based on the absence of large coronary artery pathology in their diabetic animals. Similar to our studies, Fischer et al (169) have also reported focal myofibrillar disarrangement with mitochondrial damage and increased glycogen pooling in streptozotocin-diabetic rat hearts. Studies by Regan et al (79) and Haider et al (32,170) have shown that there is increased myocardial collagen and l i p i d deposition in hearts of diabe-t i c dogs and monkeys with no alterations in either the mitochondria or the myofilaments. However, in both these studies, the animals were mildly diabetic as compared to the severe diabetic state of our ani-mals. The heart normally derives most of its energy for contraction from non-esterified fatty acids. The accumulation of triglycerides in left - 168 -ventricles of diabetic animals may reflect lipase inhibition, result-ing in a diminished supply of fatty acid and impaired contractile fun-ction of the diabetic myocardium (171). Scar formation and diffuse fibrosis could also impair left ventricular function. In addition, blood viscosity, platelet adhesion and erythrocyte deformabi1ity are also abnormal in diabetes; these rheological disturbances may reduce local tissue perfusion and further compromise the coronary circulation in vivo (172,173). In addition, the increased concentration of glyco-sylated hemoglobin with its increased affinity for oxygen may contri-bute to a decreased ratio of oxygen availability to demand, or a state of relative hypoxia (173). A spectrum of abnormalities of left ventricular function ranging from normal to severely depressed contractile function exists in dia-betes. Whether these are a result of one disorder with a common etio-logy or of multiple unrelated disorders with differing pathogenesis is at present unknown. Finally the important question of how much myocardial pathology may be the consequence of the metabolic disturbances of diabetes per  se, and how much is secondary to the coronary vascular disease is not known. Recent work in diabetic dogs (79) suggests that mild diabetes can alter myocardial composition and function in the absence of chan-ges in coronary flow and lesions in large or small coronary vessels. Our data also indicate that there is absence of atherosclerosis in the diabetic rabbit, and regional blood flow measurements in the left ven-t r i c l e do not indicate a disturbance in coronary flow. - 169 -In light of these studies, the traditional view that cardiac disease in the human diabetic is essentially the result of coronary artery disease may need to be revised. Our experiments do provide preliminary confirmation for the con-tention that diabetes produces significant myocardial morphological and biochemical damage. Because the diabetic state in these animals is associated with sustained elevations in blood glucose and serum l i p i d s , and since rabbits are highly prone to developing atherosclero-s i s , the present experimental model may prove useful in more closely approximating human insulin-dependent diabetes mellitus and, there-fore, provide a convenient means of investigating the long term eff-ects of diabetes on various physiological systems. 6.5 ISO-Induced Mil This thesis has also described the development of a model of chro-nic Mil induced by ISO in the rabbit, a species exhibiting a high sen-s i t i v i t y to repeatedly administered low doses of ISO, thus mimicking a state of chronically elevated sympathetic tone. As in previous stu-dies on rats (174,175,176,177,178), injections of ISO to rabbits in-duced grossly detectable hemorrhagic infarct-like lesions in the heart. Although the severity of these lesions was not directly quan-titated, studies in rats have demonstrated that the severity of damage increases with the dose of ISO. One major difference between our study in rabbits and the studies previously done with ISO in the rat relates to the frequency of dosing and the total dose of ISO administered. Rabbits are highly sensitive - 170 -to catechol amine-induced arrhythmias and the maximum dose administered subcutaneously on the f i r s t day was 0.5 mg/kg. In addition, the treatment schedule used to produce Mil was 15 days. On the other hand, rats tolerate massive doses of ISO (200-400 mg/kg, administered subcu-taneously) given over only 2-days. Because rabbits are more sensitive to catecholamines, they may be more likely to resemble the human in response to catecholamines and therefore be more appropriate as a model to study stress-induced pathological disorders (179). Measurements in serum indicated significant elevations in blood glucose and creatine phosphokinase activity. The marked increase in blood glucose levels following ISO administration is similar to the development of hyperglycemia or transitory glucose tolerance observed in patients with myocardial infarction (180,181). The effects of ISO on the rabbit heart were extremely pronounced. In agreement with data reported in the literature for rat hearts, we observed decreases in myocardial ATP and in subcellular organelle ATPase activities (182,183,184). The decrease in ATP may, in part, be related to calcium influx. The increased intracellular calcium accu-mulation has been suggested to increase ATP consumption by stimulating myofibrillar and sarcolemmal ATPase a c t i v i t i e s , thereby enhancing the hydrolysis of high energy phosphates (185). In addition, the intra-cellular calcium overload could result in excessive mitochondrial cal-cium accumulation which would lead to a decreased ATP production due to impairment of oxidative phosphorylation (186). Recent data from our laboratory indicate that mitochondria isolated from hearts - 171 -following ligation and reperfusion of the coronary artery (a condition where, calcium influx in the heart is significantly increased) show a greatly diminished abil i t y to generate ATP (unpublished observations). In addition to decreased ATP levels, we also observed an ISO-in-duced decrease in cardiac glycogen. The depletion of glycogen is likely the result of glycogenolysis stimulation by ISO-induced activa-tion of phosphorylase (187). Depletion of glycogen has also been shown to occur after coronary ligation in animals and in conditions of fulminating systemic anoxia (188). The electrolyte changes encountered in ISO-induced Mil