Proper heart cell function depends on the intimate association between Ca2+ signaling elements in t-tubules and the conduction macromolecular complexes at gap junctions. These crucial molecules are locally organized and controlled by signaling cascades that interact with the continuous, dynamic cytoskeleton. The functional importance of this intricate network has been recently emphasized by its links to acquired and inherited forms of human dilated cardiomyopathies. Despite the emerging appreciation for the crucial role of cardiac cytoskeletal networks, these structures, and the signaling cascades that control their organization, are barely understood. Through the coordinated efforts of the project leaders, this PPG seeks to understand how Ca2+ signaling depends upon dynamic cytoskeletal organization and how they are both regulated by local, targeted signaling cascades in heart. Project 1 will examine the role of phosphatases in control of cytoskeletal architecture at t-tubules (link to Project 2) and L-type Ca2+ channel function (link to Project 4). The importance of phosphatases targeting will be further examined through ectopic expression of targeting motifs and tested in animal models of cardiomyopathies (link to Project 3). Project 2 will examine the cytoskeleton of cardiac myocytes, particularly the spectrin and dystrophin networks that organize and stabilize the sarcolemma and t-tubular membranes. The role of these networks in organizing gap junctions (link to Project 3) and in maintaining proper t-tubule/SR communication (link to Project 4) will be addressed, as will the effects of phosphorylation on these functions (link to Project 1). Project 3 will examine the role of stress-activated signaling in disruption of cytoskeletal structures (link to Project 2) and loss of connexin-43 at gap junctions through the use of transgenic mouse heart failure models. A goal will be to identify molecular mechanisms of stress-activated signaling on phosphatase retargeting (link to Project 1) and compromised EC coupling (link to Project 4). Project 4 will examine how Ca2+ signaling depends on filament proteins of t-tubule cytoarchitecture (link to Projects 1 and 2) and on the phosphorylation state of signaling proteins (link to Projects 1 and 3). Studies in transgenic animals will explore how the reorganization of the cytoskeleton contributes to altered Ca2+ signaling seen in cardiomyopathies (link to Projects 2 and 3). Thus, the well-integrated plans of the Project Leaders will provide new information on the fundamental molecular themes that link mechanical stress to local cellular signaling and ?cytoskeletonopathies? that underlie human heart disease.
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