Bridging the gap between mutation & cellular effects: Defining the mechanisms of hypertrophic cardiomyopathy

弥合突变之间的差距

• 批准号: 10459570
• 负责人: Masataka Kawana
• 金额: $ 16.2万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10459570
• 关键词: ATP phosphohydrolase; Actins; Affect; Affinity; American; Arrhythmia; Behavior; Binding; Biochemical; Biological Assay; Biological Models; Biomechanics; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Clinical; Cytoplasm; DNA Sequence Alteration; Development; Dilated Cardiomyopathy; Disease; Disease model; Electron Microscopy; Ensure; Event; Fiber; Future; Gene Expression Profiling; Gene Targeting; General Population; Generations; Genetic; Genetic Diseases; Goals; Grant; Head; Heart; Heart failure; Human; Hypertrophic Cardiomyopathy; Hypertrophy; In Vitro; Kinetics; Knowledge; Laboratories; Lead; Learning; Light; Link; Measures; Mechanics; Mitochondria; Molecular; Morbidity - disease rate; Mutation; Myosin ATPase; Myosin Heavy Chains; Myosin Regulatory Light Chains; Oryctolagus cuniculus; Oxygen Consumption; Pathogenesis; Patients; Pharmacotherapy; Phenotype; Preparation; Property; Protein Biochemistry; Proteins; Recombinants; Reproducibility; Research; Research Personnel; Sarcomeres; Secondary to; Seminal; Signal Pathway; Signal Transduction; Skin; Sturnus vulgaris; Sudden Death; System; Testing; Therapeutic Intervention; Tissues; Training; Work; Writing; biophysical properties; biophysical techniques; career; design; gain of function; genetic approach; improved; indexing; induced pluripotent stem cell derived cardiomyocytes; innovation; mitochondrial dysfunction; myosin-binding protein C; novel; novel therapeutic intervention; protein function; protein protein interaction; risk stratification; skills; symposium; targeted treatment; therapeutic target; tool

Hypertrophic cardiomyopathy (HCM) affects more than 1 in 500 Americans with an extensive burden of morbidity in the form of arrhythmia, heart failure, and sudden death. More than 25 years since the discovery of the genetic underpinnings of HCM, we continue to have limited understanding of the primary effect of genetic mutation on protein function and it is unclear how the genetic mutation leads to hypertrophic signaling in cardiomyocytes. This lack of understanding limits the development of effective pharmacotherapy for HCM. The objective of this proposal is to further advance the knowledge of how mutations affect sarcomere function using biochemical and biophysical approach using purified protein and skinned fiber, as well as human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) as tools for disease modeling to assess the triggers leading to hypertrophy. Given the findings from prior biochemical assessment of myosin heavy chain mutation that cause HCM, it is hypothesized that HCM mutations result in gain of function in sarcomere by increase in number of myosin heads available for cross-bridge formation (Na) through protein interaction and activation state of myosin. It is further hypothesized that increase in Na result in energy imbalance in cells due to increased ATP usage, leading to altered Ca2+ dynamics and mitochondrial dysfunction. In this proposal, 3 mutations in regulatory light chain (RLC) of myosin that are linked to HCM are chosen to test the above hypothesis further: E22K, R58Q and D166V. In addition, D94A mutation that is linked to dilated cardiomyopathy (DCM) is also chosen to assess the effect of mutation that causes the opposite cardiac phenotype for comparison. Aim 1 measures the impact of HCM mutations on myosin's folded state, by assessing protein-protein interaction between the RLC and other sarcomere components (including myosin binding protein C) using novel binding affinity assay. Aim 2 quantifies the effect of HCM mutations on RLC using skinned myofiber from rabbit and purified recombinant human protein, with respect to myosin activation state by measuring the kinetics of myosin using fluorescent ATP. Finally, Aim 3 defines the cellular effect of RLC mutations using hiPSC-CM, by measuring cell mechanics, Ca2+ dynamics and mitochondrial function. I will particularly focus on obtaining properly matured hiPSC-CM by rigorous structural assessment. The current proposal is designed to gain further understanding of molecular pathogenesis of HCM from protein level to myofiber level, focusing on myosin's structural change leading to altered activation state. It will also link these biochemical findings to biomechanical property at the cellular level, and transcriptional profiling will be performed to identify new gene targets involved in hypertrophic signaling pathway. The proposal will also allow me to learn further skills in myofiber and hiPSC-CM as new platforms for performing functional analysis of cardiomyopathy model systems. Moving forward, this proposal will be the basis of my independent R01 grant using these innovative approaches.

肥厚型心肌病(HCM)在美国每500人中就有1人患病，负担广泛 以心律失常、心力衰竭和猝死的形式出现的发病率。距离这一发现已有25年之久 对于HCM的遗传基础，我们对其主要作用的了解仍然有限。 基因突变对蛋白质功能的影响，目前尚不清楚基因突变是如何导致肥大信号的 在心肌细胞中。这种认识的缺乏限制了肥厚型心肌炎有效药物治疗的发展。 这项建议的目的是进一步加深对突变如何影响肌节功能的了解。 使用生化和生物物理方法，使用纯化的蛋白质和皮肤纤维，以及人类 诱导多能干细胞来源的心肌细胞(hiPSC-CM)作为疾病模型的工具 导致肥大的诱因。考虑到先前对肌球蛋白重链进行生化评估的结果 导致HCM的突变，假设HCM突变通过以下方式导致肌节功能增强 通过蛋白质相互作用增加可用于跨桥形成(Na)的肌球蛋白头部的数量 肌球蛋白的激活状态。进一步推测，钠的增加会导致细胞内能量失衡。 增加ATP的使用，导致钙动力学改变和线粒体功能障碍。 在这项建议中，肌球蛋白调节轻链(RLC)中与HCM相关的3个突变是 选择E22K、R58Q和D166V进一步检验上述假设。此外，D94A突变与 扩张型心肌病(DCM)也被选择来评估导致相反的突变的影响 心脏表型以供比较。目的1测量HCM突变对肌球蛋白折叠状态的影响，通过 评估RLC和其他肌节成分(包括肌球蛋白)之间的蛋白质-蛋白质相互作用 结合蛋白C)使用新的结合亲和力分析。目的2量化HCM突变对RLC的影响 兔皮肌纤维和纯化的重组人蛋白对肌球蛋白的影响 用荧光三磷酸腺苷测定肌球蛋白的激活状态。最后，目标3定义了细胞 用HiPSC-CM检测细胞力学、钙动力学和线粒体对RLC突变的影响 功能。我将特别关注通过严格的结构评估获得适当成熟的hiPSC-CM。 目前的建议旨在进一步了解肥厚性心肌炎的分子发病机制。 从蛋白质水平到肌纤维水平，重点关注肌球蛋白的结构变化导致激活状态的改变。会的 也将这些生化发现与细胞水平的生物力学特性和转录 将进行图谱分析，以确定与肥大信号通路有关的新基因靶点。这个 建议书还将让我学习肌纤维和HiPSC-CM方面的进一步技能，作为表演的新平台 心肌病模型系统的功能分析。展望未来，这项提议将成为我的 使用这些创新方法的独立R01赠款。