parallel those reported by Lehr et al (189) who also observed increases in water content, sodium and calcium in rats treated with ISO. According to Rona et al (190), increased sarcolemmal membrane permeability is an important early event in catecholamine-induced Mil. These authors demonstrated the presence of the macromolecular tracer, horseradish peroxidase, within damaged cardiac cells of rats after ISO administra-tion. This increased permeability, together with inhibition of the various ATPases, may lead to the excessive accumulation of sodium and calcium. Numerous studies have shown a correlation between the inhi-bition of various ATPases and damage to the myocardium in different models of ischemic injury (115,116,184). ISO also induced a significant increase in the heart cell mass. It is known that ISO (in high doses) will induce cardiac hypertrophy with massive tissue lesions (174,175,191). The rapid growth of the - 172 -heart must ultimately represent an imbalance between protein synthesis and degradation. Studies by Taylor and Pang (192) have shown that ISO-induced hypertrophy in rats is associated with an increase in ven-tricular RNA content, suggesting an increase in protein synthesis. In addition, we have also found an increase in hydroxyproline in the ISO-treated animals, an observation previously made in the rat by many investigators (175,180,192). The mechanism for the enhanced collagen in ISO-induced Mil has been related to hypertrophy in the early phase and to scarring/repair during the later phase of the injury. Measurements of cardiac lysosomal enzyme latency indicated an in-crease in the non-sedimentable activity of cathepsin D and N-acetyl e-glucosaminidase. Such changes have also been shown to accompany Mi l , although their role in such injury is unclear. Such changes are conventionally viewed as reflecting either increased f r a g i l i t y of ly-sospmes (as a result of increased size of the organelles and/or reduced integrity of the membrane structure) during in vitro homogeni-zation or actual translocation (leakage) of enzyme in vivo from damag-ed lysosomes into the cytosol or both. Only in the latter case would lysosomal hydrolases become more accessible to endogenous substrates. In line with earlier suggestions by DeDuve and Beaufay regarding hep-atic ischemia (159), i t has been suggested that lysosomal alterations of this type could play an important role in producing myocardial nec-rosis. Thus, according to the "lysosomal hypothesis", an ischemic insult might lead to labilization of lysosomes with activation of their hydrolases, which in turn might lead to abnormal degradation - 173 -of vital cellular components and thus, death of the c e l l . However, there are several problems in evaluating the possible importance of changes in lysosomal l a b i l i t y during ischemia after ISO-treatment. Not the least of these is the di f f i c u l t y in specifying whether lysosomal alterations precede cell death. If such is the case they might play a role in causing the necrosis. However, i f irreversible damage occurs f i r s t , the observed lysosomal changes would be secondary consequences of the events relating to necrosis. Which of the many metabolic events that accompany ISO-induced Mil might produce lysosomal membrane alterations remains a crucial question, as does the problem of determining whether the observed lysosomal alterations are a cause or a consequence of ISO-induced myocardial damage (193). The ultrastructural and functional alterations occuring in ISO-induced Mil are different in some aspects from those resulting from coronary artery ligation. The ultrastructural and functional altera-tions in the ISO-model were slow in onset as were previously described chemical changes, such as ATP and glycogen depletion (194). The ul-trastructural changes seen in the occlusion model are characterized by severe disruption of myofibrils, oedema, loss of glycogen and dilata-tion of the endoplasmic reticulum (115,195,196). Qualitatively similar changes were seen in the ISO-model, but the magnitude of these changes is much less. In addition in the latter model, li p i d droplets are observed throughout the myocardium. One distinct change in the ISO-model is the relative lack of electron-dense deposits in the - 174 -mitochondria. These precipitates are prominent after coronary artery ligation and reperfusion. In the ISO-induced injury, the rapid removal of necrotic debris and the repair processes which occur within days after the injury are probably facilitated by the patency of coronary arteries, whereas this process may take weeks following infarction produced by coronary artery ligation. The functional and ECG changes in the ISO-model are somewhat simi-lar to those found following coronary occlusion. After coronary artery ligation, a small drop in blood pressure is observed which is sometimes followed by an increase in heart rate. These changes are seen almost immediately upon occlusion. In the ISO-model, the blood pressure drop and heart rate increase are significantly marked. Again, the development of ST-segment elevation in the ISO-model is seen much later compared to the occlusion model, presumably reflecting the slower onset of myocardial cellular necrosis (197,198). Our overall observations are consistent with findings reported by others (199,200) on ultrastructural and functional changes of cardiac muscle cells in rats treated with ISO. The major difference observed is in the rate of development of lesions. In rabbits, the earliest detectable ultrastructural changes are seen only after 5-7 days of treatment with ISO. However, the ultrastructural alterations in mito-chondria, myofibrils, glycogen granules and sarcoplasmic reticulum in rats and rabbits appear to be qualitatively very similar. Our data on glycogen granule depletion agree closely with results - 175 -obtained in our biochemical study which also indicated a decreased glycogen content of hearts from ISO-treated animals (194). Swelling of the endoplasmic reticulum was also apparent after ISO treatment. The swelling of this system apparently reflects the influx of a large amount of extracellular f l u i d into myocardial cells as the result of altered sarcolemmal membrane permeability (190). In our biochemical study, we also showed that