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

肥厚型心肌病（HCM）影响超过1/500的美国人，具有广泛的负担 心律不齐、心力衰竭和猝死的发病率。自25年前发现 HCM的遗传基础，我们仍然有有限的了解的主要影响， 基因突变对蛋白质功能的影响，目前还不清楚基因突变如何导致肥大信号传导 在心肌细胞中。这种缺乏了解限制了HCM有效药物治疗的发展。 该建议的目的是进一步推进突变如何影响肌节功能的知识 使用生物化学和生物物理方法，使用纯化的蛋白质和带皮纤维，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， 引发肥大。鉴于先前肌球蛋白重链生化评估的结果， 突变导致HCM，假设HCM突变导致肌节功能的获得， 通过蛋白质相互作用可用于交叉桥形成（Na）的肌球蛋白头数量增加， 肌球蛋白的激活状态。进一步假设Na的增加导致细胞中的能量不平衡， 增加ATP的使用，导致改变Ca 2+动力学和线粒体功能障碍。 在这个提议中，与HCM相关的肌球蛋白调节轻链（RLC）中的3个突变是 选择进一步测试上述假设：E22 K、R58 Q和D166 V。此外，D94 A突变与 扩张型心肌病（DCM）也被选择来评估突变的影响，导致相反的 用于比较的心脏表型。目的1测量HCM突变对肌球蛋白折叠状态的影响， 评估RLC和其它肌节组分（包括肌球蛋白）之间的蛋白质-蛋白质相互作用 结合蛋白C）。目的2量化HCM突变对RLC的影响 使用来自兔的去皮肌纤维和纯化的重组人蛋白质，关于肌球蛋白 激活状态通过使用荧光ATP测量肌球蛋白的动力学。最后，目标3定义了蜂窝 使用hiPSC-CM，通过测量细胞力学、Ca 2+动力学和线粒体 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。 目前的建议是为了获得进一步了解分子发病机制的HCM从 蛋白质水平到肌纤维水平，关注肌球蛋白的结构变化导致激活状态改变。它将 还将这些生化发现与细胞水平的生物力学特性和转录水平联系起来， 将进行谱分析以鉴定参与肥大信号传导途径的新基因靶标。的 我的建议也将使我能够进一步学习肌纤维和hiPSC-CM作为新的表演平台的技能 心肌病模型系统的功能分析。展望未来，这项建议将是我的基础， 独立R 01赠款使用这些创新的方法。

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.