Patient Specific Induced Pluripotent Stem Cell Derived Cardiomyocytes to Define Mechanisms of Electrical-Mechanical Dysfunction in DilatedCardiomyopathy

这份提案描述了一项为期五年的有指导的医生-科学家培训计划，供候选人开发 他在科学方法、指导、写作和资质方面的熟练程度。此外，候选人将 掌握基因组编辑、基因表型分析和心肌钙离子方面的独立技能 发信号。这将在研究晚期钠电流在扩张性疾病中的作用的背景下完成。 心肌病。晚期钠电流由功能失调的人类门控电压通道引起，已知 有助于其他心脏疾病，但晚期钠电流在扩张型心肌病中的作用仍然存在 不完全理解，主要基于动物模型。这里检验的假设是增强的 晚期钠电流进一步导致扩张型心肌病的机械和电功能障碍 钙离子转运加剧，导致去极化后延迟。这项提案旨在审查 人诱导多能干细胞在晚期钠电流疾病中的细胞机制 心肌细胞(IPSC-CM)。这项提议中的所有IPSC-CMS将使用一种新的协议来成熟，该协议增强了 IPSC-CMS的电学、机械和细胞信号特性更接近成年心肌细胞的特性。 首先，来自三名携带SCN5A变异的扩张型心肌病患者的心肌细胞将被检查以 确定晚期钠电流是否是这些病例心肌病的驱动机制。后来， 类似的实验将在IPSC-CMS中重复进行，这些患者来自非由 影响钠通道基因座的变异。晚期钠通道电流将被阻断和增强以评估 对机械和电学表型的影响。最后，兰尼定受体在调节 去极化后的延迟将被测试以确定这种细胞机制是否是 晚期钠电流导致心律失常。这将揭示与基因相关的特定基因疾病机制。 到晚期钠电流，这可能解释了电和机械功能障碍在病例中的不均一性 扩张型心肌病和确定新的治疗靶点。这项提议采用了一种新颖的方法来使用 IPSC-CM来源于扩张型心肌炎患者的多种病因变异，以研究疾病的差异 机制。这些实验的结果将为今后的工作检验提供初步的数据和专业知识 其他基因变异在影响扩张型心肌病电和机械功能障碍中的作用及识别 针对心肌病患者的基因特异性治疗。这些调查将根据 候选人咨询委员会的指导，该委员会在以下方面拥有丰富的专业知识 这项提议。斯坦福大学的环境非常适合这项研究计划，并为 候选人作为一名独立调查员的成长。在这项提议达到顶峰时，候选人将拥有 竞争R01资金并追求内科科学家职业生涯的专业知识和初步数据。

This proposal describes a five-year mentored physician-scientist training program for the candidate to develop his proficiency in the scientific method, mentorship, writing and grantsmanship. Furthermore, the candidate will acquire independent skills in areas of genome editing, genotype-phenotype analysis and cardiac calcium signaling. This will be accomplished in the context of investigating the role of late sodium current in dilated cardiomyopathy. Late sodium current arises from dysfunctional human gated voltage channels and is known to contribute to other cardiac disorders, but the role of late sodium current in dilated cardiomyopathy remains incompletely understood and largely based on animal models. The hypothesis examined here is that enhanced late sodium current results in mechanical and electrical dysfunction in dilated cardiomyopathy by further aggravation of calcium handling and gives rise to delayed after depolarizations. This proposal aims to examine the cellular mechanism of disease of the late sodium current using human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM). All iPSC-CMs in this proposal will be matured using a novel protocol that augments electrical, mechanical and cell signaling properties of iPSC-CMs closer to properties of adult cardiomyocytes. First, cardiomyocytes from three dilated cardiomyopathy patients with SCN5A variants will be examined to determine if the late sodium current is the driving mechanism of cardiomyopathy in those cases. Subsequently, similar experiments will be repeated in iPSC-CMs from patients with dilated cardiomyopathy not caused by variants affecting sodium channel loci. Late sodium channel current will be blocked and enhanced to evaluate the impact on mechanical and electrical phenotype. Lastly, the role of the ryanodine receptor in mediating delayed after depolarizations will be tested to determine if this cellular mechanism is the pathway by which the late sodium current contributes to arrhythmia. This will uncover genotype-specific disease mechanisms related to late sodium current that may explain heterogeneity in electrical and mechanical dysfunction across cases of dilated cardiomyopathy and identify novel therapeutic targets. This proposal takes a novel approach of using iPSC-CM derived from patients with multiple causative variants of DCM to study differences in disease mechanism. Results of these experiments will provide preliminary data and expertise to future work examining role of other genetic variants in influencing electrical and mechanical dysfunction in DCM and identifying genotype-specific therapies for patients with cardiomyopathy. These investigations will be performed under the guidance of the candidate’s advisory committee, which possesses significant expertise in the skills applied in this proposal. The environment at Stanford is ideal for this research proposal and provides a rich setting for the candidate’s growth as an independent investigator. At the culmination of this proposal, the candidate will have the expertise and preliminary data to compete for R01 funding and pursue a career as a physician scientist.