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

患者特异性诱导多能干细胞来源的心肌细胞以确定扩张型心肌病中电机械功能障碍的机制

• 批准号: 10227999
• 负责人: Karim Sallam
• 金额: $ 16.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227999
• 关键词: Action Potentials; Adult; Advisory Committees; Affect; Anemone; Animal Model; Animals; Applied Skills; Area; Arrhythmia; Automobile Driving; Bypass; CRISPR/Cas technology; Calcium; Calcium Signaling; Calmodulin; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cause of Death; Cell model; Cyclic AMP-Dependent Protein Kinases; Data; Dilated Cardiomyopathy; Disease; Effectiveness; Environment; Equilibrium; Evaluation; Foundations; Functional disorder; Funding; Future; Genetic; Genotype; Growth; Heart Diseases; Heart failure; Heterogeneity; Human; Incidence; Inherited; Investigation; Knowledge; Lead; Measures; Mechanics; Mediating; Mentors; Mentorship; Messenger RNA; Metabolic; Methods; Microscopy; Modeling; Modification; Molecular Abnormality; Other Genetics; Pathogenesis; Pathway interactions; Patients; Peptides; Pharmacology; Phenotype; Phosphorylation; Physicians; Play; Predisposition; Property; Proteins; Protocols documentation; Publishing; Recording of previous events; Research; Research Personnel; Research Proposals; Resolution; Risk; Role; RyR2; Ryanodine Receptor Calcium Release Channel; Scientist; Signal Transduction; Small Interfering RNA; Sodium; Sodium Channel; Sudden Death; Testing; Toxin; Training; Training Programs; Transfection; Treatment Efficacy; Variant; Ventricular Arrhythmia; Work; Writing; base; biobank; career; causal variant; disease phenotype; experimental study; genetic variant; genome editing; heart cell; heart rhythm; human model; improved; individualized medicine; induced pluripotent stem cell; knock-down; new therapeutic target; novel; novel strategies; phospholamban; ranolazine; rare variant; receptor; response; risk stratification; skills; targeted treatment; therapeutic evaluation; therapeutic target; voltage; voltage gated channel

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.

该提案描述了一个为期五年的指导医师科学家培训计划，供候选人发展 他在科学方法、指导、写作和资助方面的熟练程度。此外，候选人将 获得基因组编辑、基因型-表型分析和心脏钙领域的独立技能 这将在研究晚钠电流在扩张中的作用的背景下完成。 晚期钠电流是由功能失调的人类门控电压通道引起的，已知会导致心肌病。 导致其他心脏疾病，但晚钠电流在扩张型心肌病中的作用仍然存在 不完全理解并且主要基于动物模型。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 该提案旨在检查钙处理的加剧并导致去极化延迟。 利用人诱导多能干细胞研究晚期钠电流疾病的细胞机制 该提案中的所有 iPSC-CM 将使用一种增强的新方案来成熟。 iPSC-CM 的电学、机械和细胞信号传导特性更接近成人心肌细胞的特性。 首先，将对三名携带 SCN5A 变异的扩张型心肌病患者的心肌细胞进行检查，以了解 随后确定晚期钠电流是否是心肌病的驱动机制。 类似的实验将在来自非以下原因引起的扩张型心肌病患者的 iPSC-CM 中重复进行： 影响钠通道位点的变异将被阻断并增强以评估 最后，兰尼碱受体在介导中的作用。 将测试去极化后延迟的信号，以确定这种细胞机制是否是 晚钠电流导致心律失常，这将揭示与基因型特异性相关的疾病机制。 晚钠电流可能解释不同病例中电气和机械功能障碍的异质性 扩张型心肌病并确定新的治疗靶点该提案采用了一种新的方法。 iPSC-CM 源自患有 DCM 多种致病变异的患者，用于研究疾病差异 这些实验的结果将为未来的工作检查提供初步数据和专业知识。 其他遗传变异在影响 DCM 电气和机械功能障碍中的作用并识别 这些研究将在心肌病患者的基因型特异性治疗下进行。 候选人咨询委员会的指导，该委员会在所应用的技能方面拥有丰富的专业知识 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 在本提案的最终阶段，候选人将拥有 竞争 R01 资金并追求医师科学家职业生涯的专业知识和初步数据。