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

我们最近发现了一种新的心脏蛋白myscape /FAM40B/Strip2，它在小鼠和人类心脏中高度富集。我们已经确定了l型钙通道（LTCC）的远端c端是myscape的直接分子相互作用伙伴。心肌细胞myscape的下调降低了细胞钙，损害了l型钙通道电流，导致心肌细胞收缩性降低。相反，myscape的过表达增加了全局Ca2+瞬态，增强了l型Ca2+通道电流，并且能够完全恢复衰竭心肌细胞中减少的电流。在体内，myscape基因缺失的斑马鱼和新产生的myscape基因敲除小鼠表现出心功能损害，进展为晚期心力衰竭。当这些小鼠遭受慢性压力过载（TAC）时，心力衰竭加速。在机制上，由于LTCC表面表达受损，肌景观缺陷小鼠表现出l型ca2 +电流、细胞容量和钙电流密度降低。由于myscape过表达能够挽救心力衰竭中LTCC电流的减少，我们现在的目标是进一步研究myscape在心力衰竭的发病和进展中的作用。因此，我们提出了几种互补的实验方法：(1)利用新生成的心脏限制性肌景观过表达转基因小鼠系，我们计划评估TAC期间心肌肌景观水平增加可能的保护作用。该小鼠系已在基尔成功建立。(2)使用位点链诱变技术，我们能够成功生成2.1个特异性腺病毒构建体，表达突变的myscape变体（鼠myyoscape D147N， R383Q， V629D和R217Ter），这些变体先前在扩张型心肌病患者以及Qtc间隔改变的患者队列中发现。在这里，我们计划分析肌景观序列变异如何影响肌景观表面表达并干扰心功能。基于这些体外研究结果，我们计划在斑马鱼细胞系中产生敲击(3)，以阐明myscape如何在体内和体外调节肥大的确切机制。(4)通过分子和超微结构分析（EM）、Di 8 ANEPPS、超分辨率膜片钳和SICM，我们计划评估小泡相关LTCC和t小管相关LTCC对myscape的影响。(5)我们还计划分析针对内源性MiRNAs的特异性拮抗剂对myscape序列的治疗潜力。

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.