Proper cardiac function relies on the stability and coordinated organization and activity of the sarcolemma, the transverse tubules and the sarcoplasmic reticulum, yet the structures that organize and stabilize these membranes are barely understood. The long-range goal of this research proposal is to understand how the membrane systems of cardiac muscle cells are organized and how they interact to promote proper cardiac function. To this end, we are studying the spectrin family of proteins in the heart. Spectrin is the prototypical member of a large group of filamentous cytoskeletal proteins called the 'spectrin superfamily' that are responsible for supporting and stabilizing the sarcolemma and internal membrane systems in the heart, their organization into distinct domains, and the ability of the sarcolemma to transduce the force of contraction. Surprisingly, the spectrins have been studied only cursorily in the heart, especially as their key roles are likely to be regulated by local signaling cascades involving phosphorylation and dephosphorylation. Here we propose to explore the hypothesis that the cytoskeletal structures created by the spectrin superfamily of proteins at the sarcolemma and t-tubule membranes, and regulated by protein kinases, are responsible for the formation and stabilization of membrane domains necessary for proper cardiac function. Our studies show that members of the spectrin superfamily, including dystrophin, bI-, aII- and bII-spectrin, form a highly crosslinked network on the inner surface of the cardiac sarcolemma. Spectrin networks for a different composition associate with transverse tubule (t- tubule) membranes. Molecular characterization of the spectrins indicates that their diversity, which results from alternative splicing as well as from the use of different gene projects, targets them to different membrane domains in cardiac muscle. Phosphorylation selectively controls the organization or stability of these domains, in part by regulating their association with spectrin. We propose to pursue thee preliminary observations through four specific aims: (1) to characterize further the spectrin-based membrane skeletal complex that organizes and supports the cardiac sarcolemmal membrane;(2) to identify and characterize the specialized spectrin network at gap junctions; (3) to determine the organization and function of spectrin networks associated with t-tubule membranes; and (4) to define the effect of phosphorylation on the spectrin network at t-tubules. As mutations in membrane-cytoskeletal proteins underlie dilated cardiomyopathies, our results should elucidate some of the basic cell biological mechanisms of heart disease.
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