Understanding the Mechanisms of Ventricular Dysfunction in Hypoplastic Left Heart Syndrome

项目摘要/摘要 左心发育不全综合征(HLHS)是美国新生儿心源性死亡的主要原因 各州占所有先天性心脏病的2%-3%。而“HLHS”则用来描述一系列 困扰左心室的异常，定义为左心严重发育不全和上行。 主动脉伴随二尖瓣和主动脉瓣闭锁或狭窄。目前，长期存在的关于 HLHS的病因学认为，主动脉或二尖瓣阻塞损害了通过左心室的血流。 导致心脏左侧结构发育不全。虽然临床试验试图舒缓血液 子宫内血流障碍，这些研究在挽救人类左心室生长和血流方面取得的成功有限。 HLHS胎儿。在这里，我提出的初步发现表明，来自HLHS患者的心肌细胞- 来源的人诱导多能干细胞(HiPSC)表现出显著的收缩能力降低 一代人认为固有的心肌功能障碍可能是导致脑室血液供应中断的原因之一 流。这些发现提出了开发潜在改善的治疗策略的令人兴奋的可能性 预防宫内左室发育不全的心肌功能。 这项提议的总体目标是确定引起 与HLHS心肌细胞收缩功能受损有关。假设是HLHS的脑室 心肌细胞表现出核心转录调节因子的异常表达 维护用于健康心肌功能的收缩机械。使用HiPSC模型系统， 我将使用健康的和HLHS患者来源的HiPSCs来检验中心假设，并实现这一目标 申请。具体目标1：确定引起心肌收缩功能受损的分子机制 HLHS-HiPSC来源的心室肌细胞。具体目标2：确定心肌细胞的作用 HLHS-hiPSC来源的心肌细胞中细胞功能和存活的转录调节。vbl.使用 CRISPR/Cas9基因组编辑，我将介绍一种遗传报告系统，该系统将允许分离 体外诱导hPSC向左、左室心肌细胞分化。这将为研究 特异性肌节和电生理机制驱动收缩功能受损并评估 这些缺陷是否是LV特有的。使用最先进的单细胞RNA测序技术，我将 对HLHS-HiPSC-心肌细胞进行广泛的转录图谱分析以确定异常表达 参与调控心肌细胞功能和存活所需基因的关键转录调控因子。 拟议工作的成功执行将带来三个重大贡献：1)将确定 HLHS心肌收缩功能障碍的特定分子驱动因素，2)将首次揭示 心肌收缩功能缺陷和机制是左心室特有的，这可能解释了左心室特有的 HLHS的损伤将识别驱动HLHS收缩功能受损的新的转录途径。

PROJECT SUMMARY/ABSTRACT Hypoplastic Left Heart Syndrome (HLHS) is the leading cause of neonatal cardiac death in the United States accounting for 2-3% of all congenital heart disease. While “HLHS” is used to describe a spectrum of anomalies afflicting the left ventricle, it is defined as the severe underdevelopment of the left heart and ascending aorta along with atresia or stenosis of the mitral and aortic valves. Currently, the long-standing theory for the etiology of HLHS proposes that aortic or mitral valve obstructions impair blood flow through the left ventricle leading to hypoplasia of the left sided structures of the heart. While clinical trials have attempted to relieve blood flow obstructions in utero, these studies have had limited success in rescuing LV growth and blood flow in human HLHS fetuses. Here, I present preliminary findings that reveal that cardiomyocytes derived from HLHS patient- derived human induced pluripotent stem cells (hiPSC) demonstrate significantly reduced contractile force generation suggesting that intrinsic myocardial dysfunction may contribute to the disruption ventricular blood flow. These findings raise the exciting possibility of developing therapeutic strategies to potentially improve myocardial function to prevent LV hypoplasia in utero. The overall objective of this proposal is to identify molecular and transcriptional mechanisms giving rise to impaired contractile function in HLHS ventricular cardiomyocytes. The hypothesis is that HLHS ventricular cardiomyocytes display dysregulated expression of core transcriptional regulators necessary for the maintenance of the contractile machinery used for healthy myocardial function. Using the hiPSC model system, I will use healthy and HLHS patient derived hiPSCs to test the central hypothesis and attain the objective of this application. Specific Aim 1: Identify the molecular mechanisms giving rise to impaired contractile function in HLHS hiPSC-derived ventricular cardiomyocytes. Specific Aim 2: Determine the role of cardiomyocyte transcriptional regulators in cell function and survival in HLHS-hiPSC derived cardiomyocytes. Using CRISPR/Cas9 genome editing, I will introduce a genetic reporter system that will allow for the isolation of ventricular and left ventricular cardiomyocytes from hiPSC differentiation in vitro. This will allow for the study of specific sarcomeric and electrophysiologic mechanisms driving impaired contractile function and to assess whether these defects are specific to the LV. Using state of the art single cell RNA-sequencing technology, I will conduct extensive transcriptomic profiling of HLHS hiPSC-cardiomyocytes to identify dysregulated expression of key transcriptional regulators involved in regulating genes necessary for myocyte function and survival. Successful execution of the work proposed will lead to three significant contributions: 1) Will identify specific molecular drivers of myocardial contractile dysfunction in HLHS, 2) Will reveal, for the first time, whether myocardial contractile deficits and mechanisms are specific to the LV potentially explaining the left side specific lesions of HLHS 3) Will identify novel transcriptional pathways driving impaired contractile function in HLHS.