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Cardiac Myofilament Calcium Sensitivity in Health and Disease Dissertation

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Cardiac Myofilament Calcium Sensitivity in Health and Disease Dissertation CARDIAC MYOFILAMENT CALCIUM SENSITIVITY IN HEALTH AND DISEASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kenneth D. Varian ***** The Ohio State University 2008 Dissertation Committee: Approved by Dr. Paul M.L. Janssen, Ph.D. Advisor Dr. Jack Rall, Ph.D. _________________________________ Dr. George Billman, Ph.D. Advisor Integrated Biomedical Sciences Dr. William Carson III, M.D. Graduate Program Dr. David Feldman, M.D., Ph.D. ABSTRACT Heart failure is the leading cause of death in much of the western world. Progressive deterioration of cardiac contractility and/or relaxation, resulting in cardiac output below metabolic demands is the central theme in heart failure. Although treatments are improving, we still do not have a full understanding of the fundamental pathophysiology that impairs contractility and relaxation. The force frequency relationship (FFR) is ubiquitously altered in failing myocardium regardless of the etiology. The FFR, which is normally positive (greater contractile force at higher contraction frequency) in healthy myocardium, is blunted, flat, or negative in dysfunctional myocardium. While the FFR has been studied extensively, the role myofilament properties play in this effect is unknown. To address deficiencies in our understanding of cardiac contraction, we worked from the following hypothesis: Alterations in myofilament calcium sensitivity significantly contribute to blunted force frequency response, contractile dysfunction, and impairment of myocardial relaxation of failing myocardium. First, we set out to develop a method which would enable quantitative analysis of shifts in myofilament calcium sensitivity due to changes in frequency in isolated, yet intact muscles at physiologic temperature. We found that we could induce slowly forming contractures by superfusing the intact isolated rat trabeculae with a solution of ii high potassium and lower sodium. The contractures allowed for development of a myofilament calcium sensitivity curve sensitive to interventions known to shift myofilament calcium sensitivity (beta adrenergic simulation and pH). We then aimed at determining if myofilament calcium sensitivity shifted with changes in frequency. We hypothesized that an increase in frequency would lead to a decreased in myofilament calcium sensitivity which was needed for frequency dependent acceleration of relaxation. We found that myofilament calcium sensitivity did decrease with frequency in intact rabbit trabeculae. This effect correlated with an increase in the phosphorylation status at Troponin I and Myosin Light Chain-2, as well as relaxation acceleration. Using staurosporine (non-specific kinase inhibitor), we found inhibition of the myofilament calcium sensitivity shift between 1 and 4 Hz. With the exception of blunting this shift in myofilament calcium sensitivity, staurosporine did little else to the force frequency relationship, suggesting that most of the effect was not due to phosphorylation of key proteins. To examine how inter-beat duration independent of posttranslational modifications occurs, we performed an analysis of twitch contractions in rabbit trabeculae stimulated randomly at 5 different cycle lengths; this was done in order to isolate the effect of cycle time on contractility and relaxation. We found that the primary cycle length correlated positively with force, while the secondary and tertiary cycle lengths correlated negatively with force. Finally, we set out to determine how frequency dependent modulations of force, relaxation, and myofilament calcium sensitivity differed in a model of right ventricular hypertrophy. We used pulmonary artery banded rabbits and examined twitch iii contractions, intracellular calcium, and myofilament calcium sensitivity (potassium contractures) at 1 through 4 Hz. We found that the shift in myofilament calcium sensitivity observed in control animals was nearly abolished in animals with right ventricular hypertrophy. This blunting of myofilament desensitization was accompanied by elevated diastolic force compared to controls, possibly reflecting some diastolic dysfunction in the hypertrophied tissue. In conclusion, myofilament calcium desensitization in response to elevated heart rate is a regulated, kinase specific effect that may be necessary to prevent insufficient diastolic filling at times of high heart rates. iv Dedicated to my grandparents: Ken and Virginia Varian, and George and Myrtle Davidson v ACKNOWLEDGMENTS In completion of my dissertation, there are several groups of people I wish to mention. First I thank my advisor Paul Janssen for his careful guidance and support throughout