Understanding the Mechanisms of Ventricular Dysfunction in Hypoplastic Left Heart Syndrome

了解左心发育不全综合征心室功能障碍的机制

• 批准号: 10017702
• 负责人: Francisco Xavier Galdos
• 金额: $ 3.83万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2022-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017702
• 关键词: Accounting; Affect; Automobile Driving; Biological Models; Biology; Blood; Blood Circulation; Blood flow; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Death; Cardiac Myocytes; Cardiac development; Cell Maintenance; Cell Survival; Cell physiology; Cells; Clinical; Clinical Trials; Congenital Heart Defects; Core Protein; Data; Defect; Development; Electrophysiology (science); Etiology; Fetus; Flow Cytometry; Functional disorder; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Goals; Growth; Heart; Heart Diseases; Heart failure; Human; Hypoplastic Left Heart Syndrome; Impairment; In Vitro; Intervention; Ion Channel; Lead; Left; Left ventricular structure; Lesion; Live Birth; Maintenance; Mediating; Mission; Mitral Valve; Mitral Valve Stenosis; Molecular; Muscle Cells; Mutation; Myocardial; Myocardial dysfunction; Myocardium; National Heart, Lung, and Blood Institute; Neonatal; Obstruction; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Physicians; Physiology; Play; Proteins; Pump; Regulation; Reporter; Research; Role; Sarcomeres; Scientist; Side; Signal Transduction; Small Interfering RNA; Structure; System; Technology; Testing; Therapeutic; Time; Training; Trees; United States; Ventricular; Ventricular Dysfunction; Work; aortic valve; ascending aorta; base; career; congenital heart disorder; genome editing; genomic locus; impaired driving performance; improved; in utero; induced pluripotent stem cell; knock-down; malformation; novel; novel therapeutic intervention; prevent; programs; single-cell RNA sequencing; stem cell differentiation; stem cell model; stem cells; success; theories; therapeutic target; transcription factor; transcriptomics

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.

项目概要/摘要 左心发育不良综合征 (HLHS) 是美国新生儿心源性死亡的主要原因 占全部先天性心脏病的2-3%。虽然“HLHS”用于描述一系列 左心室异常，定义为左心和上行心严重发育不全 主动脉以及二尖瓣和主动脉瓣闭锁或狭窄。目前，长期以来的理论 HLHS 的病因学认为主动脉瓣或二尖瓣阻塞会损害通过左心室的血流 导致心脏左侧结构发育不全。虽然临床试验试图缓解血液 由于子宫内血流阻塞，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，我提出的初步研究结果表明，来自 HLHS 患者的心肌细胞- 衍生的人类诱导多能干细胞 (hiPSC) 表现出收缩力显着降低 一代表明内在的心肌功能障碍可能导致心室血液的破坏 流动。这些发现提出了开发治疗策略以潜在改善的令人兴奋的可能性 心肌功能，防止子宫内左室发育不全。 该提案的总体目标是确定产生的分子和转录机制 HLHS 心室心肌细胞收缩功能受损。假设 HLHS 心室 心肌细胞表现出核心转录调节因子的表达失调 维护用于健康心肌功能的收缩机制。使用 hiPSC 模型系统， 我将使用健康和 HLHS 患者衍生的 hiPSC 来检验中心假设并实现此目标 应用。具体目标 1：确定导致收缩功能受损的分子机制 HLHS hiPSC 来源的心室心肌细胞。具体目标 2：确定心肌细胞的作用 HLHS-hiPSC 衍生心肌细胞的细胞功能和存活的转录调节因子。使用 CRISPR/Cas9 基因组编辑，我将介绍一个基因报告系统，可以分离 来自 hiPSC 体外分化的心室和左心室心肌细胞。这将允许研究 驱动收缩功能受损的特定肌节和电生理机制并评估 这些缺陷是否是 LV 所特有的。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组分析，以确定表达失调 参与调节心肌细胞功能和存活所需基因的关键转录调节因子。 成功执行所提议的工作将带来三项重大贡献：1）将确定 HLHS 中心肌收缩功能障碍的特定分子驱动因素，2) 将首次揭示是否 心肌收缩缺陷和机制是 LV 特有的，可能解释左侧的具体情况 HLHS 的病变 3) 将识别驱动 HLHS 收缩功能受损的新转录途径。