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

基本信息

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
项目类别：
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

项目摘要

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“倦怠型”肥厚型心肌病是常见的遗传性心肌病，可导致目前，每年有超过 115,000 名退伍军人接受 VA Health 的心力衰竭护理。护理系统。尽管努力实施指南指导的药物治疗，但 5 年总体死亡率仍为诊断后约 50%，因此显然，这不仅对退伍军人而且对一般人都很重要人口。肌球蛋白重链 7 (MYH7) 突变是肥大和扩张的常见原因 MYH7 变异的基因检测因频繁识别 MYH7 变异而受到限制。当致病变异被发现时，其意义未知，并且缺乏疾病缓解疗法。该提案将确定会导致收缩功能障碍的 MYH7 变异，这是开发心力衰竭，并研究人类诱导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，最近被证明可以导致射血分数降低的心力衰竭这导致了 MYH7 的一个子集的假设。突变诱发的心肌病是由于这两种蛋白质之间的相互作用受损所致。工作使用饱和诱变和高通量改良酵母双杂交测定来识别几乎所有 MYH7 S2 结构域中的突变破坏了与 C1C2 结构域的正常蛋白质-蛋白质相互作用 cMyBPC。这将有助于识别所有易受感染的临床相关 MYH7 S2 变体。开发心力衰竭并生成“查找”表，以便能够自信地识别患者可能受益于治疗干预（目标 1）。最近的研究证明了肌球蛋白降解增加的可能性。肌肉环指蛋白-1 (MuRF1)，一种针对 MYH7 和其他肌节蛋白的 E3 连接酶表达致病性 MYH7 的人诱导多能干细胞来源的心肌细胞中的降解 E848G 变体导致 MYH7 突变诱导 MuRF1 上调的假设。心肌病会导致收缩功能障碍，降低 MuRF1 水平会增加收缩力。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 突变诱发的心肌病（目标 2）如果成功，将决定 MuRF1 是否可以成为一种新型心肌病。这些遗传性心肌病的治疗靶点。拟议的工作使用了多种创新技术。基因编辑人类诱导多能干细胞衍生的功能测定和机制研究心肌细胞识别具有发生心力衰竭风险的 MYH7 突变患者，然后确定潜在的新型疾病缓解干预措施的适用性。目标 1 中的方法可以与计算基因组学中提出的训练很好地结合起来。 MYH7 和其他肌节蛋白之间的相互作用，有可能识别所有临床相关的 MYH7 Aim 2 中人类诱导多能干细胞的机制研究将有助于阐明 MYH7 突变诱导的心肌病的发病机制以及与 Aim 1 一起将构成 CDA2 奖项第四年期间的未来优异奖提案总体而言，CDA2 将提供以下奖项。被提名者需要接受额外的培训才能使用基于 iPSC 的疾病模型和计算基因组学通过精准医学方法发现新的疾病缓解疗法。