the sarcolemmal Na+,K+-ATPase was inhi-bited in rabbits treated with ISO. The decreased activity of this enzyme is another indication that ISO treatment leads to alteration in sarcolemmal membrane integrity and thus the associated abnormalities in cellular water and electrolyte content resembling those associated with acute coronary artery ligation (195,201). Changes in the fine structure of mitochondria after ISO-adminis-tration were particularly prominent. This was not surprising because mitochondria are known to be extremely sensitive to hypoxia (201). The presence of a few electron-dense particles in the mitochondria of damaged cardiac muscle cells remains an unexplained phenomenon. The morphological changes appear similar to the large dense precipitates observed by Bloom and Cancilla (202) and thought to represent deposits of calcium and possibly magnesium. We have shown that mitochondria isolated from ISO-treated animals have greatly increased levels of calcium as compared with controls (194). We have also observed l i p i d droplets in the ISO-treated rabbit hearts. Lipid accumulation in the myocardium as a result of hypoxia has been reported in patients with angina pectoris, diphtheria and - 176 -particularly during overdoses of catecholamines (203,204). Lipid mo-bilization by catecholamines depends on lipolysis of depot t r i g l y c e r i -des, with a resulting increase in serum free fatty acids. The exact biochemical mechanisms whereby plasma lipids are taken up into the injured myocardium are as yet unknown. To date, the exact pathological mechanism determining myocardial cellular injury following ISO treatment remains unsolved. The basic histological lesion is necrosis of the myocardial fibers and inter s t i -t i a l structures. The electron microscopic studies of Korb (205) and Ferrans et al (206) in rats would seem to argue against the involve-ment of direct coronary obstruction in the effects of ISO treatment. ISO has a cardiostimulatory effect that greatly increases the oxygen requirements of the heart. This cardiostimulatory effect, as manifes-ted, for example, in the increased heart rate, was quite pronounced in our studies. In addition, ISO decreased the systemic blood pressure, presumably as the result of peripheral vasodilatation. This could result in a temporary relative ischemia of the myofibers as the result of decreased coronary perfusion pressure. Rona et al (177,178) from their studies in the rat suggest that ISO-induced Mil is a true ische-mic infarct. Its pathogenesis, they suggest, is related to a vasodi-lating effect of ISO, resulting in dilatation of pre-existing by-pass channels between coronary arteries and veins. This, in turn, would lead to a drop in intramyocardial capillary pressure below that neces-sary for adequate tissue perfusion. However, our studies on blood flow measurements in the left ventricles of control and ISO-treated - 177 -animals revealed no difference in the regional coronary blood flow, suggesting that the necrosis induced reflects biochemical and chemical alterations affecting the metabolism of myocardial c e l l s . At this point, i t is relevant to compare occlusive and non-occlu-sive (or metabolic) models of experimental MIL It has been observed by us (115,194) that the cardiac necrosis provoked by occlusion or by injection of ISO differ from each other not only in rate of develop-ment but also in the magnitude of the biochemical alterations. The rate of development of M i l with ISO is a process occuring over several days whereas the development of M i l after occlusion develops within minutes to hours. One characteristic difference between the occlusion and ISO-induced M i l is that the former has a distinct occluded zone and the latter is a diffuse form of necrosis involving not only the left but also the right ventricle. When the ISO model of M i l is compared to the acute coronary artery ligation model, qualitatively similar biochemical changes are observ-ed, but their magnitude is greater in the latter model. After 40-min-utes of ligation and 60-minutes of reperfusion of the left circumflex coronary artery in the rabbit, an accumulation of lactate, decreases in ATP (60%) and glycogen (83%) are observed. Although decreases are also seen following ISO-treatment, their magnitude is much less. Me-asurements of subcellular organelle enzyme markers are indicative of drastic decreases in the mitochondrial (55%), sarcoplasmic reticulum (33%) and sarcolemmal ATPase (35%) activities in the occlusion model ( - 178 -after 40 minutes ligation. Qualitatively similar changes are also present in the ISO-induced model, but the decreases are more gradual and dependent on the duration of the ISO treatment. It should be noted that the accumulation of calcium which was so marked in the ISO-model was not seen in the occlusion model unless reperfusion was instituted following ischemia (115). Other changes seen in the ISO-model but not following coronary ligation were hypertrophy of the myocardium, collagen deposition and increases in blood glucose and serum free fatty acids. The experimental observations presented in this study are compati-ble with the concept that hypoxia plays a role in the pathogenesis of ISO induced Mil. Baroldi et al (207) have shown that the ISO-induced Mil is morphologically similar to that of coagulative myocytolysis described in human myocardial infarction, suggesting that the ISO-model may be relevant to some types of c l i n i c a l cardiomyopathy. In addition, unlike the acute "open-chest" models, this model allows the long term follow-up of drug interventions. 