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

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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.