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: 10421247
• 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/10421247
• 关键词: Accounting; Address; Affect; American; Award; Biological Assay; Cardiac Myosins; Cardiomyopathies; Caring; Cells; Clinical; Co-Immunoprecipitations; Data; Diagnosis; Dilated Cardiomyopathy; Disease; Disease model; Dominant-Negative Mutation; EFRAC; Emotional; Functional disorder; Future; General Population; Genetic; Genetic Databases; Genomics; Goals; Guidelines; Healthcare; Healthcare Systems; Heart failure; 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; Play; Process; Protein Isoforms; Proteins; Research; Ring Finger Domain; Risk; Rodent; Role; Techniques; Testing; Therapeutic Intervention; Thick Filament; Training; Two-Hybrid System Techniques; Up-Regulation; Variant; Veterans; Work; Yeasts; base; cardiac tissue engineering; cardiogenesis; cellular 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.

扩张和“烧毁”肥厚性心肌病是常见的遗传性心肌病，领导 心力衰竭。目前，每年有超过115,000名退伍军人因VA Health而受到心力衰竭的护理 护理系统。尽管努力实施指导指导的医疗疗法，但总体5年死亡率是 诊断后〜50％，因此显然这不仅对退伍军人，而且对一般都是一种重要的疾病 人口。 肌球蛋白重链7（MYH7）突变是肥厚和扩张的常见原因 心肌病。 MYH7变体的基因测试受到了经常识别的变体的限制 当发现致病性变异时，未知的显着性和缺乏改良疾病的疗法。这 提案将确定会导致收缩功能障碍的MYH7变体，这是开发的第一步 心力衰竭，研究人类诱导多能干细胞衍生的疾病发病机理 心肌细胞。 MYH7 S2结构域或心脏肌球蛋白结合蛋白C的C1C2结构域中的突变 （CMYBPC）破坏S2/C1C2之间正常蛋白质蛋白质相互作用的（最近已显示为 通过减少的射血分数诱导心力衰竭。这导致了一个假设，即myh7的一个子集 突变引起的心肌病是由于这两种蛋白质之间的相互作用受损。提议 工作使用饱和诱变和高通量改良的酵母双杂交测定法来识别几乎所有 MYH7 S2结构域中的突变破坏了正常蛋白质 - 蛋白质与C1C2结构域的相互作用 cmybpc。这将有助于确定所有容易受到影响的临床相关MYH7 S2变体 发展心力衰竭并产生“查找”桌子，以使患者有信心识别 这可能会受益于治疗干预措施（AIM 1）。功能异常功能的突变体MyH7蛋白提高了 肌球蛋白降解增加的可能性。最近的工作证明了这一点的支持 肌肉环蛋白-1（MURF1），一种靶向MYH7和其他肌肉蛋白的E3连接酶 降解，在人类诱导的多能干细胞衍生的心肌细胞中表达致病性MYH7 E848G变体。这导致了以下假设：MyH7突变引起的Murf1上调 心肌病有助于收缩功能障碍，而降低MURF1水平将增加收缩力。 拟议的工作将利用功能收益和功能丧失实验来阐明Murf1在 MYH7突变引起的心肌病（AIM 2）。如果成功，它将确定Murf1是否可以是小说 这些遗传性心肌病的治疗靶标。 拟议的工作使用了几种创新技术。它结合了尖端的高通量 在一般编辑的人类诱导的多能干细胞衍生的机械研究的功能测定 心肌细胞鉴定患有患心力衰竭风险的MYH7突变患者，然后 确定潜在的新型疾病改良干预的适用性。高通量测定法 与计算基因组学的拟议培训很好地整合了。 AIM 1中的方法稍后可以应用 MYH7与其他肉类蛋白之间的相互作用，以识别所有临床相关的MYH7 变体。人类诱导的多能干细胞在AIM 2中的机械研究将有助于阐明 MYH7突变引起的心肌病的发病机理以及AIM 1将构成A的基础 CDA2奖的第四年未来的绩效奖励提案。总体而言，CDA2将提供 提名人使用基于IPSC的疾病建模所需的其他培训 基因组学以精确的医学方法发现新的疾病改良疗法。