Defining the molecular regulators of valvular delamination via multi-omic dissection of Ebstein’s Anomaly

项目摘要/摘要 心脏瓣膜是维持心脏单向血流的关键。先天和后天 瓣膜病是儿童和成人发病率和死亡率的主要来源。当前 治疗策略是有限的，通常需要用不太理想的假体进行手术替代。 瓣膜形成始于上皮细胞向间充质细胞转化形成心内膜垫 (EMT)。许多调节EMT事件的信号通路已经被定义；然而，人们知之甚少 关于EMT后瓣膜形成的分子调控机制。埃布斯坦畸形，一种罕见的先天性心脏缺陷， 以不同发育不良、肌肉化的三尖瓣叶为特征，这些叶通常拴在 下层心肌，导致瓣环心尖移位和右室房化。 这种缺陷通常归因于瓣膜分层的失败？？瓣膜分层是EMT后的一个关键的形态发生步骤。 瓣膜发生过程中，原始瓣叶从下层心肌中分离出来。上一首 研究表明，瓣膜间质细胞(VICs)分化动力学的改变可能有助于 这种瓣膜缺陷的发病机制。我建议定义瓣膜分层的分子调节器 通过对艾布斯坦畸形的多组学解剖，利用艾布斯坦异常小鼠模型系统， 原始人体组织和人类遗传学数据。在我的第一个目标中，我将使用时空单细胞RNA 测序和人类遗传学分析确定房室垫信号相互作用调节 瓣膜剥离。在我的第二个目标中，我将使用集成的单细胞RNA/ATAC测序方法来 定义驱动VIC命运决定的基因调控网络。我的主要赞助人迪帕克·斯里瓦斯塔瓦博士 在发育心脏生物学、人类遗传学和干细胞生物学方面拥有广泛的专业知识。我的同事- 发起人叶春博士在实验和计算单细胞基因组学方面拥有专业知识。他们的 集体专业知识将确保我获得必要的培训和指导，以完成拟议的 研究。为了获得我的项目所需的稀有组织样本，我成立了一个多中心合作 有两个高容量的艾布斯坦畸形患者中心。此外，我还获得了人力资源方面的专业知识 来自李晶晶博士的遗传学和机器学习，以支持我的 项目。同时，我还在从事儿科心胸外科的临床指导工作。 与我的临床培训共同发起人，Peter Kouretas医生，加州大学旧金山分校儿科心脏外科主任 移植计划。总体而言，拟议的研究将阐明以前未描述的机制 对晚期房室瓣膜形态发生的研究，并为今后新的开发工作提供信息 以再生医学为基础的先天性和获得性瓣膜病治疗。F30 NRSA奖学金 支持将培养我在发育心脏生物学和计算基因组学方面的专业知识，进一步促进我 朝着成为一名学术儿科心胸外科医生兼科学家的终极职业目标迈进。

PROJECT SUMMARY/ABSTRACT Cardiac valves are critical to the maintenance of unidirectional blood flow in the heart. Congenital and acquired valvulopathies are a major source of morbidity and mortality in both the pediatric and adult populations. Current therapeutic strategies are limited, often requiring surgical replacement with suboptimal prostheses. Valvulogenesis begins with formation of the endocardial cushions via epithelial-to-mesenchymal transition (EMT). Many of the signaling pathways regulating this EMT event have been defined; however, little is known about the molecular regulators of post-EMT valvulogenesis. Ebstein’s Anomaly, a rare congenital heart defect, is characterized by variably dysplastic, muscularized tricuspid valve leaflets that are often tethered to the underlying myocardium, resulting in apical displacement of the annulus and atrialization of the right ventricle. This defect is often attributed to a failure of valvular delamination – a critical morphogenetic step of post-EMT valvulogenesis during which the primordial valve leaflets separate from the underlying myocardium. Previous studies suggest that altered differentiation dynamics of valvular interstitial cells (VICs) may contribute to the pathogenesis of this valvular defect. I propose to define the molecular regulators of valvular delamination via a multi-omic dissection of Ebstein’s Anomaly, leveraging an Ebstein’s Anomaly murine model system, primary human tissue, and human genetics data. In my first aim, I will use spatiotemporal single cell RNA sequencing and human genetics analyses to identify the atrioventricular cushion signaling interactions regulating valvular delamination. In my second aim, I will use an integrated single cell RNA/ATAC sequencing approach to define the gene regulatory networks driving VIC fate determination. My primary sponsor, Dr. Deepak Srivastava, has extensive expertise in developmental cardiac biology, human genetics, and stem cell biology. My co- sponsor, Dr. Chun (Jimmie) Ye, has an expertise in experimental and computational single cell genomics. Their collective expertise will assure that I receive the necessary training and mentorship to complete the proposed research. To obtain the rare tissue samples required for my project, I have formed a multi-center collaboration with two high volume Ebstein’s Anomaly patient centers. Additionally, I have secured expertise in human genetics and machine learning from Dr. Jingjing Li, Ph.D., in support of the human genetics component of my project. Concurrently, I am engaging in a longitudinal clinical preceptorship in pediatric cardiothoracic surgery with my clinical training co-sponsor, Dr. Peter Kouretas, Surgical Director of the UCSF Pediatric Heart Transplantation Program. Overall, the proposed research will elucidate previously uncharacterized mechanisms of late atrioventricular valvular morphogenesis and inform future efforts toward the development of novel regenerative-medicine based therapeutics for congenital and acquired valvulopathies. F30 NRSA fellowship support would foster my expertise in developmental cardiac biology and computational genomics, furthering me toward my ultimate career goal of becoming an academic pediatric cardiothoracic surgeon-scientist.