SUBSTRATE ENHANCEMENT UTLILIZING RIBOSE PRETREATMENT IN NORMAL AND PATHOLOGIC STATES William Jack Wallen A thesis submitted in conformity with requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto Q Copyright by William Jack Wallen L 997 National Library Bibliothèque nationafe 1+1 ofcalada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wdiïngton Street 395, nie Wellington OttawaON K1AON4 OttawaON K1AW Canada Canada The euthor has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Libmy of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts kom it Ni la thèse ni des extraits substantiels may be printed or othenwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. SUBSTRATE ENHANCEMENT UTILIZING RIBOSE PRETREATMENT IN NORMAL AND PATHOLOGIC STATES- William Jack Wailen, Master of Science, Department of Physiology, University of Toronto, 1997. These studies investigated the effects of advanced intravenous (IV) and intramuscuiar 0 pretreatment with D-ribose on energy stores in myocardium, skeletal muscle (SKM), and liver (LG), and myocardial tolerance to global ischemia Ribose significantly (ps0.05) elevated myocardial glycogen when administered N but not M,but did not alter SKM or LG. Myocardial tolerance to global ischemia (TIC) was significantiy improved by both routes, associated with a reduced net consumption of myocardial energy stores during early ischemia When administered in the presence of the inhalation anesthetic halothane, TIC was shortened (p0.05) by ribose pretreatment, associated with an increased net consumption of myocardiai energy stores. No differences in SKM were observed, while TV ribose appeared to prevent a significant depression in LG. In the presence of hypertensionfrnyocardial hypertrophy, IV ribose pretreatment significantly (ps0.05) improved in vivo Ieft venûicular performance, but did not alter energy stores or TIC. I cannot adequately express my thanks to my graduate supervisor, Dr. Carin Wittnich, for her guidance throughout the course of this work. She provided endless encouragement and enthusiasn, knew when to crack the whip, and brought out the best of my abilities. Her insights and knowledge of research helped to bring these studies to fhition. 1 must also thank Michad Belanger for his assistance in ail aspects of this research, from surgical techniques to biochemical analyses. However, 1 owe my biggest debt to him for his fiiendship and sense of humor over the years, which helped keep me sane through the trials and tribulations of this work. My thanks also to the other graduate students in the lab, Shona Torrance, Cathy Carlyle, and Cathy Boscarino, whose assistance in the performance of these studies, discussions of data, and fellowship helped immensely. A special acknowledgement to Shona and Cathy Carlyle for setting high standards, which 1 hope I have lived up to. I also wish to thank Drs. Uwe Ackermann and Roy Baker for their participation on my program cornmittee. Their patience, suggestions, and guidance during the course of my research are greatly appreciated. Finally, 1 must thank the members of my family for their love and support. To my stepfather, George Harris, my brother Dave, his wife hue, and their children, Christopher, Kathryn, and Nicholas, and my sister Beth; 1 know you dl think that I'm gohg to be a student forever, but see, there is an end in sight! 1 must also mention rny mother, who encouraged me throughout my education, but who did not live to see the completion of this thesis. 1 hope she would be pleased with the results. Portions of this work have been published previously (CmJ Surg, l995;38(4):A6, and J Mol Ce11 Cardiol, l995;27(6):Al84). iii TABLE OF CONTENTS Abstract Acknowledgements Table of Contents iv List of Abbreviations vii List of Figures ix List of Tables Chapter One: INTRODUCTION............oa.a...o.-................o.......o..... I................ ......-.......-0. 