Dissecting the mechanisms of how MYH7 S2 mutations lead to genetic hypertrophic cardiomyopathy

家族性肥厚型心肌病(HCM)是一种遗传性心肌病，每500名美国成年人中就有1人患病。 肌球蛋白重链7(MYH7)是一种肌节粗丝蛋白，它的突变占肥厚性心肌病的20%-40% 案例3，4然而，目前的理解缺乏一种可概括的机制，通过这种机制可使具体的多年期高峰期7 变异会导致HCM，并且不存在特定的疾病修改疗法。5 MYH7 S2结构域，宿主 许多已发现的致病变异体与心肌肌球蛋白结合蛋白C的C1C2结构域相互作用 (cMyBP-C)。6我们从一名患有MYH7的肥厚性心肌病患者体内培养出了HiPSC来源的心肌细胞(hiPSC-CMS)。 变种E848G。我们的初步数据表明，E848G扰乱了S2/C1C2的相互作用，并减少了MYH7 富足。因此，我的中心假设是破坏S2/C1C2相互作用的致病MYH7 S2变异 减少MYH7蛋白丰度导致收缩功能；恢复S2/C1C2相互作用 (目标1)或MYH7丰度(目标2)可能会改善收缩功能。我的具体目标是：(1) 证明MYH7 S2/C1C2相互作用的中断会导致收缩功能障碍；以及(2)阐明 E848G诱导MYH7蛋白丰度丧失的机制及能否恢复正常MYH7蛋白的检测 富足可以挽救收缩功能。我将用CRISPR/Cas9产生致病的MYH7 S2变体 在哺乳动物双杂交方法中用于量化S2/C1C2相互作用的功能障碍。我会恢复的 致病MYH7 S2变异体通过互补氨基靶向突变破坏S2/C1C2相互作用 C1C2中的酸性物质，理想地恢复了相应的工程心脏组织(EHTS)的收缩功能。这些 研究结果将应用于意义未知的MYH7 S2变种，以测试S2/C1C2完整性，并在 HiPSC-CMS。这一目标将建立一种新的快速方法，从功能上重新分类未知的MYH7 S2变体 意义。在第二个目标中，我将表征来源于MYH7-E848G HiPSC的EHTS的收缩能力低下。 CMS，并与观察到的MYH7蛋白丰度的丢失相关。之后我会用荧光恢复 光漂白(FRAP)技术在使用和不使用E848G的肌瘤中捕捉MYH7循环的动力学 变种。MYH7的过表达和突变等位基因的沉默将检验MYH7与MYH7的关系 丰度和收缩功能。这些发现将在其他MYH7致病变种中得到证实。 这一目标将建立一种可推广的机制，通过该机制，MYH7变体将失去MYH7蛋白丰度 并因此丧失收缩功能。总而言之，这些实验将使诊断工具能够预测 MYH7 S2变异的致病性和解决收缩功能的治疗方法。这个项目将 在华盛顿大学系高度支持和协作的环境中举行 医学。在我的赞助人和共同赞助人(Daniel Yang博士和Charles E.Murry博士)的指导下， )，这个项目将为我提供所需的培训，以实现我建立 位于细胞生物学、组织工程和临床应用的十字路口的独立研究实验室。

Familial hypertrophic cardiomyopathy (HCM) is a genetic cardiomyopathy affecting 1 in 500 US adults.1,2 Mutation in myosin heavy chain seven (MYH7), a sarcomeric thick filament protein, accounts for 20-40% of HCM cases.3,4 However, current understanding lacks a generalizable mechanism through which specific MYH7 variants result in HCM, and no specific disease-modifying therapy exists.5 The MYH7 S2 domain, host to numerous identified pathogenic variants, interacts with the C1C2 domain of cardiac myosin binding protein C (cMyBP-C).6 We have generated hiPSC-derived cardiomyocytes (hiPSC-CMs) from an HCM patient with MYH7 variant E848G. Our preliminary data suggest E848G disrupts the S2/C1C2 interaction and reduces MYH7 abundance. Thus, my central hypothesis is pathogenic MYH7 S2 variants that disrupt S2/C1C2 interaction and reduce MYH7 protein abundance result in contractile function; restoration of S2/C1C2 interaction (Aim 1) or MYH7 abundance (Aim 2) may improve contractile function. My specific aims are to: (1) demonstrate disruption of MYH7 S2/C1C2 interaction results in contractile dysfunction; and (2) elucidate the mechanism of E848G-induced loss of MYH7 protein abundance and test whether restoring normal MYH7 protein abundance can rescue contractile function. I will generate pathogenic MYH7 S2 variants with CRISPR/Cas9 for use in a mammalian two-hybrid approach to quantify dysfunction of the S2/C1C2 interaction. I will restore S2/C1C2 interaction disrupted by pathogenic MYH7 S2 variant by targeted mutation of the complementary amino acid in C1C2, ideally recovering contractile function in corresponding engineered heart tissues (EHTs). These findings will be applied to MYH7 S2 variants of unknown significance to test for S2/C1C2 integrity and verify in hiPSC-CMs. This aim will establish a novel rapid method to functionally reclassify MYH7 S2 variants of unknown significance. In the second aim, I will characterize hypocontractility in EHTs derived from MYH7-E848G hiPSC- CMs and correlate with observed loss in MYH7 protein abundance. I will use fluorescent recovery after photobleaching (FRAP) to capture the dynamics of MYH7 cycling in sarcomeres with and without the E848G variant. Overexpression of MYH7 and silencing of the mutant allele will test the relationship between MYH7 abundance and contractile function. These findings will then be corroborated in other MYH7 pathogenic variants. This aim will establish a generalizable mechanism through which MYH7 variants lose MYH7 protein abundance and consequently lose contractile function. In sum, these experiments will enable diagnostic tools for predicting pathogenicity of MYH7 S2 variants and therapeutic approaches to address contractile function. This project will take place in the highly supportive and collaborative environment of the University of Washington Department of Medicine. With the mentorship of my Sponsor and Co-Sponsor (Dr. Daniel Yang and Dr. Charles E. Murry, respectively), this project will provide the training required for me to realize my goal of establishing an independent research laboratory at the crossroads of cellular biology, tissue engineering, and clinical application.