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

• 批准号: 10463127
• 负责人: Alexander Loiben
• 金额: $ 6.72万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 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/10463127
• 关键词: Address; Adult; Affect; Alleles; Amino Acids; Benign; Biopsy; Blood; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cellular biology; 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; Sum; Testing; Therapeutic; Thick Filament; Tissue Engineering; Training; Universities; Variant; Washington; Work; Yang; 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

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.

家族性肥厚型心肌病（HCM）是一种遗传性心肌病，影响1/500的美国成年人。 肌球蛋白重链7（MYH 7）突变是一种肌节粗丝蛋白，占HCM的20- 40 3，4然而，目前的理解缺乏一种可推广的机制，通过这种机制，特定的MYH 7 MYH 7 S2结构域是HCM的宿主， 许多已鉴定的致病性变体，与心肌肌球蛋白结合蛋白C的C1 C2结构域相互作用 6我们已经从患有MYH 7的HCM患者产生了hiPSC衍生的心肌细胞（hiPSC-CM）。 变体E848 G。我们的初步数据表明，E848 G破坏了S2/C1 C2相互作用，降低了MYH 7 丰饶。因此，我的中心假设是破坏S2/C1 C2相互作用的致病性MYH 7 S2变体 降低MYH 7蛋白丰度导致收缩功能;恢复S2/C1 C2相互作用 (Aim 1）或MYH 7丰度（Aim 2）可改善收缩功能。我的具体目标是：（1） 证明MYH 7 S2/C1 C2相互作用的破坏导致收缩功能障碍;和（2）阐明MYH 7 S2/C1 C2相互作用的机制。 E848 G诱导MYH 7蛋白丰度丧失的机制以及是否恢复正常MYH 7蛋白的测试 丰富可以挽救收缩功能。我将用CRISPR/Cas9产生致病性MYH 7 S2变体， 在哺乳动物双杂交方法中用于定量S2/C1 C2相互作用的功能障碍。我也必复还 致病性MYH 7 S2变体通过互补氨基酸的靶向突变破坏S2/C1 C2相互作用 C1 C2中的酸，理想地恢复相应工程化心脏组织（EHT）中的收缩功能。这些 研究结果将应用于意义未知的MYH 7 S2变体，以检测S2/C1 C2完整性，并验证 hiPSC-CM。这一目标将建立一种新的快速方法，对未知的MYH 7 S2变体进行功能重新分类。 意义在第二个目标中，我将表征来自MYH 7-E848 G hiPSC的EHT中的收缩功能减退。 CM，并与观察到的MYH 7蛋白丰度的损失相关。我将使用荧光恢复后 光漂白（FRAP），以捕获有和没有E848 G的肌节中MYH 7循环的动力学 变量。MYH 7的过表达和突变等位基因的沉默将测试MYH 7与基因突变之间的关系。 丰度和收缩功能。这些发现将在其他MYH 7致病性变体中得到证实。 该目标将建立MYH 7变体失去MYH 7蛋白丰度的可推广机制 从而失去收缩功能。总之，这些实验将使诊断工具能够预测 MYH 7 S2变体的致病性和解决收缩功能的治疗方法。该项目将 发生在华盛顿系的大学的高度支持和协作的环境 药在我的赞助商和共同赞助商（丹尼尔杨博士和查尔斯E。默里， 本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交叉点的独立研究实验室。