6.6 ISO-Induced Mil in Alloxan Diabetic Rabbits It is well known that diabetics are more prone than is the general population to cardiovascular disease (2,4,5,6), and in particular to ischemic heart disease and congestive heart failure (10,11). A rela-tionship between diabetes and early onset of atherosclerotic heart disease is easily demonstrable epidemiologically, c l i n i c a l l y and at autopsy (8,12,15). However, the cardiovascular complications of dia-betes may not be referable only to atherosclerosis. The neurogenic 179 and humoral factors that may adversely affect the cardiovascular sys-tem in diabetic patients are frequently overlooked. Catecholamines may play some as yet poorly undefined role in this regard. In this study, the possible adverse effects of the potent catecholamine, ISO, were examined in the al loxan-diabetic rabbit, a species which we have shown to exhibit a high sensitivity to repeatedly administered low doses of ISO (mimicking a state of elevated sympathetic tone). The biochemical and ultrastructural changes resulting from the treatment of rabbits with alloxan or ISO individually have already been charac-terized previously in this thesis (145,194,208,209). Treatment of control and diabetic animals with ISO resulted in a number of sim i l a r i t i e s , including comparable incidence of arrhythmias, percent increase in oedema of the left ventricles, decreases in mito-chondrial and sarcolemmal ATPase ac t i v i t i e s , ATP depletion, hemodyna-mic changes ana lysosomal enzyme alterations. However, the diabetic animals showed a number of distinct changes relative to controls which included greater glycogen depletion and calcium accumulation as well as a somewhat greater decrease in sarcoplasmic reticulum ATPase acti-v ity. Previous work with ^calcium has shown that a number of cardio-myopathies lacking coronary involvement result from an overload of calcium (185). This applies particularly to cardiac lesions caused by large doses of sympathomimetic amines, including ISO. In our study, calcium accumulation was particularly prominent in the ISO-treated diabetic animal hearts. Such an uncontrolled rise in calcium is - 180 -known to activate a number of processes, including activation of calcium-stimulated myofibrillar and sarcolemmal ATPase ac t i v i t i e s , thus enhancing hydrolysis of ATP and accentuating the ischemia-induced decrease in myocardial ATP content (185). Why the myocardial calcium levels are more pronounced in diabetic hearts is not really known but may result from an impairment in the mechanisms responsible for pumping calcium out of the myocardium aga-inst a very large electrochemical gradient and/or from damage to the membrane system that normally limits calcium entry into the cell (210). Alternatively, depletion of tissue ATP stores might result in a localized accumulation of calcium at the inner surface of the cell membrane (112). This localized accumulation of calcium could cause an 2+ activation of the Ca -activated ATPase located at the inner surface of the sarcolemma leading to a further depletion of the tissue stores of ATP (112,210). Another reason why calcium influx is more pronounc-ed in diabetic hearts, compared to control hearts, could be related to magnesium depletion. We have found that alloxan-diabetic rabbits show significant magnesium depletion in the right ventricles, aorta, red blood cells and pancreas (211). Magnesium depletion has been suggest-ed to be an important factor in the etiology of ischemic heart disease (212). In support of this association are reports of reduced serum magnesium in patients with chronic heart disease and myocardial infar-ction (213). Reduced myocardial magnesium has also been found in isc-hemic hearts at autopsy (213). A number of investigators have shown that a deficiency of magnesium in the diet can lead to both myocardial - 181 -necrosis and atherogenesis (213). It could be that during magnesium deficiency, the calcium influx would be f a c i l i t a t e d , thereby leading to an activation of various calcium-dependent energy consuming reactions. However, to what extent this magnesium depletion is responsible for initiating or promoting ISO-induced Mil in diabetes is at present unknown. Considering the fact that low magnesium levels have been reported in sera and erythrocytes of diabetic patients (155), i t would not be surprising to see enhanced myocardial necrosis during elevated sympathetic activity. • Another prominent alteration in our study was the depression of cardiac sarcoplasmic reticulum ATPase activity in the ISO-treated dia-betic hearts. Decreased sarcoplasmic reticulum ATPase activity has been observed in a number of physiological and pathological states where contractile activity and relaxation are impaired. These include myocardial failure due to pressure overload, diabetes and cardiomyo-pathy due to hypothyroidism (81,166). The reason for the greater de-pression of sarcoplasmic reticulum ATPase activity in the ISO-treated diabetic animal hearts is not really known but has been linked to the accumulation of long-chain acyl carnitines/CoA. It has been demons-trated that long-chain acyl CoA accumulation does occur in diabetic rats and during ischemia (90). Whether or not these acyl compounds have a detrimental effect on cellular function and contribute to cell death is not known for certain, but there is a good possibility that they do so when present in high concentrations. These compounds - 182 -have been shown to affect several enzymatic reactions and they are powerful detergents. These compounds also inhibit several enzymatic processes, including adenine nucleotide translocase, acyl CoA synthetase and triglyceride lipase (214). In addition, these compounds have also been shown to inhibit cellular functions such as the sarcolemmal and sarcoplasmic reticulum ATPase activities and mito-chondrial respiration (215). It is also possible that the detergent action of the accumulated fatty acids can disrupt membrane structure and intefere with normal function (214). This may be particularly true for mitochondrial membranes since 95% of total CoA present in heart cells is located in the mitochondrial membranes. We have found that mitochondria isolated from diabetic hearts show a reduced ability to generate ATP and in addition, the morphology of the mitochondria is somewhat abnormal (145,216). Unlike our study in diabetic rabbits, Fein et al (217) did not show any greater damage in the ISO-treated diabetic rats compared to controls. Similarly, Gotsche (218) failed to demonstrate any s i g n i f i -cant difference in the ultrastructural or ECG changes after ISO treat-ment of 7-day streptozotocin diabetic rats. In the studies by Gotsche, i t is the possibile that B-receptor desensitization accounts for the lack of ISO-toxicity in diabetic rats and that certain strains of rats are resistant to ISO (218). 