Dissecting the mechanism of how dominant negative MYH7 mutations lead to genetic cardiomyopathies

剖析 MYH7 显性失活突变导致遗传性心肌病的机制

• 批准号: 10515348
• 负责人: Kai-Chun Daniel Yang
• 金额: --
• 依托单位: VA PUGET SOUND HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10515348
• 关键词: Accounting; Address; Affect; American; Award; Biological Assay; Cardiac Myosins; Cardiomyopathies; Caring; Cells; Clinical; Co-Immunoprecipitations; Data; Development; Diagnosis; Dilated Cardiomyopathy; Disease; Disease model; Dominant-Negative Mutation; EFRAC; Emotional; Functional disorder; Future; General Population; Genetic; Genetic Databases; Genomics; Goals; Guidelines; Healthcare Systems; Heart failure; Hospitalization; Human; Hypertrophic Cardiomyopathy; Impairment; Induced Mutation; Intervention; Knock-out; Lead; Mammalian Cell; Medical; Methods; Modeling; Morbidity - disease rate; Muscle; Mutagenesis; Mutation; Myosin ATPase; Myosin Heavy Chains; Pathogenesis; Pathogenicity; Patients; Process; Protein Isoforms; Proteins; Research; Ring Finger Domain; Risk; Rodent; Role; Susceptibility Gene; Techniques; Testing; Therapeutic Intervention; Thick Filament; Training; Two-Hybrid System Techniques; Up-Regulation; Variant; Veterans; Work; Yeasts; cardiac tissue engineering; clinically relevant; experimental study; familial dilated cardiomyopathy; gain of function; genetic testing; heart cell; high throughput screening; human disease; improved; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; inherited cardiomyopathy; innovation; loss of function; mortality; mutant; myosin-binding protein C; new therapeutic target; novel; overexpression; precision medicine; preservation; protein degradation; protein protein interaction; ubiquitin-protein ligase; variant of unknown significance; yeast two hybrid system

Dilated and “burnt out” hypertrophic cardiomyopathies are common genetic cardiomyopathies that lead to heart failure. Currently over 115,000 Veterans annually receive care for heart failure from the VA Health Care System. Despite efforts to implement guideline-directed medical therapy, the overall 5 year mortality is ~50% after diagnosis, so clearly this is a disease important to not only Veterans but also the general population. Myosin heavy chain 7 (MYH7) mutations are common causes of hypertrophic and dilated cardiomyopathies. Genetic testing for MYH7 variants have been limited by frequent identification of variants of unknown significance and the lack of disease-modifying therapies when pathogenic variants are identified. This proposal will identify MYH7 variants that will cause contractile dysfunction, the first step to the development of heart failure, and study the disease pathogenesis in human induced pluripotent stem cell-derived cardiomyocytes. Mutations in either the MYH7 S2 domain or the C1C2 domain of cardiac myosin binding protein C (cMyBPC) that disrupt the normal protein-protein interaction between S2/C1C2 have recently been shown to induce heart failure with reduced ejection fraction. This leads to the hypothesis that a subset of MYH7 mutation-induced cardiomyopathies are due to impaired interaction between these two proteins. The proposed work uses saturation mutagenesis and high-throughput modified yeast two-hybrid assays to identify nearly all mutations in the MYH7 S2 domain that disrupt normal protein-protein interaction with the C1C2 domain of cMyBPC. This will assist in identifying all clinically relevant MYH7 S2 variants that are susceptible to developing heart failure and generate a “look up” table that would enable the confident identification of patients that could benefit from therapeutic intervention (Aim 1). Abnormally functioning mutant MYH7 protein raises the possibility of increased myosin degradation. This is supported by recent work demonstrating an upregulation of muscle RING-finger protein-1 (MuRF1), an E3 ligase that targets MYH7 and other sarcomeric proteins for degradation, in human induced pluripotent stem cell-derived cardiomyocytes expressing the pathogenic MYH7 E848G variant. This leads to the hypothesis that MuRF1 upregulation in MYH7 mutation-induced cardiomyopathies contributes to systolic dysfunction and that reducing MuRF1 levels will increase contractility. The proposed work will use gain-of-function and loss-of-function experiments to elucidate the role of MuRF1 in MYH7 mutation-induced cardiomyopathies (Aim 2). If successful it will determine if MuRF1 can be a novel therapeutic target for these genetic cardiomyopathies. The proposed work uses several innovative techniques. It combines cutting-edge high-throughput functional assays with mechanistic studies in genetically-edited human induced pluripotent stem cell-derived cardiomyocytes to identify patients with MYH7 mutations that are at risk of developing heart failure and then determines the suitability of a potential novel disease-modifying intervention. The high-throughput assays will integrate well with the proposed training in computational genomics. The method in Aim 1 can later be applied to interactions between MYH7 and other sarcomeric proteins to potentially identify all clinically relevant MYH7 variants. The mechanistic studies in human induced pluripotent stem cells in Aim 2 will help elucidate the pathogenesis of MYH7 mutation-induced cardiomyopathies and together with Aim 1 will form the basis for a future Merit Award proposal during the 4th year of the CDA2 award. Overall, the CDA2 will provide the additional training necessary for the nominee to use iPSC-based disease modeling with computational genomics to discover new disease-modifying therapies with precision medicine approaches.

