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

剖析MYH7 S2突变导致遗传性肥厚型心肌病的机制

基本信息

批准号：
10687810
负责人：
Alexander Loiben
金额：
$ 6.95万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10687810
关键词：
Address Adult Affect Alleles Amino Acids Benign Biopsy Blood CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiac Myosins Cellular biology Classification ClinVar Clinical Data Diagnosis Diagnostic Disease Familial Hypertrophic Cardiomyopathy Functional disorder Genes Genetic Genetic Transcription Goals Heart Heart failure Human Hypertrophic Cardiomyopathy Intervention Laboratory Research Lead Left Ventricular Hypertrophy Medicine Mentorship Methods Modeling Mutation Myosin ATPase Myosin Heavy Chains Outcome Pathogenesis Pathogenicity Patients Phenotype Photobleaching Protein Deficiency Protein Isoforms Proteins Pump Recovery Reporting Risk Rodent Model Role Sarcomeres Severities Site Testing Therapeutic Thick Filament Tissue Engineering Training Universities Variant Washington Work cardiac tissue engineering clinical application collaborative environment diagnostic strategy diagnostic tool economic impact experimental study improved induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes inherited cardiomyopathy middle age mutant myosin-binding protein C novel novel diagnostics novel therapeutics overexpression predictive tools rapid technique restoration supportive environment variant of unknown significance yeast two hybrid system

项目摘要

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.

家族性肥厚性心肌病 (HCM) 是一种遗传性心肌病，每 500 名美国成年人中就有 1 人患有这种疾病。1,2 肌球蛋白重链七 (MYH7)（一种肌节粗丝蛋白）的突变占 HCM 的 20-40% 3,4 然而，目前的理解缺乏一种通用的机制，通过该机制特定的 MYH7 变异会导致 HCM，并且不存在特定的疾病缓解疗法。5 MYH7 S2 结构域，宿主许多已鉴定的致病变异，与心肌肌球蛋白结合蛋白 C 的 C1C2 结构域相互作用 (cMyBP-C).6 我们从患有 MYH7 的 HCM 患者中产生了 hiPSC 衍生的心肌细胞 (hiPSC-CM) 变体 E848G。我们的初步数据表明 E848G 破坏了 S2/C1C2 相互作用并减少了 MYH7 丰富。因此，我的中心假设是致病性 MYH7 S2 变异会破坏 S2/C1C2 相互作用降低 MYH7 蛋白丰度导致收缩功能； S2/C1C2 相互作用的恢复（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 相互作用的破坏会导致收缩功能障碍； (2) 阐明 E848G诱导MYH7蛋白丰度丧失的机制并测试是否恢复正常的MYH7蛋白丰富可以挽救收缩功能。我将使用 CRISPR/Cas9 生成致病性 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。我会恢复通过互补氨基酸的靶向突变，致病性 MYH7 S2 变体破坏了 S2/C1C2 相互作用 C1C2 中的酸，理想地恢复相应工程心脏组织 (EHT) 的收缩功能。这些研究结果将应用于意义不明的 MYH7 S2 变体，以测试 S2/C1C2 完整性并在 hiPSC-CM。这一目标将建立一种新颖的快速方法，对未知的 MYH7 S2 变体进行功能重新分类意义。在第二个目标中，我将描述源自 MYH7-E848G hiPSC 的 EHT 的低收缩性。 CM 和与观察到的 MYH7 蛋白丰度损失相关。之后我将使用荧光恢复使用和不使用 E848G 的情况下进行光漂白 (FRAP) 以捕获肌节中 MYH7 循环的动态变体。 MYH7的过度表达和突变等位基因的沉默将测试MYH7之间的关系丰度和收缩功能。这些发现随后将在其他 MYH7 致病变异中得到证实。这一目标将建立一种通用机制，通过该机制，MYH7 变体会失去 MYH7 蛋白丰度并因此失去收缩功能。总之，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。该项目将在华盛顿大学的高度支持和协作的环境中进行药品。在我的赞助商和共同赞助商（Daniel Yang 博士和 Charles E. Murry 博士）的指导下，分别），这个项目将为我提供所需的培训，以实现我建立一个位于细胞生物学、组织工程和临床应用十字路口的独立研究实验室。