6.7 Coronary Artery Ligation and Reperfusion in Control and  Diabetic Rabbits Since direct coronary revascularization has become c l i n i c a l l y - 183 -feasible, there has been interest in the application of this approach to the treatment of acute myocardial infarction in both diabetic and non-diabetic humans (104,105,219). On f i r s t consideration, this would seem to be a logical approach. As a consequence of the heterogeneity in the degree of damage following coronary occlusion, some tissue is irreversibly damaged while other tissue is i n i t i a l l y reversibly injur-ed. Therefore, restoration, of blood flow by surgically bypassing the occluded or severely stenotic coronary artery should theoretically diminish the extent of necrosis. One of the recommended experimental interventions aimed at salvaging the jeopardized myocardium after cor-onary occlusion is reperfusion of the affected zone. Current contro-versy centers on the adviseabi1ity of, and c r i t e r i a for, reperfusion in c l i n i c a l acute myocardial infarction (219,220). The success of such an intervention is c r i t i c a l l y related to an understanding of the phy-siological mechanism(s) that influence myocardial v i a b i l i t y both dur-ing periods of ischemia and subsequent restoration of blood flow. C l i n i c a l l y , i t seemed likely that revascularization of the acutly de-veloping infarct by emergency application of coronary vein graft might abort the infarct or at least limit infarct size (219). However, most of the current evidence indicates that reperfusion following coronary artery occlusion alters the metabolism of the ischemic myocardium and may even hasten necrosis (221). Due to the greater prevalence and severity of coronary artery disease in diabetics (11,12,15), i t seems likely that the incidence of reperfusion-related damage may be more common in these patients. Thus, our study was undertaken to - 184 -investigate the consequences of reperfusion in alloxan diabetic rabbits and to compare the results with those previously obtained under the same conditions in non-diabetic animals (115). Compared to the control myocardium, diabetic hearts undergoing reperfusion showed a number of distinct biochemical, ultrastructural and hemodynamic alterations. A striking abnormality was the s i g n i f i -cant increase in the calcium content of the reperfused diabetic myo-cardium as compared to the control. The increase in calcium has been reported previously in non-diabetic animals (115,221) and probably results from the inability of the cell to remove calcium out of the 2+ cell because of a defect in the Ca -dependent ATPase of the sarco-lemmal membrane (222). The role of this calcium overload in the development of Mil during reperfusion is s t i l l uncertain. Massive calcium overload is observed in myocytes injured during ischemia and then reperfused with arterial blood (220,221). In addition, calcium overload is also a feature of catecholamine-cardiotoxicity (194) and the calcium-paradox phenomenon (223). In each of these instances, calcium enters the c e l l , through as yet unidentified mechanism(s), where i t is extensively accumulated by mitochondria (223). These observations have led to the speculation that calcium overload per se may be a lethal event in ischemic injury (222,223,224). In addition, other workers have also shown that mito-chondrial function is reduced by reperfusion after transient ischemia and the impaired function was correlated with an increase in - 185 -intramitochondrial calcium content (225). The influx of calcium may contribute to terminal depletion of ATP stores by the activation of various calcium-dependent ATPases. The breakdown of ATP is accompanied by a release of H+. However, the extent of cytoplasmic acidification will depend upon the buffering capacity of the c e l l . A sudden and large accumulation of H+ would, however, precipitate cell damage almost as an explosive event because certain phospholipases and proteases in the cytoplasm and lysosmes are 2+ + both Ca - and H -dependent. There are, of course, many other + effects of a rapid rise in H , including a slowed rate of inactiva-tion of sodium channels, thereby resulting in conduction abnormalities (80,214). The influx of calcium has also been shown to activate va-rious phospholipases in ischemic hearts resulting in cellular accumu-lation and release of lysophosphatidyl compounds (226). These com-pounds may themselves cause alterations in action potential duration and cause development of arrhythmias when added to isolated perfused rat hearts (226). Our observation that mitochondria obtained from diabetic animal hearts have a diminished a b i l i t y to generate ATP is in agreement with the recent work by Pierce and Dhalla (216). These workers have shown that mitochondria from non-ischemic diabetic rat hearts are functio-nally defective. It is not really known why the mitochondria are damaged and have an impaired function in the diabetic animals. Reasons for the depressed mitochondrial function, besides elevated long-chain acylcarnitines, could be one or more of several general - 186 -possibi1ites: 1) inhibition or destruction of mitochondrial creatine phosphokinase, 2) and damage to the structural integrity of the mito-chondria-which is confirmed from our ultrastructural studies (145). With regard to alterations in subcellular membrane systems, diabe-t i c hearts showed significantly greater inhibition of the sarcolemmal ATPase activity compared to controls following ischemia and reperfu-sion. Both the sarcolemmal ATPase and the functionally related K+-p-nitrophenylphosphatase were affected to the same degree, sug-gesting an involvement of the whole enzyme complex rather than an in-terference in one component of its function. Recent work by Wood et al (227) suggests that an ischemia-induced decrease in enzyme activity in vivo might be the result of elevated intermediate products of fatty acid metabolism, specifically long-chain acyl CoA has been shown to occur during the f i r s t 20 minutes of ischemia. In view of the fact that diabetic animals have been shown to have significantly elevated levels of myocardial long-chain acyl CoA, i t could be that the further generation of these compounds during ischemia may greatly intensify their inhibitory actions on the various ATPases and mitochondrial fun-ction. We have demonstrated that following ischemia