Modeling myosin mechanobiology towards understanding the mechanisms of hypertrophic cardiomyopathy

项目概述：本项目的重点是了解β心肌肌球蛋白的突变是如何发生的。 对肌球蛋白分子功能具有不同的和经常相反的作用，导致相似的疾病表型 肥厚型心肌病（HCM）：心肌细胞（CM）肥大、过度收缩和组织纤维化。 了解疾病机制和多尺度表型异质性 （分子、细胞和临床）将使更好和更个性化的治疗方法得以发展。 对于HCM患者。本建议的第一个目的是阐明细胞内改变的机制， 力影响细胞内信号传导和CM肥大。第二个目标将使用计算建模， 检查肌球蛋白功能的相关参数的改变如何在时间上改变力的产生， 在空间上。第三个目标将确定细胞外力学的改变如何影响CM和心脏成纤维细胞 表型该提案使用了几种创新的分子，细胞，生物工程和计算 建模工具，以检查和情境化改变的细胞力学在驱动心脏变化中的作用， 细胞表型人类诱导多能干细胞中的CRISPR/Cas9基因编辑提供了一个模型系统 研究特定突变在动态细胞环境中的影响。微图案化工程平台 将用于改善肌原纤维排列，并允许直接测量细胞内力的产生， 牵引力显微镜分子生物学和转录组学技术将用于测量 细胞内信号传导和基因表达对机械扰动的响应。另一个创新是 黏着斑蛋白张力传感器FRET探针直接测量肌节细胞内力的新应用 Z盘和细胞粘连。这些平台一起将允许直接验证时间和空间 使用计算建模（分子肌球蛋白模型和细胞特异性有限元）预测细胞力 模型）。最后，研究细胞外力学改变对CM功能和心脏成纤维细胞的影响， 在工程环境中的活化将使我们深入了解纤维化重塑的影响。研究与 职业发展培训计划将使Vander Roest博士能够过渡到一个独立的 事业在本提案的指导（K99阶段）期间，Vander Roest博士将在以下方面发展技术技能： 分子生物力学，FRET测量和转录组分析，并扩大她的技能， 在心脏病的背景下进行计算建模。培训将在斯坦福大学进行， 在伯恩斯坦和斯普迪奇博士的指导下，以及一个专家跨学科咨询委员会， 包括2名计算建模专家（Regnier博士和坎贝尔博士）。这些技能的发展将 支持该项目独立阶段的研究计划，结合新的技能和经验 Vander Roest博士过去在肌成纤维细胞机械生物学方面的经验， 成功过渡到独立。

PROJECT SUMMARY: This proposed project focuses on understanding how mutations in beta cardiac myosin with diverse and often opposing effects on myosin molecular function contribute to similar disease phenotypes of hypertrophic cardiomyopathy (HCM): cardiomyocyte (CM) hypertrophy, hypercontractility, and tissue fibrosis. Understanding disease mechanisms and the heterogeneity of phenotypes across multiple scales (molecular, cellular, and clinical) will enable the development of better and more individualized therapies for patients with HCM. The first aim of this proposal will clarify the mechanisms by which altered intracellular forces affect intracellular signaling and CM hypertrophy. The second aim will use computational modeling to examine how alterations to interrelated parameters of myosin function change force production temporally and spatially. The third aim will define how altered extracellular mechanics affect CM and cardiac fibroblast phenotypes. The proposal uses several innovative molecular, cellular, bioengineering, and computational modeling tools to examine and contextualize the role of altered cellular mechanics in driving changes in cardiac cell phenotypes. CRISPR/Cas9 gene editing in human induced pluripotent stem cells provides a model system to investigate the effects of specific mutations in a dynamic cellular context. Micropatterned engineered platforms will be used to improve myofibril alignment and allow direct measurement of intracellular force production by traction force microscopy. Molecular biology and transcriptomic techniques will be used to measure changes in intracellular signaling and gene expression in response to mechanical perturbation. Another innovation is the novel application of a vinculin tension sensor FRET probe to directly measure intracellular forces at sarcomere Z disks and at cellular adhesions. Together these platforms will allow direct validation of temporal and spatial cellular forces predicted using computational modeling (molecular myosin models and cell-specific finite element models). Finally, investigating the effect of altered extracellular mechanics on CM function and cardiac fibroblast activation in engineered environments will give insights into the effects of fibrotic remodeling. The research and career development training plans will enable the transition of Dr. Vander Roest to an independent career. During the mentored (K99 phase) of this proposal, Dr. Vander Roest will develop technical skills in molecular biomechanics, FRET measurements, and transcriptome analysis, and expand her skills in computational modeling in the context of cardiac disease. This training will take place at Stanford University, under the mentorship of Drs. Bernstein and Spudich, as well as an expert trans-disciplinary advisory committee, including 2 experts in computational modeling (Drs. Regnier and Campbell). The development of these skills will support the research plan for the independent phase of this project, combining new skills and experience gained during this award with Dr. Vander Roest’s past experience in myofibroblast mechanobiology to facilitate a successful transition to independence.