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The Effects of Cardiac Myosin Binding Protein-C and Inorganic Phosphate on Length-Dependent Activation

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THE EFFECTS OF CARDIAC MYOSIN BINDING PROTEIN-C AND INORGANIC PHOSPHATE ON LENGTH-DEPENDENT ACTIVATION By MILANA LEYGERMAN Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Adviser: Dr. Julian Stelzer Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY May, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________Milana Leygerman_________________________________ candidate for the ____Master of Science____degree *. (signed)_____________Dr. William Schilling (chair of the committee) __________________ Dr. Thomas Nosek ___________________Dr. Margaret Chandler ___________________ Dr. Saptarsi Haldar ___________________ Dr. Andrea Romani ___________________ Dr. Julian Stelzer (date) __________________03/16/2011_____ *We also certify that written approval has been obtained for any proprietary material contained therein. Acknowledgements I would like to thank my thesis advisor Dr. Julian Stelzer for all his guidance and help during my working on my thesis. I would also like to thank the lab personnel: Brian Ziese and Dr. Arthur Coulton for their help. Additionally, I would like to thank my thesis committee, including Dr. Nosek, Dr. Andrea Romani, Dr. Schilling, Dr. Chandler, and Dr. Haldar for all their support. Table of Contents LIST OF FIGURES...........................................................................................................3 ABSTRACT……………………………………………………………………………..4 INTRODUCTION………………………………………………………………………5 Actin...…………………………………………………………………………………....7 Titin……………………………………………………………………………………....8 Tropomyosin.…………………………………………………………………………….8 Troponins….……………………………………………………………………………..9 Myosin…………………………………………………………………………………...9 Myosin binding protein-C…………………………………………………………….....10 Cross-bridge cycling…………………………………………………………………….12 Frank-Starling Law of the Heart………………………………………………………...16 Role of inorganic phosphate in cross-bridge cycling…………………………………....17 EXPERIMENTAL DESIGN…………………………………………………………..20 Mouse models…………………………………………………………………………...21 Myocardial preparations………………………………………………………………...21 Experimental apparatus…………………………………………………………………22 Solutions………………………………………………………………………………...23 Phosphate studies……………………………………………………………………….27 RESULTS……………………………………………………………………………....27 DISCUSSION………………………………………………………………………….35 BIBLIOGRAPHY……………………………………………………………………..40 1 List of Figures Figure 1…………………………………………………………………………………..6 Figure 2…………………………………………………………………………………..7 Figure 3……………………………………………………………………………….....12 Figure 4………………………………………………………………………………….14 Figure 5………………………………………………………………………………….15 Figure 6………………………………………………………………………………….17 Figure 7………………………………………………………………………………….19 Figure 8………………………………………………………………………………….23 Figure 9………………………………………………………………………………….23 Figure 10………………………………………………………………………………...25 Figure 11………………………………………………………………………………...26 Figure 12………………………………………………………………………………...30 Figure 13………………………………………………………………………………...31 Figure 14………………………………………………………………………………...32 Figure 15………………………………………………………………………………...32 Figure 16………………………………………………………………………………...33 Figure 17………………………………………………………………………………...34 Figure 18………………………………………………………………………………...34 Figure 19………………………………………………………………………………...35 2 The Effects of Cardiac Myosin Binding Protein-C and Inorganic Phosphate on Length Dependent Activation Abstract By MILANA LEYGERMAN The contractile unit of a striated muscle is called the sarcomere and is composed of actin, myosin, titin, the troponin complex, tropomyosin, the myosin light chains, and cardiac myosin binding protein-C (cMyBP-C). Muscle contraction is caused by cross-bridge cycling, which involves the sliding of thick filaments past thin filaments. cMyBP-C is a constituent of the thick filament and is involved in regulation of contraction. Mutations in this protein have been known to lead to hypertrophic cardiomyopathy, an autosomal disease characterized by hypertrophy and fibrosis. Sarcomere length is an important determinant of muscle contractility as increased length results in greater overlap between actin and myosin leading to greater Ca2+-sensitivity and force generation. The Frank- Starling law of the heart is an important relationship for regulation of cardiac muscle contraction and is influenced by sarcomere length. In conditions of heart failure, there is a downward and rightward shift of the Frank-Starling relationship where an increase in end-diastolic volume generates a relatively smaller increase in stroke volume insufficient to meet the demands of the heart. Inorganic phosphate plays an important role in muscle 3 contraction as its release from the acto-myosin complex is a crucial step in the cross- bridge cycle that