and reperfusion there was an associated depression of the sarcoplasmic reticulum ATPase acti-vity, the depression being greater in the diabetic animals. This is consistent with our electron microscopic studies which indicate swel-ling and distortion of the sarcoplasmic reticulum. With the disrup-tion of the sarcoplasmic reticulum, there is less calcium - 187 -sequestered during diastole which results in a net loss of available calcium to be released by the next action potential. This results in a net decrease in the calcium binding to troponin which results in a decrease in tension development. Further, with a depression of the calcium pump, there is a slower rate of calcium reaccumulation, and in physiologic systems this would be expressed as a decrease in the rate of relaxation, a finding well documented in diabetic animals (81). In association with the inhibition of the sarcolemmal ATPase acti-vity in both diabetic and non-diabetic animals, gross alterations are also observed in the Na/K ratio and total water content of the cells during reperfusion, suggesting that there is a generalized disruption of the sarcolemmal membrane integrity. Such alterations of intracel-lular Na and decreased K are commonly seen in the f a i l i n g heart (222). This is also supported by observations that the sarcolemmal ATPase activity is depressed in heart failure in the cardiomyopathic hamster (222,228). Our results show that the severity of ultrastructural changes in control and diabetic myocardial cells increased following reperfu-sion. In both groups of animals increasing periods of ischemia were characterized by glycogen depletion, and the appearance of amorphous electron dense bodies as well as sarcolemmal breaks and chromatin clu-mping. The final and probably irreversible mitochondrial changes were vacuolization, formation of myelin figures, disappearance of mitochon-drial cristae and rupture of the outer membrane. Another characteris-t i c feature of irreversible mitochondrial damage was the - 188 -appearance of large amorphous matrix densities, which probably consist of denatured protein and/or calcium l i p i d complexes. Changes in pH caused by intracellular acidosis are reported to cause permeability changes of the cellular and mitochondrial membranes. Armiger et al (229) described the occurence of mitochondrial densities after 30-90 minutes of incubation of normal dog myocardium in lactate solution. Their results suggest that lactate at physiologic pH may produce the swelling, loss of density of the matrix and disorganization of the cristae. The loss of permeability in the membranes may cause the loss of essential factors which eventually results in the ability of the mitochondria to reconstitute cristae structure and function. The ap-pearance of nuclei is also altered by ischemia. Swelling of the nuc-leus disappears because of nuclear pyknosis, an alteration which is also believed to be irreversible. Irreversible nuclear damage is most probably incompatible with the survival of the cell because re-pair processes requiring protein synthesis are d i f f i c u l t to imagine with a damaged nucleus. After reperfusion, the cell swelling was more marked and hypercontraction bands, increased vacuolization, dense gra-nular bodies in the mitochondria and intermyofibriliar odema were also evident. The increase of intermyofibriliar space with prolonged isch-emia and reperfusion may reflect the increase in intracellular water which is detectable by gravimetric analysis. On the other hand, such myofibrillar separation may also result from the mechanical stress of surrounding viable, contracting myocardium acting on these irreversib-ly injured c e l l s . This phenomenon of explosive cell swelling has - 189 -frequently been observed during the reperfusion of ischemic tissues and reflects irreversible sarcolemmal alterations which no longer allow cells to maintain volume homeostasis (221). Studies by Moore and Godin (115) have shown that non-diabetic rab-bits also exhibit similar ultrastructural damage of the left ventricle following coronary artery ligation and reperfusion. The increase in hypercontraction bands in diabetic animals are identical with the so called "hyaline Querbander" observed long ago by light microscopists in ischemic myocardium (230). Since sarcomeres of normal cells con-tract during fixation with glutaraldehyde, we feel that the excess hypercontraction bands may be an a r t i f a c t , but the extent of the a r t i -fact is impossible to define. Hypercontraction may not be present in native myocardium but occurs upon contact with the fixative. The hig-her incidence of contracture bands may be due to an increased suscep-t i b i l i t y of diabetic cardiac muscle cells to develop this artifact under conditions of ischemia and subsequent reperfusion. This may reflect alterations of properties of the whole cell or single cell compartments occuring under these conditions, e.g. changes of the met-abolic status of the c e l l , permeability of membranes or functions of enzymes of the various ATPases, a l l which have been shown to be alter-ed in the ischemic diabetic myocardium (194,209,231,232). With regard to hemodynamics, a decline in the left ventricular and arterial pressure was seen after ligation and reperfusion. It appears that the severity of tissue damage was related to depressed - 190 -hemodynamic function, i e , tissue damage paralleled the decline in left ventricular and arterial pressures. Haider et al (170) have recently shown that occlusion of the left anterior descending coronary artery in diabetic dogs produced a greater impairment of left ventricular function in comparison to controls. In addition, the pressure-volume relationship of the left ventricle during ischemia indicated a signi-ficantly higher ratio of end-diastolic pressure to end-diastolic volume in diabetic dogs, a finding which is consistent with an inter-pretation of reduced compliance compared to the non-diabetic dogs. These authors suggest that the altered response of diabetic dogs to ischemia may be related to changes in energy production and u t i l i z a -tion. Furthermore, the accumulation of PAS-positive glycoprotein and collagen could also contribute to the enhanced diastolic stiffness manifested as a larger rise of end diastolic pressure