Many investigations have helped define the molecular elements underlying the major Ca2+ signaling cycle in heart known as the excitation-contraction (EC) coupling cascade. Since phosphorylation reactions have been a preeminent focus of regulatory mechanisms of this pathway, protein phosphatases are under-appreciated enzymes in cardiac signaling. The PI has surprising new preliminary results that inhibition of serine/threonine phosphatases, PP1 and PP2A, leads to a rapid delocalization of a transverse-tubule-associated cytoskeletal protein that is accompanied by changes in EC coupling, ion channel activity and contractile events. These results are consistent with the emerging theme that intracellular signaling specificity includes the intimate association of kinases and phosphatases with specific subcellular sites that controls the local dynamic balance of protein phosphorylation. This hypothesis will be explored from a new perspective by identifying the role of spatial segregation of protein phosphatase 2A (PP2A) in signaling in cardiac myocytes. The project is organized around three inter-related aims. (1) What are the physiological properties of local events in the excitation-contraction coupling cascade that are regulated by phosphatases? Fundamental properties of cardiac myocytes regulated by protein phosphatases will be characterized. (2) How does subcellular targeting of PP2A regulate Ca2+ signaling and excitation contraction coupling in heart cells? New viral gene transfer methods will be used to overexpress targeting subunits of PP2A. The functional impact will be assessed. (3) How does subcellular targeting of PP2A regulate signal transduction pathways in cardiac myocytes? These experiments will capitalize on a new discovery by the PI that the overexpression of a PP2A targeting subunit blunts beta-adrenergic responsiveness in cultured cardiac myocytes. A strength of this proposal is that an integrated approach will be used that exploits viral gene transfer, biochemistry, cell biology, voltage-clamp and high resolution imaging of intracellular Ca2+ and cardiac proteins. Based on the preliminary data presented, this project will define important regulatory mechanisms at local strategic sites in cardiac cells. Successful completion of the planned work will yield new insights into the molecular processes underlying fundamental signaling changes that are seen in pathological processes of heart failure and the senescent heart.
