The Role of Yes Associated Protein (YAP) in Hypertrophic Cardiomyopathy

项目总结/摘要 肥厚型心肌病（HCM）是最常见的遗传性心血管疾病， 500个人晚期临床表现为心肌收缩过度， （器官和单个心肌细胞的增大）和纤维化。在B- 肌球蛋白重链（MYH 7）、肌球蛋白结合蛋白C（MYBPC 3）和肌钙蛋白（cTn）与 并在器官水平上增加收缩力。然而，分子水平上的动力学仍然存在 不清楚，因为不同的肌节蛋白突变可导致力增加、减少或不变。 生产在理解这些在分子水平上改变的动力学如何导致 HCM在细胞水平上更晚期的肥大表型。有趣的是，河马之路 已证明在发育生长期间被激活，在心脏稳态期间静止， 在病理性生长中重新激活（即HCM）。然而，它参与疾病，特别是启动 对肥大表型的认识很少。在这里，我们的目标是了解是否稳态 通过典型的生长调节因子Hippo-YAP的机械信号传导被改变：1）通过改变细胞内的 单一HCM突变心肌细胞的生物力学和2）通过改变机械环境。我们 建议使用基因编辑的人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）， 与HCM相关的点突变，作为一个降序模型来研究 机械信号和肥大生长。我们将调节机械应力（即患病条件） 在健康和患病心肌细胞中通过用正性肌力药物处理和在纤维样硬组织中培养 条件下，并通过荧光标记的关键调控蛋白， Hippo途径（雅普）。为了进一步阐明雅普促进表型的机制， HCM我们已经开发了一种新的光遗传学工具，称为OptoYAP，它提供了完整的时间和空间信息。 控制河马路径最后，我们的目标是了解雅普在细胞内重新激活的机制。 通过扰乱细胞核的机械信号来治疗病理状况。我们假设1）变化 力的产生改变了Hippo通路的稳态机械信号传导，以启动细胞内的 肥大和2）细胞外环境的后续变化（硬化）复合了这种效应 导致前馈信号推进疾病表型。3)病理性雅普信号传导是由 细胞骨架过度的力传递导致细胞核变形。我们的结果将提供 深入了解HCM进展，并为治疗HCM的治疗方案提供试验平台。

Project Summary/Abstract Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease – affecting 1 in 500 individuals. Advanced forms of the disease clinically present with hypercontractility, hypertrophy (enlargement of the organ and individual cardiomyocytes) and fibrosis. Several single-point mutations in b- myosin heavy chain (MYH7), Myosin Binding Protein C (MYBPC3), and Troponin (cTn) have been associated with HCM and increased contractility at the organ level. However, the kinetics at the molecular level remain unclear, as different sarcomeric protein mutations can result in increased, decreased, or unchanged force production. A knowledge gap persist in understanding how these altered kinetics at the molecular level lead to the more advanced hypertrophic phenotype of HCM at the cellular level. Interestingly, the Hippo Pathway has been demonstrated to be activated during developmental growth, quiescent during cardiac homeostasis, and reactivated in pathological growth (i.e. HCM). However its involvement in the disease, in particular the initiation of the hypertrophic phenotype, is poorly understood. Here, we aim to understand whether homeostatic mechanical signaling through the canonical growth regulator, Hippo-YAP, is altered 1) by changes in the biomechanics of single HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We propose to use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) genetically edited to harbor point mutations associated with HCM, as a reduce ordered model to study the relationship between mechanical signaling and hypertrophic growth. We will modulate mechanical stresses (i.e. diseased conditions) in healthy and diseased cardiomyocytes by treatment with inotropic drugs and culture in fibrotic-like stiff conditions and track the resulting signaling events by fluorescently labeling the key regulatory protein of the Hippo pathway (YAP). To further elucidate the mechanism by which YAP is contributing to the phenotypes of HCM we have developed a novel optogenetic tool, termed OptoYAP, which provides full temporal and spatial control of the Hippo pathway. Lastly we aim to understand the mechanism behind the reactivation of YAP in pathological conditions by perturbing the mechanical signaling by the nucleus. We hypothesize that 1) changes in force production alter the homeostatic mechano-signaling of the Hippo pathway to initiate cellular hypertrophy and 2) subsequent changes to the extracellular environment (stiffening) compounds this effect leading to a feedforward signal progressing the disease phenotypes. 3) pathological YAP signaling is driven by excessive force transmission by the cytoskeleton resulting in nuclear deformation. Our results will provide insights into HCM progression and provide a testbed for therapeutic options in treating HCM.