in relation to end-diastolic volume. Our results also indicate that coronary artery ligation and re-perfusion in the diabetic rabbit is associated with an increased i n c i -dence of ventricular f i b r i l l a t i o n . The reason(s) for the increased incidence of electrical instability of diabetic hearts is not known but the altered Ca/Mg ratio could be one important factor. Magnesium loss during ischemia and a sustained magnesium deficit during reperfu-sion may contribute to the worsening of tissue injury and the develop-ment of arrhythmias during reperfusion. In the context of surgically-induced global ischemia, magnesium supplementation has been found to provide substantial tissue protection and i t is speculated that - 191 -part of the protection by magnesium might involve the suppression of reperfusion arrhythmias (233). Characteristic changes in the QRS complex and the ST-segment occ-ured following occlusion and reperfusion in control and diabetic ani-mals. Whereas ST-segment elevations and increases in R-wave amplitude are ECG expressions of myocardial ischemic injury, and are reversible when coronary occlusion is released during the early phase of ische-mia: development of Q-waves, on the other hand, are the electrocardio-graphic expressions of irreversible injury (234). The depth of Q-waves on the epicardial surface of hearts after coronary artery ligation have been found to be closely related to the depletion of creatine phosphokinase activity and to the degree of histologic damage in the myocardium (235). The sequence of events described in our experiments in both con-trol and diabetic animal hearts, is similar to that described by Cooley et al (236) in the stone heart syndrome, which occasionally develops in patients placed on cardiopulmonary-bypass with normother-mic ischemic arrest and may account for the paradox of necrosis in areas of revascularization (reperfusion) after bypass surgery. Whether or not reperfusion is beneficial or deterimental to the ischemic myocardium would appear to depend upon the severity and na-ture of the ischemic damage and the extent to which cellular and sub-cellular membrane systems can withstand the associated sudden ion f l u -xes. Currently, i t is only possible to state that the damage which 192 occurs between 20-40 minutes after the onset of severe ischemia might not be reversed by reperfusion and as the duration of ischemia increa-ses the chance of recovery is reduced and that of reperfusion damage is increased. Under certain conditions, such as marginally reduced flow or hypothermia, i t is of course possible for ischemic cells to survive for many hours. In relation to time and injury, i t is perhaps important to consi-der the possibility that reperfusion damage may not be a true exten-sion of damage but a compression or acceleration of that which would otherwise have occured. The possibility becomes particularly relevant to ischemic injury i f reperfusion damage occurs only after, or c o n f i -dent with the onset of irreversible damage. Our study leads to the conclusion that reperfusion in both the control and diabetic animals may not be capable of reversing hemodyna-mics, biochemical or ultrastructural alterations induced by coronary artery ligation, but the consequences of reperfusion in the diabetic animal are much more severe than those observed in the control animals. 6.8 Coronary Artery Ligation in Diabetic Rats Our experiment on coronary artery ligation in diabetic rats pro-duced dramatic results. Our studies show that diabetic rats were more prone to post-1igation arrhythmias (especially ventricular f i b r i l l a -tion) from which mortality rate was 100%. Similarly Feuvray et al (90) studied the effects of ischemia on the isolated diabetic rat heart and showed that ischemia resulted in a faster rate of 193 ventricular failure in diabetic hearts which was also associated with a rapid rise in tissue long-chain acyl CoA and acylcarnitine esters. The exact reason(s) as to why ventricular f i b r i l l a t i o n is greater in diabetic animals is not really known but a number of hypotheses have been put forward. In seeking to define mechanisms underlying the genesis of serious ventricular arrhythmias (particularly ventricular f i b r i l l a t i o n ) during both ischemia and reperfusion, i t is probably f a i r to state that the establishment of reentry circuits and possibly enhanced automaticity are the ultimate electrophysiologic aberrations responsible for the manifestations of the arrhythmia. It is equally well established that regional heterogeneity of tissue injury or recovery is probably a c r i -tical determinant of the establishment of reentry c i r c u i t s . What re-mains to be established is which of the many complex molecular changes occuring during ischemia is c r i t i c a l to the initiation of electrophy-siological i n s t a b i l i t y . In recent years, many processes have been suggested as potential culprits. These include unfavorable r e d i s t r i -bution and accumulation of ions (particularly calcium, magnesium and potassium), release of catecholamines, elevated c/WP, changes in avai-l a b i l i t y of ATP, accumulation and utilization of fatty acids and a c t i -vity of membrane lysophosphatidic acids (237). The higher incidence of arrhythmias in diabetic rats could be due to either a l l or none of the above or due to magnesium depletion. Recent observations indicate that the incidence of ventricular f i b r i l -lation in the isolated rat heart was directly related to the 194 magnesium concentration in the perfusing medium and that f i b r i l l a t i o n could be totally eliminated by increasing the magnesium concentration of the perfusate to 4-8 nM, even when this was increased only 3 minu-tes prior to perfusion (233). The exact mechanism by which increased magnesium decreases disturbances in rhythm is not known. However, magnesium is known to exert a number of important biological effects. It can induce vasodilation, decrease heart rate and, in some species such as the rat, alter transmembrane calcium fluxes by displacing cal-cium from low affinity extracellular binding sites (238). Each of these affects might result in a reduction of vulnerability to arrhyth-mias. Finally magnesium bound to ATP is a prerequisite for energy transfer reactions. The provision of magnesium during reperfusion may act to compensate for its loss during ischemia and so improve the ene-rgy utilization status of the heart during the c r i t i c a l early moments of reperfusion, another factor which may contribute to the electrical stabilization of the heart (238). 