THE CARDIAC FATTY ACID METABOLIC PATHWAY IN HEART FAILURE by Eric E. Morgan Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. William C. Stanley Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY May, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Eric E. Morgan candidate for the Ph.D degree *. (signed) Richard Eckert (chair of the committee) William C. Stanley Brian D. Hoit Steve Fisher Matthais Buck (date) 01/20/06 *We also certify that written approval has been obtained for any proprietary material contained therein. ii DEDICATION This work is dedicated to Drs. William Stanley, Margaret Chandler and Brian Hoit, whose unique perspectives on research collectively afforded me a very rich and well- rounded educational experience. iii TABLE OF CONTENTS Dedication...........................................................................................................................ii Table of Contents……………………………………………………………………..…iii List of Tables…………………………………………………………...……………….vii List of Figures……………………………………………………………………………ix Acknowledgments…………………………………......………………………………...xi List of Abbreviations………………………………………………………………...…xii Abstract…………………………………………………………..………...…………....xv Chapter 1: Energy Metabolism in the Heart……………………………….…….........1 1.1. Introduction……………………………………………………………………......1 1.2. Myocardial Carbohydrate and Fatty Acid Metabolism…………………....……...2 1.2.1. Overview…………………………………………………………...……...2 1.2.2. Carbohydrate Metabolism………………………………………………....3 1.2.3. Fatty Acid Metabolism……………………………………………………5 1.3. Regulation of Expression of Fatty Acid Oxidation Enzymes by Nuclear Receptors…………………………………………………………………….……8 1.4. Overview of Heart Failure……………………………………………………….11 1.5. Myocardial Lipid Accumulation and Heart Failure……………………………...13 1.6. Metabolic Phenotype of the Failing Heart……………………………………….14 1.7. Substrate Selection and Contractile Function in Heart Failure……………..........17 1.8. Rationale and Hypothesis……………………………………………………..…18 iv Chapter 2: Validation of Echocardiographic Methods for Assessing Left Ventricular Dysfunction in Rats with Myocardial Infarction….......….24 2.1. Introduction………………………………………………………………………24 2.2. Materials and Methods…………………………………………………………...25 2.2.1. Study Design and Induction of Myocardial Infarction…………………..25 2.2.2. Echocardiography………………………………………………………..26 2.2.3. Hemodynamic Measurements……………………………………………30 2.2.4. Statistical Analysis……………………………………………………….30 2.3. Results……………………………………………………………………………31 2.3.1. Hemodynamics…………………………………………………………..31 2.3.2. Echocardiography………………………………………………………..31 2.3.3. Intra- and Inter-observer error………………………………………...…31 2.3.4. WMSI and MPI Correlations…………………………………………….35 2.4. Discussion………………………………………………………………………..35 2.5. Limitations……………………………………………………………………… 40 2.6. Conclusions………………………………………………………………………40 Chapter 3: Effects of Coronary Artery Ligation Induced Heart Failure on Cardiac Metabolic Enzyme Gene and Protein Expression…............42 3.1. Introduction…………………………………………………………….………...42 3.2. Methods………………...……………………………………………….………..44 3.2.1. Study Design and Induction of Myocardial Infarction…………………..44 2.3.2. Echocardiography………………………………………………………..44 3.2.3. Hemodynamic Measurements……………………………………………45 v 3.2.4. RNA Extraction and Quantitative RT-PCR……………………………...46 3.2.5. Western Immunoblot Analysis…………………………………………..46 3.2.6. Metabolites and Enzyme Activities………………………………….......47 3.2.7. Statistical Analysis……………………………………………………….47 3.3. Results………………….……………………………….………………………..47 3.3.1. Body and Heart Mass……………………...………………….………….47 3.3.2. Cardiac Function……………………..….…………………….…………48 3.3.3. mRNA Expression…………………………...……………..……………52 3.3.4. Enzyme Activity and Protein Expression…………..….….……………..52 3.3.5. Metabolite Levels……………………………………………...………...52 3.4. Discussion………………………………………………………………………..60 Chapter 4: Effects of Chronic Activation of Peroxisome Proliferator Activated Receptor Alpha or High Fat Feeding in a Rat Infarct Model of Heart Failure…………………………………………………………………..….63 4.1. Introduction……...……………………………………………………...…….....63 4.2. Methods………………………………………………………………………….64 4.2.1. Study Design and Induction of Myocardial Infarction…………………..64 4.2.2. Echocardiography………………………………………………………..65 4.2.3. Hemodynamic Measurements……………………………………………66 4.2.4. Metabolic Products and Enzyme Activity……………………………….67 4.2.5. RNA Extraction and Quantitative RT-PCR……………………………...67 4.2.6. Western Immunoblot Analysis…………………………………………..68 4.2.7. Statistical Analysis……………………………………………………….68 vi 4.3. Results………………………………..…………………………………………...69 4.3.1. Body and Heart Mass………………………………………………….