The contraction of cardiac muscle is modulated by Ca2+/calmodulin- dependent myosin light chain kinase (MLCK), Ca2+ dependent protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) through the phosphorylation of myofibrillar proteins such as troponin T (TnT), troponin I (TnI) and myosin light chain 2 (MLC2). The overall goal of this proposal is to determine the concerted role(s) of myosin light chain kinase and protein kinase C isozymes in phosphorylating cardiac MLC2 and the function of the phosphorylation in cardiac physiology. Phosphorylation, activity assay, and contractile movement of myocytes will be used to determine the role(s) of MLCK and PKC isozymes in cardiac function. The effect of phosphorylation on Ca2+ stimulated MgATPase activity will be determined. Site-specific phosphorylation patterns of cardiac MLC2 by MLCK and PKC isozymes will also be determined through two-dimensional phosphopeptide mapping. Functional significance of phosphorylation of cardiac MLC2 by MLCK and PKC isozymes will be determined using cardiac recombinant MLC2 mutants in which the serine residues at the phosphorylation sites will be replaced by alanine residues. Contractility of the cardiomyocytes will be measured with and without MLCK and PKC inhibitors to determine the biochemical and physiological relevance of MLCK and PKC phosphorylation of the cardiac MLC2. It is anticipated that the data collected from this study will provide new information on the regulation of cardiac physiology by MLCK and PKC isozymes. Specific roles of MLCK and PKC isozymes in regulation cardiac function will be determined. The role(s) MLCK and PKC isozymes play in site-specific phosphorylation and in producing biochemical and physiological changes in the myofibrils will also be determined.
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