6.9 Summary and Conclusion 1) Injection of alloxan monohydrate to rabbits led to the development of a diabetic state characterized by polyuria, polydipsia, glycosuria and hyperglycemia. A stable non-ketotic form of diabetes was achieved and the growth rate of the animals was near normal over the 10-week study period. 2) Examination of hearts from 10-week diabetic rabbits, revealed signi-ficant glycogen, hydroxyproline and cholesterol accumulation. In add-it i o n , significant decreases in myocardial function, magnesium - 195 -content and sarcoplasmic reticulum ATPase activity were observed in the diabetic animals. Ultrastructural alterations were most evident after 10-weeks of diabetes. 3) Myocardial ischemic injury produced by injection of isoproterenol to the rabbit was associated with depletion of ATP and glycogen and increases in Ca, Na and hydroxyproline. In addition to ultrastructu-ral alterations, significant depression of sarcolemmal, mitochondrial and sarcoplasmic reticulum ATPases activities were observed. 4) Injection of isoproterenol to diabetic rabbits produced significan-tly greater glycogen depletion, depression of the sarcoplasmic reticu-lum ATPase activity and increased Ca accumulation compared to isopro-terenol treated non-diabetic animals. 5) Coronary artery ligation for 40 minutes in control animals produced depression of the subcellular organelle enzyme ATPase activities and ATP levels with no significant changes in hemodynamics. 6) Reperfusion of the coronary artery following ligation in control animals resulted in a significant accumulation of calcium and sodium, a further depression of sarcolemmal, and sarcoplasmic reticulum ATPase activities and ATP content. In addition, reperfusion was associated with a further worsening of the hemodynamics and increased incidence of arrhythmias. 7) Coronary artery ligation and reperfusion in diabetic rabbits resul-ted in a significantly greater Ca accumulation, depressed mitochon-drial ATP generation, depressed subcellular organelle ATPase a c t i v i -ties and severe ultrastructural alterations compared to non-diabetic animals. In addition, diabetic rabbits had a higher incidence of - 196 -ventricular f i b r i l l a t i o n and cardiogenic shock during reperfusion com-pared to non-diabetic animals. 8) Coronary artery ligation in 6 and 12 week diabetic rats resulted in a higher incidence of ventricular f i b r i l l a t i o n and mortality compared to non-diabetic rats. Both 6 and 12-week diabetic rat hearts showed significant magnesium depletion. In conclusion, we are arguing for a very central role for altera-tions in the calcium/magnesium ratio in the pathogenesis of ischemic heart damage in experimental diabetes. The alteration in this ion ratio may not only be mediating the biochemical alterations that are responsible for ischemic injury, but may also worsen the functional pi-consequences of this injury. The influx of Ca ions prevents res-toration of mitochondrial function lost, in part, as a result of a change in the inner membrane initiated again by calcium ions potentia-ted by long-chain acyl CoA-esters. The excessive influx of calcium may also be responsible for the depletion of high energy phosphates (ATP) by activating the various ATPases. The depletion of magnesium in diabetes may be responsible not only for the initiation/progression of ischemic injury but may also account for the higher incidence of elec-t r i c a l disturbances observed in the diabetic animals. The present study has provided evidence for an increased vulnera-b i l i t y of the diabetic myocardium to ventricular arrhythmias following myocardial ischemic injury. Our data implicate abnormalities in the handling and/or activity of intracellular calcium as one possible fac-tor determining the greater risk and severity of complications from ischemic injury in the diabetic complication. Although the extent to - 197 -which our findings in experimental diabetes are applicable to the c l i -nical situation is d i f f i c u l t to assess at the present time, our work does suggest possible pharmacological approaches (most obviously, the use of calcium channel blockers and/or magnesium supplementation) to be explored in the ultimate search for means to reduce the serious and frequently fatal cardiovascular complications of chronic diabetes. These conclusions are clearly based on normal cellular physiology and the expected alterations in these functions that result from isch-emia. 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Lange Medical Publications. Los Altos. 235. Shell, W.E., Sobel, B.E. (1976) Biochemical markers of ischemic injury. Circulation. Supp 53: 98-106. 236. Cooley, D.A., Reul, G.L., Wukash, D.C. (1962) Ischemic contractures of the heart: "stone heart". Am J Cardiol. 29:575. 237. Opie, L.H., Nathan, D., Lubbe, W.F. (1979) Biochemical aspects of arrhythmogenesis and ventricular f i b r i l l a t i o n . Am J  Cardiol. 43:131-148. 238. Manning, A.S., Hearse, D.J. (1984) Reperfusion-induced arrhythmias: mechanisms and prevention. J Molec Cell Cardiol. 16:497-518. - 217 -Addendum In this thesis we have suggested the role of relative ischemia in the pathogenesis of isoproterenol-induced myocardial necrosis brought about by the interaction of direct myocardial stimulation and depres-sor effect on coronary circulation. Despite the fact that biochemical and chemical changes in ISO-induced myocardial necrosis resemble those following coronary artery ligation and reperfusion, recent studies indicate that ISO has a multifactorial pathogenesis and may not be exclusively due to hypoxia. Among the contributory pathogenetic fac-tors suspected of playing a role in ISO-induced myocardial injury are coronary microcirculatory effects, altered membrane permeability a l -2+ terations, Ca overload and catecholamine oxidation products (Rona, 1985). Rona, G. (1985). Catecholamine cardiotoxicity. J Molec Cell Cardiol. 17: 291-306. 

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