The present application is a request, to extend the Pi's MERIT Award for a further 5 years of support. The goal of this project is to elucidate mechanisms by which Ca^*, intermolecular cooperativity, and protein phosphorylations regulate myocardial contraction in health and disease. The objective of this proposal is to determine the roles of myosin binding protein-C (cMyBP-C), with emphasis on the regulation of contraction by cMyBP-C phosphorylation. We propose: Hypothesis 1, cMyBP-C modulates contraction by binding to myosin subfragment 2 (S2), thereby controlling the availability of cross-bridges to actin;Hypothesis 2, reduced systolic function in our cMyBP-C null mouse results from accelerated cross- bridge kinetics due to deletion of cMyBP-C;and Hypothesis 3, the positive inotropy induced by ^ff-adrenergic agonists is due in part to PKA-mediated phosphorylation of cMyBP-C. Considerable progress has been made in the current grant period in testing each of these principal hypotheses, including the following results: 1) cMyBP-C is the primary regulator of myofibrillar contractile kinetics due to PKA phosphorylation of contractile proteins during )?-adrenergic stimulation of myocardium, 2) cMyBP-C binds to myosin along the long axis of the thick filament, thereby refuting the widely held """"""""collar model"""""""" of cMyBP-C binding, 3) phosphorylation or ablation of cMyBP-C causes cross-bridges to move toward the thin filament, and 4) CAMKII phosphorylation of cMyBP-C mediates the positive force-frequency response in myo-cardium. These and other results set the stage for studies of the molecular mechanisms of these findings. We will develop new mouse lines expressing phosphorylation mutants of cMyBP-C to identify the PKA and CAMKII sites in cMyBP-C, the order of these phosphorylations, and the effects of each on myocardial function. We will determine whether the effects of cMyBP-C are due to its interactions with myosin or if, as proposed by some, its putative binding to actin is also involved. Further studies will focus on the functional and structural roles of cMyBP-C in living nnuscle by studying the effects of its ablation or phosphorylation on contraction in vivo and in isolated muscle. The possibility that the disease phenotypes of cMyBP-C knock-out and phosphorylation mutant mice are due in part to compensatory mechanisms will be studied by reconstitution of null myocardium with wild-type and mutant proteins, by conditional expression of null and mutant alleles, and by re-expression of wild-type alleles. These results promise to provide insights into the mechanisms by which contractile state is modulated in healthy myocardium and also the basis for functional deficits in diseased hearts.
Work from this laboratory and others has demonstrated that myosin binding protein C is a critical regulator of heart muscle function in health and in diseases such as heart failure and hyper- trophic cardiomyopathies. The studies proposed in this project are designed to understand the types of cardiac function that are mediated by this protein and also the changes in cardiac function due to disease mutations in the gene encoding the protein. Elucidating the molecular mechanisms of action of myosin binding protein C will lead to the identification of new therapeutic targets and strategies for the treatment of diseases of heart muscle and the heart.
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