1 Section 1: Clinical Relevance 2 Section II: Myocardial Hypertrophy 4 Myocardial Mechanics 5 Volume Overload 7 Pressure Overload 8 Cellular Morphology and Ultrastructural Changes 9 Section III: Myocardial Metabohm 14 Overview 14 Energy Stores 15 (i) Creatine Phosphate 15 (ii) Myocardial Glycogen 17 Glycogen Synthesis 18 Glycogen Breakdown 19 Pathways of ATP Synthesis 2 1 Glycolysis 22 Lipid Metabolism 26 (i) p-oxidation 27 (ii) Tricarboxylic Acid Cycle (TCA) 29 (iii) The Electron Transport Chain 3 1 The Pentose Phosphate Pathway 35 Adenine Nucleotide De Novo Synthesis 38 Adenine Nucleotide Salvage 40 (i) Purine Nucleotide Degradation 42 (ii) Purine Salvage 43 Section IV: Myocardial Function 45 Systolic Function 45 Diastolic Function 47 Section V: Liver and Skeletal Muscle 49 Liver 49 Skeletai Muscle 50 Section VI: Myocardial Ischemia 5 1 Ischernic Contracture 53 Section VLI: Substrate Enhancement Glucose Adenosine Triphosphate (An) Adenosine (i) lahibition of adenosine transport (ii) Adenosine administration Ribose Chapter Three: MATERIALS AND METHODS.oo..mo.....oo~ooo~-~oo~~~~~~oo~~oooooo 67 Animal Care 68 Study Protocol 68 (i) Acute Protocol 68 (ii) Ischemic Tolerance 69 (iii) Biochemical Analyses 70 (iv) Myocardial Function Studies 70 Pretreatment Protocol 72 (i) Ribose Administration - Route of Administration 72 (ii) Ribose Administration - Role of Inhalation on Route of 73 Administration (iii) Ribose Administration - Impact of Pathology 73 (Hypertension and Myocardial Hypertrophy) Statisticd Analysis 74 Chapter Four: RESULTS., ..........o.o.o.o.ooo..o....o.o....o..oo...oooo.o.ooo.o...........ooo..o...o........oo..o.o... 75 1: Effect of Route of Administration 76 II: Administration of Ribose in the Presence of Inhalation Anesthesia 83 ïII: Cornparison of Saline Pretreatment Groups 87 IV: Ribose Administration in a Pathologie State 91 V: Effects of Ribose Administration on Myocardial Function in the SHR 95 Chapter Five: DISCUSSION~~~~~~~oooo~oo~ooooooo~~oooo~oo~ooooo~~~~ooooooooooooooooo99 Choice of Mode1 1O0 Effects of Bolus Ribose Administration - IV vs. IM 102 Effects of Bolus Ribose Administration in the Presence 113 of Halothane Effects of Ribose in the Presence of Chronic Hypertension and 117 Myocardial Hypertrophy Chapter Seven: LIMITATIONS AND FUTURE DIRECnONS............................... 129 1. Animal Mode1 130 II. Metabolic Meams 130 III. In Vivo Myocardial Function 131 IV: Mechanisrn of Ribose Effects 133 Appendices....e..,..œ...............................................e.œ.......................................................... 149 Appendix 1: Biochemical Methodologies 150 Tissue Metabolite Extraction 150 Fluorescence Spectrophotometry ("Fluorometry") 151 ATP/CP Analysis 152 Lactate Analysis 154 Glycogen Analysis 155 High Performance Liquid Chromatography (HPLC) 156 Appendix II: Ribose - Duration Study 159 LIST OF ABBREVIATIONS Ad0 adenosine GSSG oxidized giutathione ADP adenosine GTP guanosine triphosphate diphosphate BK hexokinase AMP adenosine HPLC high perCormance iiquid monophosphate chromatography AMP-S adenylosuccinate HPRT hypoxanthine AnER anaerobic energy reserve phosphoribosyl- ANOVA analysis of variance transferase APRT adenine phosphoribosyl- Em heart rate transferase FIxn hyporanthine ATP adenosine tri- IMP inosine phosphate monophosphate bpm beats per minute IM intramuscular CABG coronary artery bypass Ino inosine grafting IV intravenous CAD coronary artery disease LDH lactate dehydrogenase CAMP cyclic AMP LG liver glycogen CK creatine kinase LV left ven tricle CKc cytosolic creatine kinase LVDP left ventricular diastotic CKm mitochondrial creatine pressure kinase LVSP left ventricular systoiic CO cardiac output pressure CoA coenzyme A MG myocardial glycogen CP creatine phosphate mc myosin heavy chain CPB cardiopulmonary MLC myosin light chah bypass NADH reduced nicotinamide ETC electron transport adenine dinucleotide chain NADPH reduced nicotinamide F-I,6-BP €NC~OS&I,~- adenine dinucleotide bisphosphate phosphate F-2,6-BP fmctose-2,6- PCA perchloric acid bisphosphate PDH pyruvate dehydrogenase FAD flavin adenine PEX. phosphofructo- dinucleotide kinase FBPase2 fructose bisphosphatase 2 PFK2 phosphof~cto- FFA free fatty acid kinase 2 G-1-P glucose-1-phosphate Pi inorganic phosphate G-3-P DH glyceraldehyde-3- PK pyruvate kinase phosphate dehydrogenase PPi inorganic pyrophosphate G-6-P glucose-6-phosphate PPP pentose phosphate G-6-P DR glucose-6-p hosp hate pathway dehydrogenase vii PRPP phosphoribosyl pyrophosphate PTCA percutaneous transluminal coronary angioplasty QH2 reduced ubiquinol R-5-P ribose-5-phosphate RV right ventricle sec seconds SEm spontaneously hypertensive rat Sm skeletal muscle giycogen SR sarcoplssmic reticulum SV stroke volume TAN total adenine nucleotides TCA tricarboxylic acid TIC time to ischemic contracture TIC0 time to onset of ischemic contracture ~CP time to peak ischemic contracture UA uric acid UDP uridine diphosphate UTP uridine triphosphate WKY Wistar-Kyoto rat