扩张型和“耗尽型”肥厚型心肌病是常见的遗传性心肌病 心力衰竭。目前，每年有超过115,000名退伍军人接受退伍军人管理局的心力衰竭护理 护理系统。尽管努力实施指导方针的药物治疗，但总的5年死亡率为 ~50%，所以显然这不仅对退伍军人，而且对普通民众都是一种重要的疾病 人口。 肌球蛋白重链7(MYH7)突变是肥大和扩张的常见原因 心肌病。对MYH7变种的基因检测一直受到频繁识别MYH7变种的限制 在确定致病变种时，未知的意义和缺乏疾病修改疗法。这 提案将确定会导致收缩功能障碍的MYH7变体，这是开发 心力衰竭，并研究人类诱导的多能干细胞来源的疾病发病机制 心肌细胞。 心肌肌球蛋白结合蛋白C的MYH7 S2结构域或C1C2结构域突变 (CMyBPC)破坏了S2/C1C2之间正常的蛋白质-蛋白质相互作用，最近被证明 降低射血分数，导致心力衰竭。这导致假设MYH7的一个子集 突变引起的心肌病是由于这两种蛋白之间的相互作用受损。建议数 Work使用饱和诱变和高通量改良酵母双杂交试验来鉴定几乎所有 MYH7 S2结构域的突变破坏了正常的蛋白质与C1C2结构域之间的相互作用 CMyBPC。这将有助于识别所有临床上相关的MYH7 S2变异，这些变异对 发展为心力衰竭，并生成一个“查找”表，从而能够自信地识别患者 这可受益于治疗性干预(目标1)。功能异常的突变MYH7蛋白增加了 肌球蛋白降解增加的可能性。这一点得到了最近一项研究的支持，该工作证明了 肌肉环指蛋白-1(MuRF1)，一种针对MYH7和其他肌瘤蛋白的E3连接酶 表达致病MYH7的人诱导多能干细胞来源的心肌细胞的降解 E848G变种。这导致了MYH7突变导致MuRF1上调的假设 心肌病导致收缩功能障碍，降低MuRF1水平将增加收缩能力。 这项拟议的工作将使用功能获得和功能丧失实验来阐明MuRF1在 MYH7突变引起的心肌病(目标2)。如果成功，它将决定《MuRF1》能否成为一部小说 这些遗传性心肌病的治疗靶点。 这项拟议的工作使用了几种创新技术。它结合了尖端的高吞吐量 基因编辑的人诱导多能干细胞的功能分析和机制研究 心肌细胞鉴定MYH7突变患者有发生心力衰竭的风险，然后 确定一种潜在的新型疾病修正干预措施的适宜性。高通量化验将 与拟议的计算基因组学培训很好地结合在一起。目标1中的方法可以稍后应用 MYH7与其他肌瘤蛋白之间的相互作用，以潜在地识别所有临床相关的MYH7 变种。在AIM 2中对人类诱导多能干细胞的机制研究将有助于阐明 MYH7突变引起的心肌病的发病机制，以及Aim 1将构成 在CDA2奖的第四个年头期间的未来功勋奖提案。总体而言，CDA2将提供 被提名者使用基于IPSC的计算疾病建模所需的额外培训 基因组学通过精确医学方法发现新的疾病修改疗法。