Molecular characterization of Myoscape, a novel L-type Calcium channel binding protein involved in cardiac Calcium cycling, contractility and pathological hypertrophy

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We recently discovered a new cardiac protein named Myoscape/FAM40B/Strip2 which is highly enriched in the murine and human heart. We have identified the distal C-terminus of the L-type calcium channel (LTCC) as a direct molecular interaction partner for Myoscape. Knockdown of Myoscape in cardiomyocytes decreased cellular Calcium and impairs L-type Calcium-channel currents resulting in reduced contractility of cardiomyocytes. Conversely, overexpression of Myoscape increased global Ca2+ transients and enhanced L-type Ca2+channel-currents, and it was able to fully restore decreased currents in failing cardiomyocytes. In vivo, Myoscape-null zebrafish as well as newly generated Myoscape-knockout mice displayed impairment of cardiac function progressing to advanced heart failure. Heart failure was accelerated when these mice were subjected to chronic pressure overload (TAC). Mechanistically, Myoscape-deficient mice showed reduced L-type-Ca2+currents, cell capacity, and calcium current densities as a result of impaired LTCC surface expression. Since Myoscape overexpression was able to rescue decreased LTCC currents in heart failure, we now aim to further examine the role of Myoscape in the pathogenesis and progression of heart failure. We thus propose several complementary experimental approaches: (1) Utilizing a newly generated heart restricted Myoscape overexpression transgenic mouse line, we plan to assess possible protective roles of increased myocardial Myoscape levels during TAC. This mouse line has already been successfully established in Kiel. (2) Using site strand mutagenesis, we were able to successfully generate 2.1 specific adenoviral constructs expressing mutated Myoscape variants (murineMyoscape D147N, R383Q, V629D and R217Ter), previously identified in patients with dilated cardiomyopathy as well as in a cohort of patient with altered Qtc intervals. Here we plan to analyse, how Myoscape sequence variants affect Myoscape surface expression and interfere with cardiac function. Based on these in vitro findings we plan to generate knock in zebrafish lines (3) To clarify the precise mechanism, how Myoscape modulates hypertrophy in vivo and in vitro. (4) Via molecular and ultrastructural analysis (EM; Di 8 ANEPPS; Super resolution patch clamp and SICM, we plan to evaluate the involvement of caveolae associated LTCC and t-tubule associated LTCC for the effects of Myoscape (5) We also plan to analyse the therapeutic potential of specific antagomirs targeting endogenous MiRNAs against Myoscape sequence.