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

Myoscape 的分子特征，一种新型 L 型钙通道结合蛋白，参与心脏钙循环、收缩性和病理性肥大

• 批准号: 387388507
• 负责人: Dr. Matthias Eden
• 金额: --
• 依托单位: Klinik für Kardiologie, Angiologie und Pulmologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/387388507?language=en
• 关键词: Molecular; characterization; Myoscape; novel; type

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.

我们最近发现了一种新的心脏蛋白，命名为Myoscape/FAM40B/Strig2，它在小鼠和人的心脏中高度丰富。我们已经确定L型钙通道(LTCC)的远端C末端是Myoscape的直接分子相互作用伙伴。敲除心肌细胞上的Myoscape可降低细胞内钙离子，损害L型钙通道电流，导致心肌细胞收缩能力降低。反之，Myoscape的过表达增加了整体钙瞬变，增强了L型钙通道电流，并能够完全恢复衰竭心肌细胞减少的电流。在体内，Myoscape基因缺失的斑马鱼以及新产生的Myoscape基因敲除小鼠表现出心功能受损，进展为晚期心力衰竭。当这些小鼠处于慢性压力超负荷(TAC)时，心力衰竭加速。从机制上讲，由于LTCC表面表达受损，Myoscape缺陷小鼠表现出L型钙电流、细胞容量和钙电流密度的减少。由于Myoscape的过度表达能够挽救心力衰竭时LTCC电流的减少，我们现在的目标是进一步研究Myoscape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)利用新建立的心脏限制性Myoscape过表达转基因小鼠系，我们计划评估在TAC期间增加心肌Myoscape水平可能的保护作用。这一小鼠品系已经在基尔成功建立。(2)利用定点链突变技术，我们成功地产生了2.1个特异的腺病毒载体，表达先前在扩张型心肌病患者和QTC间期改变患者中发现的突变的MyoscapeD147N、R383Q、V629D和R217Ter。在这里，我们计划分析Myoscape序列变异是如何影响Myoscape表面表达并干扰心脏功能的。基于这些体外发现，我们计划在斑马鱼品系中产生敲击(3)，以阐明准确的机制，即Myoscape如何在体内和体外调节肥大。(4)通过分子和超微结构分析(EM；Di 8 ANEPPS；超分辨膜片钳和SICM)，我们计划评估小凹相关的LTCC和T管相关的LTCC对Myoscape的影响。(5)我们还计划分析针对内源性miRNAs的特定反义体对Myoscape序列的治疗潜力。