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: 10246981
• 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/10246981
• 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; Structure; 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; 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.

肥厚的心肌病（HCM）影响了500名以上的美国人，负担很大 心律不齐，心力衰竭和猝死的发病率。自发现以来已有25年以上 在HCM的遗传基础中，我们继续对 蛋白质功能上的基因突变，尚不清楚遗传突变如何导致肥厚的信号传导 在心肌细胞中。缺乏理解限制了HCM有效药物疗法的发展。 该提案的目的是进一步促进有关突变如何影响肌节功能的知识 使用纯化的蛋白质和皮肤纤维以及人类使用生化和生物物理方法以及人 诱导多能干细胞衍生的心肌细胞（HIPSC-CM）作为疾病建模的工具评估 导致肥大的触发因素。考虑到肌球蛋白重链的先前生化评估的发现 引起HCM的突变，假设HCM突变导致Saromere的功能增长 通过蛋白质相互作用和 肌球蛋白的激活状态。进一步假设NA的增加导致细胞的能量不平衡 增加ATP的使用情况，导致CA2+动力学和线粒体功能障碍改变。 在此提案中，与HCM相关的肌球蛋白调节轻链（RLC）中的3个突变是 选择进一步检验上述假设：E22K，R58Q和D166V。另外，链接的D94a突变 还选择了扩张的心肌病（DCM）来评估引起相反的突变的作用 心脏表型进行比较。 AIM 1通过 评估RLC与其他肌动成分之间的蛋白质 - 蛋白质相互作用（包括肌球蛋白 结合蛋白c）使用新型结合亲和力测定。 AIM 2量化了HCM突变对RLC的影响 相对于肌球蛋白，使用兔子的皮肤肌纤维和纯化的重组人蛋白 通过使用荧光ATP测量肌球蛋白的动力学来激活状态。最后，AIM 3定义了细胞 通过测量细胞力学，CA2+动力学和线粒体的RLC突变的影响 功能。我将特别专注于通过严格的结构评估获得正确成熟的HIPSC-CM。 当前的建议旨在进一步了解HCM的分子发病机理 蛋白质水平达到肌纤维水平，重点是肌​​球蛋白的结构变化，导致激活状态改变。会 还将这些生化发现与细胞水平的生物力学特性联系起来和转录 将进行分析以识别肥厚信号通路涉及的新基因靶标。这 提案还可以使我能够在Myofiber和HIPSC-CM中学习进一步的技能，作为表演的新平台 心肌病模型系统的功能分析。向前迈进，该提议将是我的基础 独立R01使用这些创新方法。