Molecular mechanisms of load-induced t-tubule regulation in the mammalian heart

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Project Summary Heart failure is most commonly associated with poor contractile function due to multi-level pathologic remodeling, including excitation-contraction coupling (ECC). This depends upon the proximity between membrane-bound L-type Ca2+ channels (LTCC) within the transverse (t)-tubule network and intracellular ryanodine receptors (RyR), which are normally very tightly colocalized. The PI and others have shown that abnormal mechanical load in vivo damages the t-tubule network, which results in uncoupling of LTCC and RyR. Junctophilin (JPH2), BIN 1 and Telethonin (TCAP), in interaction with the microtubule network, regulate t- tubule structure, but how they do so in response to load variation is not known. Prior experimental strategies have been unable to assess the effect of direct mechanical loading upon isolated cardiomyocytes, nor have they had the experimental flexibility to allow facile genetic manipulation of the pathways involved. Using new methods to directly modulate mechanical load on isolated cardiomyocytes and intact human myocardium in vitro, this K99/R00 seeks to test the hypothesis that t-tubule structure is normally regulated by a microtubule dependent JPH2, BIN1 and TCAP pathway, which in conditions of direct mechanical overload is deranged by microtubule mediated redistribution of JPH2, and reduced expression of JPH2, BIN 1 and TCAP. In Aim 1, using a novel magnetorheological elastomer (MRE) culture system, isolated cardiomyocytes will be subjected to pathological overload and undergo comprehensive characterization of ECC and t-tubule structure to test the hypothesis that cardiomyocyte-autonomous mechanisms are sufficient to mediate the load-dependent remodeling of the t-tubule system observed in heart failure. Because the phenotype arises in 48 hours, comprehensive dissection of the underlying molecular mechanisms will be performed by combined live cell imaging and adenoviral mediated genetic manipulations. Second, the novel but well-validated cardiac slice method will be used to specifically control pre-load and after-load in order to vertically integrate insights from cardiomyocyte-autonomous experiments in understanding the role of mechanical load regulation of the t- tubule system at the level of the isolated myocardium, including in human control and diseased myocardium. Mechanical unloading of failing hearts in vivo rescues t-tubule structure and ECC, which has been associated with significant contractile improvements. Using the tools developed in Aims 1 and 2, failing cells and slices will undergo mechanical unloading to determine the biomechanical and molecular mediators of this reverse remodeling. The completion of this work will significantly add to the PI's post-doctoral training in cellular electrophysiology, advanced super-resolution imaging and translational cardiovascular research and will be essential for his transition to independence.