Multiscale investigation of cardiomyopathy-associated mutations in metavinculin

项目总结/摘要 家族性心肌病是一种遗传性心脏病，涉及心室重塑和心脏功能改变。 收缩性这些疾病通常是由肌节内的蛋白质突变引起的，肌节内的基本蛋白质突变是导致这些疾病的主要原因。 心肌细胞的收缩单位。参与机械传导的非肌节蛋白的突变， 细胞感知机械力并作出反应的过程也与心肌病有关 但受到的关注却少得多。例如，对人类患者的研究发现， 心肌病相关突变的metavinculin，肌肉特异性亚型的普遍存在的 mechanotransducer粘着斑蛋白，但这些突变如何导致疾病表型还没有得到很好的理解。 结构研究表明，将后黏着斑蛋白与黏着斑蛋白区分开的68个氨基酸插入物取代了 肌动蛋白结合纽蛋白尾部结构域中的第一个α-螺旋。虽然这种α-螺旋不直接结合肌动蛋白， 后黏着斑蛋白的插入导致肌动蛋白丝组织的剧烈变化， 黏着斑蛋白，这表明肌动蛋白结合的变构效应。这项拟议中的研究将检验以下假设： 由metavinculin突变引起的心肌病的致病机制涉及心脏 通过损伤后黏着斑蛋白与肌动蛋白的力依赖性结合来进行机械转导。单- 后黏着斑蛋白与肌动蛋白结合的分子力测量将直接解决力是否稳定 如先前已经证明的粘着斑蛋白与肌动蛋白的结合，以及 致病性突变对这种力依赖性结合。力依赖性改变的细胞后果 将在携带这种疾病的干细胞衍生的心肌细胞中研究metavinculin-actin结合， 使用肌节发生的活细胞成像（新肌节的建立）引起突变， 牵引力显微镜这些实验也将在无后黏着斑蛋白的心肌细胞上进行， 阐明metavinculin在肌角化和细胞收缩性中的作用。根据这项规定提供的培训 奖学金将在华盛顿大学医学院进行，该学院是生物医学教育的世界领导者 与研究拟议的研究符合NIH的战略目标，通过解决正常的 后黏着斑蛋白的生物学功能和发病和进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NIH使命的科学队伍的战略目标， 支持PI建立所需的研究，教学和专业技能的发展， 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.