z 3'+' I THE INFLUENCE OF DIETARY FATTY ACIDS ON CARDIAC FUNCTION A thesis suhmitted for the degree of DOCTOR OF PHILOSOPY by Salvatore Pepe, B.Sc. (Hons.Med.) Department of Physiology University of Adelaide South Australia November 1991 l1 I'd give all wealth thnt years have piled, Th¿ slow result of lífe's decay, To be once more a little child For onc bríght swnmcr-day. Rev. Cha¡les Lutwidge Dodgson to tlæ chíld. wln always oslcs qucstions to the power of a child's dreatn iii CONTENTS ABBRBVIATIONS vii ABSTRACT ix DECLARATION nt ACKNOWLEDCIEMENTS nü CHAPTER I. GENERAL INTRODUCTION I I.1. Ischaemic Coronary Heart Disease 1 I.2. Defining Myocardial Ischaemia 2 I.3. Consequenies of Ischaemia 3 I.4. Coronary Risk Factors For Sudden Cardiac Death 5 I.5. Dietary Fatty Acids 8 I.6. Fatty Acids In Myocardial Ischaemia 9 I.7. Models of Myocardial Ischaemia 10 I.8. Thesis Aims 13 CHAPTER tr. GENERAL METHODS. The Maintained Afterload 16 Model of Global Ischaemia in the Ervthrocyte Perfused Isolated 'Workinq Rat Heart II.1. Introduction 16 tr.2. Methods 24 tr.2.a) Perfusion Media 24 tr.2.b) Working Heart Preparation 25 tr.2.c) Bxperimental Design 26 tr.2.d) Calculations 29 tr.2.e) Statistical Analysis 30 tr.3. Results 31 tr.3.a) Performance Of Unpaced Heârts 3l tr.3.b) Conection For Heart Rate: Paced Hearts 33 II.4. Discussion 43 II.5. Conclusioo 47 CHAPTER ltr. EFFECT OF DIETARY FATTY ACID SUPPLE- 49 MENTS ON CARDIAC PERFORMANCE: Ischaemic Iniurv & Post-lschaemic Recoverv III.1. Introduction 49 Itr.2. Methods 55 Itr.2.a) Diets 55 m.2.b) Animals 58 il.2.c) Perfusion Protocol 58 lV m.2.d) Creatine Kinase Analysis 60 Itr.2.e) Lacta;tþ Release Analysis 6l m2.Ð K* Flamephotometry 61 [.2.Ð Data Handling & Statistical Analysis 61 Itr.3. Rezults 62 Itr.3.a) Animal Growth and Tissue Composition 62 Itr.3.b) Cardiac Output 64 Itr.3.c) Ventricular Function 64 m.3.d) Myocardial Oxygen Consumption 70 Itr.3.e) Metabolism 70 m.3.0 Creatine Phosphokinase 7L III.4. Discussion 72 CHAPTER ry. TI{E INFLT]ENCE OF DIETARY FAT 81 SI]PPLEMBNTATION ON CARDIAC ARRTIYTHMIA INCIDENCE AND VT]LNERABILITY fV.l. Introduction 81 IV.2. Methods 86 IV.2.a) Animals 86 IV.2.b) Experiment A: Perfusion Protocol 87 IV.2.c) Experiment B: Programmed Electrical Stimulatioo protocol 88 IV.2.d) Ventricular Arrhythmia Assessment 89 IV.2.e) Søtistical Analysis 89 fV.3. Results 90 fV.4. Discussion 95 CHÄPTER V. TIIE INFLIJENCE OF CORONARY FLOW ON t02 MYOCARDIAL OXYGEN CONSIJMPTION V. 1. Introduction 102 V.2. Methods 103 V.2.a) Animals & Dietary Supplementation 103 V,2.b) Perfusion Proûocol 103 V.2.c) Vasodilator Treatment 105 V.2.d) Analysis Of Coronary Effluent Contents 106 V.2.e) Data llandling and Statistical Analysis 106 V.3. Rezults 107 V.3.a) Vasodilator Influence on Coronary Flow & Cardiac Ouþut 107 V.3.b) Vasodilator Influence on Ventricular performance 107 v V.3.c) Vasodilator Influence on Oxygen Utilisation 108 V.3.d) Vasodilator Influence on Ischaemic Injury 110 V.4. Discussion LI7 CHAPTBR VI. TIIE INFLI]ENCE OF CAI.CIUM IN DIETARY 123 LIPID & ISCIIAEMIA INDUCED CHANGES IN OXYGEN METABOLISM AND VENTRJCI]LAR PBRFORMANCE. V[.1. Introduction 123 VI.2. Methods t29 VI.2.a) Animals & Diets t29 VI.2.b) holated Working Heart Preparation & Perfusion Protocol 130 VI.2.c) Experiment A:Determi¡ation of Basal Metabolism:K* A¡rest 131 *l VI.2.d) Experiment B:The Modification of Extracellular [Ca* t3L VI.2.e) Analysis of Coronary Effluent Contents r33 VL2.