Multiscale investigation of cardiomyopathy-associated mutations in metavinculin

项目摘要/摘要 家族性心肌病是一种遗传性心脏病，涉及心室重构和心脏改变 伸缩性。这些疾病通常是由肌节内的蛋白质突变引起的，肌节是最基本的 心肌细胞的收缩单位。参与机械转导的非肌瘤蛋白的突变， 细胞感知和响应机械力的过程也与心肌病有关 但受到的关注要少得多。例如，对人类患者的研究已经确定 心肌病相关的Metavinculin基因突变，这是一种无处不在的肌肉特异性亚型 机械转导纽蛋白，但这些突变如何导致疾病表型还不是很清楚。 结构研究表明，区分Metavinculin和vinculin的68个氨基酸插入片段取代了 肌动蛋白结合纽蛋白尾部区域的第一个α-螺旋。虽然这个α-螺旋不直接结合肌动蛋白， 与之相比，Metavinculin插入导致肌动蛋白细丝的组织发生了剧烈的变化 到纽蛋白，暗示了肌动蛋白结合的变构效应。这项拟议的研究将检验这一假设 Metavinculin突变引起的心肌病的发病机制与心脏功能紊乱有关 通过损害Metavinculin与肌动蛋白的力依赖结合而进行的机械转导。单人- Metavinculin与肌动蛋白结合的分子力测量将直接解决作用力是否稳定的问题 Metavinculin与肌动蛋白的结合，就像以前对vinculin所证明的那样，以及 这种力依赖结合上的致病突变。改变力量依赖的细胞后果 将在携带这种疾病的干细胞来源的心肌细胞中研究Metavinculin与肌动蛋白的结合。 使用肌瘤发生的活细胞成像(建立新的肉瘤)和 牵引力显微镜。这些实验也将在Metavinculin缺失的心肌细胞上进行 阐明Metavinculin在肌瘤发生和细胞收缩中的作用。在此项下提供的培训 奖学金将在生物医学教育的世界领先者华盛顿大学医学院举行 和研究。拟议的研究与美国国立卫生研究院的战略目标相一致，通过解决正常 Metavinculin的生物学功能及其发生发展的病理生物学机制 由Metavinculin突变引起的心肌病。此外，拟议的培训计划将有助于 发展一支能够完成国家卫生研究院使命的科学队伍的战略目标是 支持研究、教学和专业技能的发展，以使PI建立一个 在心脏机械生物学领域成功的独立研究项目。

Project Summary/Abstract Familial cardiomyopathies are genetic heart diseases that involve ventricular remodeling and altered cardiac contractility. These diseases are often caused by mutations in proteins within the sarcomere, the fundamental contractile unit of cardiomyocytes. Mutations in non-sarcomeric proteins involved in mechanotransduction, the process by which cells sense and respond to mechanical force, have also been implicated in cardiomyopathy but have received considerably less attention. For instance, studies of human patients have identified cardiomyopathy-associated mutations in metavinculin, the muscle-specific isoform of the ubiquitous mechanotransducer vinculin, but how these mutations lead to the disease phenotype is not well-understood. Structural studies have shown that the 68-amino acid insert that differentiates metavinculin from vinculin replaces the first alpha-helix in the actin-binding vinculin tail domain. Although this alpha-helix does not directly bind actin, the metavinculin insert results in drastic changes of the organization of actin filaments by metavinculin compared to vinculin, suggesting an allosteric effect on actin binding. The proposed research will test the hypothesis that the pathogenic mechanism of cardiomyopathy caused by mutations in metavinculin involves disruption of cardiac mechanotransduction through impairment of the force-dependent binding of metavinculin to actin. Single- molecule force measurements of metavinculin binding to actin will directly address whether force stabilizes binding of metavinculin to actin, as has been previously demonstrated for vinculin, as well as the effect of pathogenic mutations on this force-dependent binding. The cellular consequences of altered force dependence of metavinculin-actin binding will be investigated in stem cell-derived cardiomyocytes that carry the disease- causing mutations using live-cell imaging of sarcomerogenesis (the establishment of new sarcomeres) and traction force microscopy. These experiments will also be carried out on metavinculin-null cardiomyocytes to elucidate the role of metavinculin in sarcomerogenesis and cellular contractility. The training provided under this fellowship will take place at the Washington University School of Medicine, a world leader in biomedical education and research. The proposed research aligns with the strategic objectives of the NIH by addressing the normal biological function of metavinculin and the pathobiological mechanism underlying the onset and progression of cardiomyopathy caused by mutations in metavinculin. In addition, the proposed training plan will contribute to the strategic objective of developing a scientific workforce capable of accomplishing the NIH’s mission by supporting the development of research, teaching, and professional skills required for the PI to establish a successful independent research program in the field of cardiac mechanobiology.