Cellular and Molecular Mechanisms of Left Ventricular Growth and Morphogenesis

描述（申请人提供）：先天性心脏病（CHD）是最常见的出生缺陷。在各种冠心病中，由心室形态发生改变引起的单心室表型具有最差的临床预后，包括三尖瓣闭锁（OMIM# 605067）、肺动脉闭锁（OMIM# 265150）和左心发育不良综合征（HLHS; OMIM# 241550,614435）。单心室心脏呈串联回路，全身静脉回流到右心室和肺动脉，再加上从肺静脉回流到左心室并流出到身体的血流，这与生存是不相容的。目前，人们对多种形式的单心室冠心病的分子机制和细胞病因了解甚少。在HLHS患者中观察到人类心脏转录因子基因NKX2.5和HAND1的突变。鉴于现有Cre系的广泛表达域，目前系统和条件敲除Nkx2.5和Hand1会导致胚胎致死，因此对可能的HLHS表型的建模和研究受到限制。缺乏限制性左心室Cre驱动程序禁止此类调查。Hand1在左心室原心野心肌中表达。我们分离了调节Hand1左心室表达的增强子，并利用它产生了一种新的左心室特异性Cre驱动子，从而可以实现对驱动左心室形态发生的细胞和分子机制的研究。我们的实验计划是从发育中的胚胎心脏中切除Hand1左心室谱系细胞，有条件地特异性删除左心室心肌内的Nkx2.5，并验证从24名不相关患者中分离出的人类Hand1突变是HLHS的病因。这项关于hand1系心肌在心脏发生过程中的作用的研究将揭示单心室表型的细胞病因学和控制心室成熟的分子程序，从而扩大对与人类疾病相关的心室形态发生的理解。相关性：导致单心室表型的冠心病具有最差的临床结果。因此，了解导致单心室心脏的冠心病的病因和分子机制，每年有可能使成千上万的儿科患者受益。hand1谱系在单心室表型的发生中起着关键作用，深入了解这一未被充分研究的心肌群体的细胞和分子机制将对冠心病患者的非手术治疗有很大的好处。

DESCRIPTION (provided by applicant): Congenital heart disease (CHD) is the most common birth defect. Among various CHDs, single ventricle phenotypes resulting from altered ventricular morphogenesis have the poorest clinical prognoses and include Tricuspid Atresia (OMIM# 605067), Pulmonary Atresia (OMIM# 265150), and Hypoplastic Left Heart Syndrome (HLHS; OMIM# 241550, 614435). The single ventricle heart presents with a series circuit such that systemic venous return to the right ventricle and pulmonary arteries combined with the flow from the pulmonary venous return into the left ventricle and out to the body is incompatible with survival. Currently, there is a poor understanding of the molecular mechanisms and cellular etiology causative of the many forms of single ventricle CHD. Human mutations in the cardiac transcription factor genes NKX2.5 and HAND1 have been observed in HLHS patients. Modeling and thus the study of possible HLHS phenotypes have been limited as current systemic and conditional knockouts of Nkx2.5 and Hand1 results in embryonic lethality given the broad expression domains of available Cre lines. The lack of a restricted left ventricle Cre driver prohibits such investigations. Hand1 is expressed within the primary heart field myocardium of the left ventricle. We have isolated the enhancer that regulates Hand1 left ventricular expression and used it to generate a novel left ventricular-specific Cre driver with which interrogation of th cellular and molecular mechanism driving left ventricular morphogenesis can be realized. Our experimental plan is to ablate the Hand1 left ventricular lineage cells from the developing embryonic heart, conditionally delete Nkx2.5 specifically within the left ventricular myocardium, and validate an identified human mutation in HAND1 isolated from 24 unrelated patients as being causative of HLHS. This study of the role that Hand1-lineage myocardium plays during cardiogenesis will shed light on the cell etiology of single ventricle phenotypes and on the molecular programs controlling ventricular maturation, thus expanding the understanding of ventricular morphogenesis as it relates to human disease. Relevance: CHDs resulting in single ventricle phenotypes have the poorest clinical outcomes. Thus, gaining an understanding of the etiology and molecular mechanisms that cause CHDs resulting in a single ventricle heart has the potential to benefit thousands of pediatric patients annually. The Hand1-lineage plays a key role in the genesis of single ventricle phenotypes and gaining insight into the cellular and molecular mechanism of this understudied myocardial population will have a great benefit to developing non-surgical treatments for CHD patients.