Ð Data Handling and Statistical Analysis r33 VI.3. Results 134 VL3. Experiment A: Basal Oxygen Metabolism t34 *] VI.3. Experiment B: The Modification of Extracellular [Ca* t34 VI.3.B:a) Pressure-Time Integral 134 VI.3.B:b) Myocardial External Work 135 VL3.B:o) Cardiac Ouþut 136 VI.3.B:d) Coronary Flow 136 VI.3.B:e) % Oxygen Extraction t37 VI.3.B:f) Myocardial Oxygen Conzumption 138 W.3.8:g) % Bfñciency 140 VI.3.B:h) Metabolites and Enzyme Content Of Coronary Effluent L44 VI.3.B:i) Effect Of Ca** On Arrhythmogenesis 143 VI.4. Discussion 144 CTIAPTER VII. EVALUATION OF FT]NCTIONAL 174 PERFORMANCE BFFICIENCY BY MYOCARDTAL LOAD TESTING: CORONARY PERFUSION PRESSURB AND PREI.OAD ALTERATIONS \ryTTH MAINTAINED AFTERLOAD VII.1. Introduction 174 VII.2. Methods t77 Ytr.2.a) Animals & Diets 177 VII.2.b) Isolated Working Heart Preparation & Perfusion Protocol tn Yn.Z.c) Experimental Design 178 VII.2.d) Analysis Of Coronary Effluent Contents t78 v1 VII.2.e) Data Handling & Statistical Analysis 179 VII.3. Results 179 VII.3.a) Myocardial External Work r79 VII.3.b) Pressure-Time Integral 180 VII.3.c) Myocardial Oxygen Consumption 180 VII.3.d) Energy Utilisation Efficiency 181 VII.3.e) Cardiac Ouþut 181 Vtr.3.0 I-actic Acid Production & Venous pH 181 Vtr.3.Ð The Effect Of Ischaemia with High Preload Stress r82 VII.3.h) The Effect Of Coronary Perfusion Preszure Reductions 183 VII.4. Discussion 185 CIIAPTER VItr. DOSE EFFECT RELATIONSHIP BET\'/EEN 205 DIETARY POLYT]NSATT]RATED FISH OIL. CARDIAC FT]NCTION AND METABOLISM VItr. 1. Introduction 205 VItr.2. Methods 207 Vltr.2.a) Animals & Diets 207 Vltr.2.b) Isolated Working Heart Preparation & Perfusion Protocol 207 Vltr.2.c) Analysis of Coronary Effluent Contents 208 Vltr.2.d) Data Ha¡dling and Ståtistical Analysis 209 VItr.3. Rezults 2IO Vltr.3.a) % Oxyget Extraction 2r0 Vltr.3.b) Myocardial Oxygen Consumption 2r0 Vltr.3.c) Myocardial External lr¡y'ork 2tt Vltr.3.d) Cardiac Ouþut 212 Vltr.3.e) Coronary Flow 2r2 VItr.3.Ð Pressure-Time Integral 212 Vltr.3.g) % Efficiercy 2t3 Vltr.3.h) Metabolites &.Etzyme Content Of Coronary Effluent 213 VItr.4. Discussion 219 CT{APTER D(. GENERAL DISCUSSION AND CONCLUSIONS 224 D(.1. Introduction 224 rrly'orking D(.2. The Erythrocyte Perfused Isolated Rat Heart 227 D(.3. Dietary Fatty Acid Influence Ou Cardiac Function 230 D(.4. Study Of Mechanisms Underlying Altered Oxygen Metabolism 237 D(.5. Potential Mechanisms Of Altered Calcium Metabolism 239 vii D(.5.a) Dietary Fatty Acid Effect On Bicosanoids 2& DLs.b) Direct Intracellular Fatty Acid Interactions 245 D(.5.c) Potential Role Of Free Radical Metabolism 249 D(.6. Future Directions 254 REFERENCES 257 ôP/ôt Pressure-Time Integral ¡rmol micro moles D zum of AA a¡achidonic acid ADP adenosine diphosphate AL afterload ATP adenosine trþhosphate bpm beats per minute c control group cccP carbonyl cya¡ide-m<hlorophenyl hydrazone CHD ischaemic coro¡rary heart disease CPP coronary perfusion pressure DHA docosahexaenoic acid DMA dimethyl aceìa;te dw dry weight E-C exc itation+ontraction BCG