the project. Paul has been an excellent motivator and mentor for me over the past 4 years. From him I have learned autonomy in research, independent thinking, and better developed skills in experimental design. My co-workers in the Janssen lab have also been supportive. Carlos Torres has given me very helpful discussions on research and acted as a mentor for me outside the lab. Michelle Monasky, Nitisha Hiranandani, Ying Xu, and Ko Bupha Intr have shared projects and ideas with me extending the breadth of my experiences in the lab. Anil Birdi, Ben Canan, Erin Shaffer, and Annemarie Hoffman have helped me immensely in preparing experiments. I am in debt to Anusak Kijtawornrat from the department of Veterinary Biosciences for his dedicated help with the pulmonary artery banding procedure. There are several faculty members outside the Janssen lab I would like to thank. Doctors Mark Ziolo and Jonathan Davis have both held extensive discussions with me about my research many of which prompted me to perform experiments that helped shape the project. My dissertation advisory committee members Doctors William Carson III, George Billman, David Feldman, and Jack Rall have all given me helpful guidance and pushed me to be more productive. The Medical Scientist program director, Allen Yates vi has also been instrumental in my success here at OSU. He acted as a research advisor and mentor when I was an undergrad at Mount Union College and provided me with sound advice and support during my years as an M.D./Ph.D. student. Finally, I would like to thank my family for their support through the years. My wife, Melinda, has been very supportive of my career and will be instrumental in my future success. My father, mother and brother have been encouraging to me my whole life and I certainly would not be here to today without their support. vii VITA February 11th, 1980………………….Born, Johns Hopkins Hospital, Baltimore Maryland 1998-2002………………………………….Mount Union College BS Biology/Chemistry 2002-2004……Medical Scientist Fellow, The Ohio State University College of Medicine 2004-2006…...Graduate Research Associate, Department of Physiology and Cell Biology 2006-2008………………...Graduate Fellow, Department of Physiology and Cell Biology PUBLICATIONS 1. Varian KD, Raman S, Janssen PML. Measurement of Myofilament Calcium Sensitivity at Physiological Temperature in Intact Cardiac Trabeculae. Am J Physiol Heart Circ Physiol 2006 May;290(5):H2092-7 2. Hiranandani N*, Varian KD*, Monasky MM, Janssen PML. Frequency-Dependant Contractile Response of Isolated Cardiac Trabeculae Under Hypo-,Normo-, and Hyper- thermic Conditions. J. Appl. Physiol 2006 May;100(5):1727-32. * these authors contributed equally. 3. Varian, KD and Janssen, PML. Frequency Dependent Acceleration of Relaxation Involves Decreased Myofilament Calcium Sensitivity. Am J Physiol Heart Circ Physiol 2007 May;292(5):H2212-9 4. Monasky MM, Varian KD, Janssen PML “Gender comparison of contractile performance and beta-adrenergic response in isolated rat cardiac trabeculae.” J Comp Physiol 2008 Mar;178(3):307-13 5. Monasky MM, Varian KD, Janssen PML “Dissociation of force decline from calcium decline by preload in isolated rabbit myocardium.” Pflugers Arch 2008 May;456(2):267- 76 viii FIELDS OF STUDY 1. Integrated biomedical sciences 2. Cardiac contractile physiology ix TABLE OF CONTENTS Page ABSTRACT........................................................................................................................ii DEDICATION.....................................................................................................................v ACKNOWLEDEMENTS...................................................................................................vi VITA.................................................................................................................................viii LIST OF TABLES............................................................................................................xiv LIST OF FIGURES...........................................................................................................xv LIST OF ABBREVIATIONS.........................................................................................xvii CHAPTERS 1. Introduction……………………………………………………………………...1 1.1 General introduction…………………………………………………..1 1.2 Excitation contraction coupling……………………………..………...4 1.2.1 The cardiac action potential………………………..………..4 1.2.2 The myocyte calcium transient……………………...…........5 1.2.3 The cardiac myofilaments………………………………..….7 1.3 Myofilament calcium sensitivity……………………………..………..9 1.4 Regulation of cardiac output and contractility…………………...…..11 1.4.1 Sympathetic modulation.......................................................12 1.4.2 The force frequency relationship..........................................13
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