drives muscle contraction. In this study, to elucidate the effects of cMyBP-C and inorganic phosphate on length dependent activation, we utilized a skinned myocardium isolated from a knockout (KO) mouse model that lacks cMyBP-C (cMyBP- C-/-). A total of 85 mechanical experiments were performed in skinned fibers isolated from WT and KO left ventricles. The results showed that increased sarcomere length increases force generation and Ca2+-sensitivity of force in WT and KO animals. The rate of force development, an index of the speed of cross-bridge function was accelerated with increased sarcomere length in the KO fibers but not WT fibers. Treatment of muscle fibers with a low concentration of inorganic phosphate (1mM) did not have an effect on maximum calcium-activated force at short and long sarcomere length in either WT or KO myocardium, but accelerated rates of force development in both WT and KO muscle fibers. These results imply that the effects of sarcomere length are enhanced in KO myocardium due to the increased proximity of myosin to actin in fibers lacking cMyBP- C. Low concentrations of inorganic phosphate may accelerate the transitions from weak to strong binding cross-bridge states in both WT and KO myocardium, thereby accelerating rates of force development. 4 Introduction: The sarcomere (Figure 1) is the contractile unit of striated muscle (skeletal and cardiac) and contains the thin filament, the thick filament, and titin. Muscle contraction and relaxation occurs via the sliding of thick filaments past thin filaments through cross- bridge cycling as can be seen in Figure 2. The thin filament consists of actin, tropomyosin, and the troponin complex, while the thick filament then consists of myosin, an asymmetric dimer that contains a globular head portion ( S1), which is associated with two hetero-dimers light-chain 1 and light-chain 2 (LC-1 and LC-2), a hinged stalk region (S2) and a rod section (de Tombe et al, 2003). The S1 head portion also consists of the actin binding domain and ATP hydrolysis domain, whereas the myosin rod section is associated with the myosin binding protein-C (MyBP-C) (de Tomble et al, 2003). MyBP- C is involved in muscle contraction by stabilizing the thick filament and regulating the number of myosin heads available for involvement in a given contractile cycle. Cardiac myosin binding protein-C (cMyBP-C) increases the stiffness of the heart muscle, which slows early contraction but then allows systole to be sustained so the heart can effectively eject after greater stiffening. Cardiac muscle contraction depends on the properties of cross-bridge cycling, and involves several thin and thick regulatory proteins, which are described below. 5 Figure 1. The structure of a sarcomere. This figure was taken from Boron and Boulpaep, Medical Physiology, 2nd Edition. Reprinted with permission from Walter Boron. 6 Figure 2. Muscle contraction. Thick and thin filaments of the sarcomere slide past one another during contraction and relaxation. Tropomyosin overlays the binding sites of actin on myosin, which inhibits contraction. The troponin complex, which has three subunits (Troponin T-tropomyosin binding, Troponin I-inhibitory, and Troponin C-Ca2+ -binding) plays a role in regulating conformational changes of tropomyosin. This figure was taken from de Tombe et al., 2003. Reprinted with permission from Pieter de Tombe and the Journal of Biomechanics. Key contractile proteins Actin Actin is a constituent of the thin filament and is polymerized to a two-stranded helical structure called F-actin. F-actin is composed of G-actin, which represents individual globular actin subunits. Sub-domain 1 of the actin helix is believed to interact with myosin (Miller et al., 1995). Actin is anchored to the Z line of the sarcomere and its interaction with myosin produces force via formation of a cross-bridge. This protein is 7 known to have interactions with other sarcomeric proteins such as titin and S100A1, which is a calcium binding homodimer protein. Titin Titin is a large sarcomeric protein that extends from Z-line to M-line. A region of titin spans the I band of the sarcomere and can develop passive force in stretched sarcomeres (Granzier and Labeit, 2007) which contributes to the passive tension of the myocardium that determines diastolic filling (Granzier and Labeit, 2007). Titin also plays a role in stabilizing the thick filament during muscle contraction. Studies have suggested that titin may play a role in the sarcomere length-dependent increased Ca2+ sensitivity of active force, which is important for the Frank-Starling law of the heart, either by enhancing acto-myosin interaction through a decrease in interfilament lattice spacing or by increasing strain on the thick filament and influencing cross-bridge
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