The goal of this proposal is to determine the molecular basis of how phosphorylation modification of cardiac proteins by cAMP dependent protein kinase A (PKA) regulate cardiac function. PKA activation under many physiological and pharmacological conditions in the heart regulates a broad spectrum of responses in cardiomyocytes by protein modification to accelerate contraction and relaxation in the intact heart. Downstream regulations of the PKA pathway are mediated by PKA regulatory subunits, such as RI and RII, and A-kinase anchoring proteins (AKAPs), which deliver PKA to specific locations within the cell through binding to R subunits. Rh is the primary PKA subunit responsible for the phosphorylation of many proteins in the heart. Alterations of PKA regulatory subunits have been found in heart failure patients. However, mechanisms underline PKA subunits alterations in failing hearts and their physiological roles in regulation of cardiac function remain unclear. Specific functions for RI and RII have also not been determined in cardiomyocytes. In this proposal, we will test the hypothesis that PKA regulatory subunits levels play important role in maintaining heart function. Using the cardiomyocyte cell culture system and cardiac specific transgenic approaches we will manipulate the RI:RII ratio in cardiomyocytes and study the consequences with molecular, cellular and pathophysiological techniques. The following specific aims are proposed:
Specific Aim 1 : To over express wild type (wt) and mutant PKA RII subunit with Ser96->Ala point mutation on its auto-phosphorylation site in cardiomyocytes.
Specific Aim 2 : To determine the biochemical and functional consequences in cardiomyocytes expressing predominantly the wt and mutant RII subunit.
Specific Aim 3 : To determine if transgenic over-expressions of the wt PKA RII and its non-phosphorylatable form affect cardiac function and lead to cardiac pathological changes. Results from these studies will help us better understand how the heart regulates its function and hence to propose more specific therapeutic strategies in treating heart diseases.
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