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

项目总结/摘要 心脏瓣膜对于维持心脏中的单向血流至关重要。先天性和后天性 瓣膜病是儿科和成人人群发病率和死亡率的主要来源。电流 治疗策略是有限的，常常需要用次优的假体进行手术置换。 瓣膜发生始于通过上皮细胞向间充质细胞转化形成内膜垫 （EMT）。许多调节EMT事件的信号通路已被确定，然而，知之甚少 关于EMT后瓣膜形成的分子调节因子Ebstein畸形，一种罕见的先天性心脏病， 其特征在于发育不良的肌化三尖瓣小叶，其通常栓系于 潜在心肌，导致瓣环心尖移位和右心室心房化。 这种缺陷通常归因于瓣膜分层失败-EMT后的关键形态发生步骤 瓣膜形成，在此期间原始瓣膜小叶与下面的心肌分离。先前 研究表明，瓣膜间质细胞（VIC）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因子 通过对Ebstein异常的多组学解剖，利用Ebstein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，以确定房室垫信号相互作用调节 瓣膜分层在我的第二个目标中，我将使用整合的单细胞RNA/ATAC测序方法， 定义驱动维克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丰富的专业知识。我的同事 赞助人Ye博士在实验和计算单细胞基因组学方面具有专长。他们的 集体的专业知识将确保我得到必要的培训和指导，以完成拟议的 research.为了获得我的项目所需的稀有组织样本，我已经形成了一个多中心合作， 有两个大容量的艾伯斯坦异常症患者中心此外，我还获得了人类方面的专业知识 来自李晶晶博士的遗传学和机器学习，支持我的人类遗传学部分 项目同时，我正在从事儿科心胸外科的纵向临床指导工作 与我的临床培训共同赞助商，彼得Kouretas博士，加州大学旧金山分校儿科心脏外科主任 移植计划。总的来说，拟议的研究将阐明以前未表征的机制 晚期房室瓣形态发生的研究，并为今后开发新的 用于先天性和获得性瓣膜病的基于再生医学的疗法。F30 NRSA研究金 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， 我最终的职业目标是成为一名学术性的儿科心胸外科医生。

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.