…69 4.3.2. Cardiac Function…………………………………………………………69 4.3.3. Triglyceride and Ceramide………………………………………………69 4.3.4. mRNA Expression……………………………………………………….75 4.3.5. Protein Expression and Enzyme Activity………………………………..75 4.4. Discussion………………………………………………………...…………..….78 Chapter 5: Discussion and Future Directions…………………………………..........82 5.1. Thesis Summary………………………………………………………………....82 5.2. Discussion and Future Directions………………………………………………83 5.2.1. Time Course of Fatty Acid Metabolic Down-regulation in Heart Failure……………………………………………….…………………...84 5.2.2. Role of Myocardial Triglyceride and Ceramide Accumulation in the Progression of Heart Failure……………………………………………..85 5.2.3. Role of Nuclear Receptor Activation in Heart Failure…………………..86 5.2.4. Pharmacologic Manipulations of Myocardial Metabolism for the Preventions of Heart Failure Following Myocardial Infarction…………90 5.3. Conclusion………………………………………………………………………91 Reference List…………………………………………………………………………...92 vii LIST OF TABLES Table 2-1: Hemodynamic variables in normal and infarcted rats……………….….......32 Table 2-2: Echocardiographic variables in normal and infarcted rats………………….33 Table 2-3: Results of linear regression analysis comparing intra- and Inter- investigator measurements of MPI and WMSI…………...…………...…….34 Table 2-4: Correlation coefficients between WMSI and MPI and left ventricular pressure measurements and echocardiographic parameters in normal and infarcted rats…………………………………………………………....37 Table 2-5: Results of forward stepwise linear regression analysis for WMSI And MPI using peak +dP/dt, cardiac index, peak end diastolic pressure, area of fractional shortening, end diastolic area and tau as independent variables…………………………………………………….38 Table 3-1: Body and heart mass in normal and infarcted rats………………………….49 Table 3-2: Left ventricular pressures and heart rates and echocardiographic data in normal and infarcted rats……….……….…...…………..………….……51 Table 3-3: mRNA expression in normal and infarcted rats…………………………….53 Table 3-4: Protein expression in normal and infarcted rats at 20 weeks post infarction or sham surgery………………………………………………….56 Table 3-5: Medium chain acyl-CoA dehydrogenase and citrate synthase activity in normal and infarcted rats………………………………………..56 Table 3-6: Plasma fatty acid and triglyceride concentrations in normal and infarcted rats………………………………………………………………...57 viii Table 4-1: Body and heart masses in infarcted, infarcted and high fat fed, and infarcted and fenofibrate treated rats…………………………………...70 Table 4-2: Hemodynamic and echocardiographic measurements in infarcted, infarcted and high fat fed, and infarcted and fenofibrate treated rats………72 Table 4-3: Plasma free fatty acids and triglycerides in infarcted, infarcted and high fat fed, and infarcted and fenofibrate treated rats…………………………...73 Table 4-4: Protein expression in infarcted, infarcted and high fat fed, and infarcted and fenofibrate treated rats………………………………………………….73 Table 4-5: Medium chain acyl-CoA dehydrogenase and citrate synthase activity in infarcted, infarcted and high fat fed, and infarcted and fenofibrate treated rats…………………………………………………………………………..77 ix LIST OF FIGURES Figure 1-1: Schematic representation of the carbohydrate and fatty acid metabolic pathways………………………………………………………….4 Figure 1-2: Schematic representation of the regulation of metabolic genes in cardiomyocytes by stimulation of the peroxisome proliferator- activated receptor α (PPARα) and retinoic X receptor (RXRα)…………….9 Figure 1-3: Major end points determined in this thesis………………………………...20 Figure 2-1: Parasternal long and short axis views of rat left ventricle…………………27 Figure 2-2: Doppler color directed pulse-wave recording of mitral and aortic flow for the determination of MPI………………………………………………29 Figure 2-3: Left ventricular peak +/-dP/dt, area fractional shortening and cardiac index plotted as a function of WMSI and MPI……………………36 Figure 3-1: Fractional area shortening and ANP expression in normal and infarcted rats at 8 and 20 weeks post infarction or sham surgery………….50 Figure 3-2: mRNA expression of non-PPARα regulated genes in normal and infarcted rats at 8 and 20 weeks post infarction or sham surgery………….54 Figure 3-3: mRNA expression of PPARα regulated genes in normal and infarcted rats at 8 and 20 weeks post infarction or sham surgery……………………55 Figure 3-4: Myocardial C16 ceramide content in normal and infarcted rats at 8 and 20 weeks post infarction or sham surgery……………………………..58 Figure 3-5: Protein expression in normal and infarcted rats at 20 weeks post infarction or sham surgery………………………………………………….59 x Figure 4-1: Left ventricular mass to body mass ratio in infarcted, infarcted and high fat fed, and infarcted and fenofibrate treated rats…………………….71 Figure 4-2: Myocardial tissue triglyceride and C16 ceramide content in infarcted,