MOLECULAR BASIS OF ABNORMAL CONDUCTION IN MICE OVER-EXPRESSING ENDOTHELIN-1 by Erin Elizabeth Mueller A thesis submitted in conformity with the requirements for the degree of PhD Graduate Department of Laboratory Medicine and Pathobiology Copyright by Erin Elizabeth Mueller (2011) MOLECULAR BASIS OF ABNORMAL CONDUCTION IN MICE OVER-EXPRESSING ENDOTHELIN-1 Erin Elizabeth Mueller Doctor of Philosophy, 2011 Department of Laboratory Medicine & Pathobiology, University of Toronto ABSTRACT Binary transgenic (BT) mice with doxycycline (DOX)-suppressible cardiac-specific over- expression of endothelin-1 (ET-1) exhibit progressive heart failure, QRS prolongation, and death following DOX withdrawal. However, the molecular basis and reversibility of the electrophysiological abnormalities in this model were not known. Here we assess the mechanisms underlying ET-1-mediated electrical remodelling, and its role in heart failure. Prior attempts to prevent this model of ET-1 induced cardiomyopathy with ET receptor antagonism were not beneficial. We now propose to evaluate the effectiveness of blocking the synthesis of ET-1 with CGS 26303, a dual inhibitor of endothelin converting enzyme (ECE) and neutral endopeptidase. BT vs. littermate control mice were withdrawn from DOX and serially studied with ultrasound biomicroscopy, octapolar catheters, multi-electrode epicardial mapping, histopathology, Western blot, immunohistochemistry and qRT-PCR. Prolonged ventricular activation and depressed rate of ventricular activation were detected as early as 4 wks after transgene activation, when structure and function of the heart remained unaffected. By 8 wks of ET-1 over-expression, biventricular systolic and diastolic dysfunction, myocardial fibrosis, cardiomyocyte hypertrophy, prolonged ventricular activation and repolarization, depressed ii rate of ventricular activation, and abnormal atrioventricular nodal function were observed. Within 4 wks of ET-1 induction, reduction were observed in connexin-43 mRNA, protein, + + and phosphorylation, Nav1.5 mRNA and protein, Na conductance, K channel interacting protein-2 mRNA and Kv4.2 mRNA. Chromatin immunoprecipitation revealed that nuclear factor κB preferentially binds to Cx43 and Nav1.5 promoters. Importantly, the associated electrophysiological abnormalities at this time point were reversible upon suppression of ET-1 over-expression and completely prevented the development of structural and functional remodelling. Treatment with CGS-26303 (5 mg/kg/day) failed to improve survival, or hemodynamic and contractile decline. ET-1-mediated ventricular conduction delays correlates with gap junction and ion channel remodelling, and precedes heart failure. The sequence and reversibility of this phenotype suggest that a primary abnormality in electrical remodelling may contribute to the pathogenesis of heart failure. CGS 26303 failed to prevent this cardiomyopathic phenotype. These data suggest that chronically high levels of bigET-1, as seen in heart failure, may induce increased ECE activity and/or non-ECE ET-1 synthesis, thus circumventing the efficacy of ECE blockade in this model. iii ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisors, Mansoor Husain and Duncan Stewart for providing insight, focus, motivation, contined support, and positive reinforcement throughout my PhD. Secondly, I would like to thank the Department of Laboratory Medicine and Pathobiology and CLAMPS for providing a great learning environment. I would also like to thank my committee members, Peter Backx and Kumaraswamy Nanthakumar for serving as mentors, and guiding my research progress. Thank you for taking an active and enthusiastic interest in my project and for providing invaluable insights, helpful discussions, and electrophysiological expertise. In particular, thank you to Stéphane Massé from Nanthakumar’s lab, for his continued assistance with electrophysiological experiments. Additionally, I would like to thank my family and friends, particularly my husband, for their continued support, encouragement, and patience throughout my graduate studies. Thank you to Peter Sabatini, Karolina Kolodziejska, Sonya Hui, Shivalika Handa, Jae Choi, Kiwon Ban, Dorota Dajnowiec, and Dan Trcka for your support, camaraderie, and all the wonderful memories over the last 7 years. I would like to thank all past and present members of the Husain lab, in particular, the surgical skills of Abdul Momen and Golam Kabir, mouse colony management and genotyping support of Haiyan Xiao and Changsen Wang, the cell culture and qRT-PCR expertise of Karolina Kolodziejska, general lab advice from Talat Afroze, primer design assistance and office antics of Omar El-Mounayri, the guidance and reliable advice of Hassan Zaidi, and the superb everyday support and administrative skills of Tracey Richards. iv I would also like to thank my MSc supervisor, Susan Howlett for instilling me with a love of science. And finally, I would like to thank the Ontario Graduate Scholarship in Science and Technology for funding throughout my PhD program. v TABLE OF CONTENTS ABSTRACT ............................................................................................................................ II ACKNOWLEDGEMENTS ................................................................................................. IV TABLE OF CONTENTS ..................................................................................................... VI LIST OF TABLES ................................................................................................................ XI LIST OF FIGURES ............................................................................................................ XII LIST OF APPENDICES ................................................................................................... XIV LIST OF ABBREVIATIONS ............................................................................................. XV CHAPTER 1. LITERATURE REVIEW .............................................................................. 1 1.1.1 Definition ........................................................................................................................ 2 1.1.2 Etiology and prevalence ................................................................................................ 2 1.1.3 Symptoms and classifications........................................................................................ 3 1.1.4 Pathophysiology ............................................................................................................. 4 1.1.4.1 Neurohormonal activation ........................................................................................ 5 1.1.4.1 LV remodelling ......................................................................................................... 7 1.1.5 Treatments ...................................................................................................................... 9 1.2 ENDOTHELIN ............................................................................................................... 10 1.2.1 Distribution, regulation and synthesis of ET-1 ......................................................... 10 1.2.2 Clearance of ET-1 ........................................................................................................ 11 1.2.3 ECE ............................................................................................................................... 13 1.2.4 ET Receptors and signal transduction ....................................................................... 13 1.2.5 Transgenic mouse models: genetic manipulation of ET-1 system ........................... 16 1.2.6. Role of ET-1 in inflammation, hypertrophy, and fibrosis ....................................... 19 vi 1.2.7 Pathophysiology of ET-1 in HF .................................................................................. 23 1.3 ELECTRICAL REMODELLING ................................................................................ 25 1.3.1 Excitation in the healthy heart .................................................................................... 25 1.3.1.1 Ionic basis of cardiac action potential ..................................................................... 26 1.3.1.2 Electrophysiological mapping ................................................................................ 26 1.3.1.3 Excitation-contraction coupling and Ca2+ cycling .................................................. 28 1.3.1.4 Ca2+ handling proteins and Ca2+ current ................................................................. 28 1.3.1.5 Sodium current ........................................................................................................ 32 + 1.3.1.6 Transient outward K current (Ito) .......................................................................... 35 + 1.3.1.7 Delayed rectifier K current (IK) ............................................................................. 36 + 1.3.1.8 Inward rectifier K current (IK1) .............................................................................. 36 1.3.1.9 Gap junctions .......................................................................................................... 37 1.3.2 Electrical remodelling and HF .................................................................................... 40 1.3.2.1 Ca+ channel remodelling ......................................................................................... 41 1.3.2.2 Na+ channel