electrocardiogram EPA eicosapentaenoic acid FO polyunsaturaÞd fish oil c grâm Hz hertz J joules K-H K¡ebs-Henseleit solution Kg kilogram vll1 LA linoleic acid LVo left ventricular filling preszure M molar mln minutes mL millilitres mmHg millimetres of mercury mmol millimoles ms milliseconds MVO, myocardial oxyger consumption NAD* nicotinamide adenine dinucleotide (oxidised) NADH nicotinamide adenine dinucleotide (reduced) P, inorganic orthophosphate PDH pyruvate dehydrogenase PL preload Pot oxygen partial preszure PTI Pressure-Time Integral PT]FA polyunsaturated fatty acids RBC red blood cell perfusate REF reference diet rpm revolutions per minute RR ruthenium red RY ryanodine SAT saturated sheep fat supplemented diet scD zudden cardiac death SD standa¡d deviation sec seconds U units VFT ventricular fibrillation threshold VPB ventricula¡ premature beats VT ventricular tachycardia w/w weight per weight lX ABSTRACT The aim was to study the direct effe¡ts of dietary fatty acids on myocardial function in rats. In particular, the effects of dietary fat intake on cardiac function stressed with myocardial ischaemia and reperfusion were investigated. Although dietary fat can influence the development of atheroscle- rosis, thrombosis, cardiac ischaemia and myocardial infarction, little is known of any direct effects by dietary fat on cardiac performance. It has also been clearly demonstrated that the fatty acid prohle of myocardial phospholipids are predominantly dependent on the qualitative properties of dietary lipid intake. Such alterations in the cellular lipid environment may be associated with a direct dietary fatty acid influence on cardiac function. Hooded-Wistar rats (4months old) were placed into 3 dietary groups: REF, a reference base diet or a L2Vo (w/w) addition of either saturated fatty acid rich sheep fat (SAT) or fish oil rich in polyunsaturated marine n-3 fatty acids (FO). Animals were maintained on the diets for a minimum of four months prior to experimental use. In order to directly study cardiac function and precisely control electrolyte and metabolic substrate availability, neural and humoral factors, preload, workload, humidity and temperature, the isolated working heart method was selected. To overcome limitations which reduce the suitability of most isolated working heart models of global ischaemia for the study of the progression of ischaemic injury, a new model of low flow global ischaemia was developed. This method did not cause total cessation of ventricular function or coronary flow and thus permitted investigation of ischaemic processes as they occurred, by simultaneous x measurement of ventricula¡ function, oxygen upøke and metabolite release in venous outflow. This new model utilised a novel placement of two valves to permit coronary perfusion pressure reductions with maintained afterload, to provide a greater ischaemic insult yet allowing simultaneous functional and metabolic evaluation. In addition, a buffer with washed porcine erythrocytes at 40% haematocrit in a modified Krebs-HenseleiUdextran solution was utilised for improved oxygen delivery, viscosity, colloid osmotic pressure (to reduce oedema) and improved mechanical performance on which to impose the ischaemic insult.