Functional Remodeling Following Myofilament Calcium Sensitization in Rats with Volume Overload Heart Failure DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kristin Diane Lewis, D.V.M. Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2014 Dissertation Committee: Pamela Lucchesi, Advisor Christopher Breuer Christopher Premanandan Lynette Rogers Copyrighted by Kristin Diane Lewis 2014 Abstract Hemodynamic volume overload (VO) is characterized by left ventricular (LV) dilation, progressive LV dysfunction, and heart failure (HF). In the aortocaval fistula (ACF) model of VO HF, LV dysfunction is accompanied by a variety of changes at the myocyte level including myofilament proteins including decreased α-to-β-myosin heavy chain (MHC) expression, decreased myofilament Ca2+ sensitivity and altered regulation of intercalated disc proteins involved in electromechanical coupling. While pharmacologic therapy alone cannot resolve the hemodynamic overload in human patients or in animal models, pre-operative pharmacologic therapy may delay time to surgical intervention, and post-operative pharmacologic therapy may accelerate functional recovery. Current therapies that target neurohormonal pathway activation provide some relief but do not reverse the decreased cardiac contractility, the central feature of systolic HF [60]. Targeting neurohormonal pathway activation can result in increased myocardial oxygen consumption and myocardial Ca2+ overload [140], and therefore it is necessary to investigate drugs that have alternative mechanisms of action. Levosimendan (Levo) stabilizes Ca2+-saturated troponin C (cTnC) prolonging its interaction with cardiac troponin I (cTnI), which promotes contractile force without increasing the intracellular Ca2+ transient amplitude or myocardial oxygen consumption [126,128,136]. Given these overall factors, my hypothesis is that therapeutic strategies such as Levo that target ii myofilament Ca2+ sensitization will preserve/improve LV function in VO HF. To test this hypothesis, three specific aims were proposed: 1) determine if short-term Levo treatment would preserve/improve LV function in rats with ACF-induced pre-HF or reversed pre- HF (Chapter 2), 2) determine if Levo could improve the detrimental effects of delayed reversal (Chapter 3), and 3) determine if chronic Levo treatment would preserve LV function when initiated in rats with ACF-induced pre-HF or established HF (Chapter 4). Sham and ACF surgery were performed at Week 0 following by Levo treatment with and without hemodynamic load reduction surgery (reversal) at various timepoints. Continued Levo improved systolic and diastolic function regardless of the treatment starting point and hemodynamic load. Improved LV function variably correlated with increased myofilament Ca2+ sensitivity, cMyBP-C/cTnI phosphorylation and normalization of α-to- β-MHC. Finally, speckle-tracking echocardiographic analysis suggests that Levo improves short-axis, but not long-axis function at end-stage HF. Because of the improved LV function with Levo, with and without hemodynamic load reduction, Levo offers a new therapeutic option in patients with VO HF. More broadly, therapeutic strategies targeting myofilament Ca2+ sensitization may provide a new therapeutic target for patients with volume overload heart failure. iii To my husband Travis, my family, and many others for your unwavering patience, love and support through the many highs and lows of this journey. iv Acknowledgments I would like to thank my advisor, Dr. Pamela Lucchesi, the members of my graduate committee, and the members of the Lucchesi lab for their support in the design and execution of the experiments done in this dissertation. I would like to thank the faculty veterinary pathologists at The Ohio State University who have been instrumental in developing and shaping my skills as a veterinary pathologist, and I would like to thank my resident-mates who walked through this journey with me. I would like those who pushed me to become a veterinary pathologist, especially NOD who taught me to ask the question, ―What’s your hypothesis?‖ Finally, I would like to thank my friends and family whose unwavering support and encouragement have helped me through the highs and lows of my educational journey. v Vita 2001................................................................B.S. Microbiology, California Polytechnic State University 2001-2006 ......................................................Research Associate, Genentech, Inc 2010................................................................D.V.M., University of California Davis, School of Veterinary Medicine 2010 to present ..............................................Veterinary Anatomic Pathology Resident, Graduate Research Associate, Department of Veterinary Biosciences, The Ohio State University Publications Wilson K, Lucchesi PA. Myofilament dysfunction as an emerging mechanism of volume overload heart failure. Pflugers Arch. 2014 Feb 1. [Epub ahead of print] PMID: 24488008 Fields of Study Major Field: Comparative and Veterinary Medicine vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi List of Tables ................................................................................................................... xiv List of Figures ................................................................................................................... xv Chapter 1: Introduction and Background ...................................................................... 1 Abstract ............................................................................................................................... 1 Introduction ......................................................................................................................... 1 Rodent and non-rodent animal models are used to study VO-HF ............................... 4 Neurohormonal activation in volume overload ........................................................... 5 Volume overload physiology and structural alterations differs from pressure overload ....................................................................................................................... 6 LV pump function in VO............................................................................................. 7 Cardiomyocyte dysfunction in VO ..................................................................................... 9 Excitation-contraction coupling .................................................................................. 9 vii VO HF, intercalated discs and electromechanical transmission ................................ 10 Gap junctions/electrical communication ................................................................... 12 Force transmission through the intercalated disc ...................................................... 13 Myofilament dysfunction in VO ....................................................................................... 14 Force generation ........................................................................................................ 14 DCM-causing mutation in thin filament proteins ...................................................... 16 Thick filament proteins in VO HF ............................................................................. 20 Passive muscle stiffness in VO ......................................................................................... 24 Extracellular matrix (ECM) alterations in VO.................................................................. 24 ECM turnover ............................................................................................................ 24 Mechanical stress-induced ECM remodeling ............................................................ 26 Mast cells and VO-induced ECM remodeling .......................................................... 28 Mast cells and paracrine-induced LV ECM remodeling ........................................... 30 Pharmacologic therapy in VO ........................................................................................... 31 Diuretics..................................................................................................................... 31 Vasodilators ............................................................................................................... 31 β-adrenergic antagonists ............................................................................................ 32 Myofilament Ca2+ sensitizers .................................................................................... 32 Myosin activators ...................................................................................................... 33 viii Phosphodiesterase inhibitors ..................................................................................... 33 Limitations and future directions .....................................................................................