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

该提案描述了一个为期五年的指导医生科学家培训计划的候选人发展 他在科学方法、指导、写作和语法方面的熟练程度。此外，候选人将 在基因组编辑、基因型-表型分析和心脏钙离子分析领域获得独立技能 信号这将在研究晚期钠电流在扩张型心肌细胞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产生，并且已知其 但晚期钠电流在扩张型心肌病中的作用仍然存在 不完全了解，主要是基于动物模型。这里检验的假设是， 晚期钠电流导致扩张型心肌病机械和电功能障碍 钙处理的恶化并引起延迟的后去极化。这项建议旨在研究 利用人诱导多能干细胞衍生的晚期钠电流疾病的细胞机制 心肌细胞（iPSC-CM）。该提案中的所有iPSC-CM都将使用一种新的协议来成熟， iPSC-CM的电、机械和细胞信号传导性质更接近于成年心肌细胞的性质。 首先，将检查来自三名具有SCN 5A变体的扩张型心肌病患者的心肌细胞， 确定在这些病例中，晚期钠电流是否是心肌病的驱动机制。随后，委员会注意到， 类似的实验将在来自扩张型心肌病患者的iPSC-CM中重复， 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 对机械和电表型的影响。最后，Ryanodine受体在介导 延迟后去极化将被测试，以确定这种细胞机制是否是途径， 迟发钠电流导致心律失常。这将揭示基因型特异性疾病相关机制 晚期钠电流，这可能解释了电和机械功能障碍的异质性， 扩张型心肌病和确定新的治疗靶点。该提案采用了一种新颖的方法， iPSC-CM来源于患有DCM的多种致病变体的患者，以研究疾病的差异 机制这些实验结果将为今后的研究工作提供初步的数据和经验 其他遗传变异在影响扩张型心肌病的电和机械功能障碍中的作用， 针对心肌病患者的基因型特异性治疗。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门知识： 这个提议。斯坦福大学的环境非常适合这项研究计划，并为研究提供了丰富的环境。 候选人作为独立调查员的成长在这一建议的高潮，候选人将有 专业知识和初步数据，以竞争R 01资金，并追求职业生涯作为一个医生科学家。

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.