Essential functions of Fibulin proteins in outflow tract morphogenesis.

PROJECT SUMMARY Congenital heart disease (CHD) is the most common birth defect in humans and patients with conotruncal defects comprise 20% of this population. Malformations of the distal aspect of the ventricle, the infundibulum, and the proximal aspect of the great arteries lead to conotruncal anomalies. Specific examples include transposition of the great vessels, double outlet right ventricle, and Tetralogy of Fallot. Moreover, in comparison to other types of CHDs, conotruncal defects are more frequently associated with genetic and syndromic abnormalities. Among this patient cohort, the mortality rate of 17% indicates a dire need for improvement in our understanding of the early developmental cues guiding these aberrations in outflow tract (OFT) morphogenesis. The purpose of this application is to uncover the molecular and cellular mechanisms that account for disruptions in OFT development and underlie human conotruncal CHDs. We identified Fibulin (Fbln) proteins as novel regulators of the extracellular matrix (ECM) essential for OFT morphogenesis. Our preliminary data demonstrate that Fblns are required for smooth muscle addition to the OFT and for TGF-β signaling in the late-differentiating progenitors that contribute to the arterial pole. In Aim 1, I will dissect the cellular and molecular mechanisms mediated by fbln genes during OFT growth. I will assess proliferation of the anterior SHF progenitor population in the Fbln loss-of-function model and evaluate differentiation and proliferation of the cardiomyocyte, endothelial cell, and smooth muscle cell contribution to anterior SHF-derived lineages at the arterial pole. I will employ EdU, apoptosis, and developmental timing assays to dissect these cell type-specific functions of Fbln genes. In Aim 2, I will probe cardiomyocyte, endothelial cell, and smooth muscle cell-type specific expression of pSmad3 in the OFT of Fbln loss-of-function embryos. Moreover, I will perform gain-of-function and loss-of-function experiments with a constitutively active Alk5 transgene and a small molecular inhibitor of Smad3 phosphorylation, respectively. These strategies will illuminate how Fbln proteins mediate OFT development via TGF-β signaling. Insights gained from these proposed studies will shed light on the mechanisms responsible for arterial compliance and elastic deformation at the arterial pole with implications for diseases involving stenosis of the aortic and pulmonary valves. Furthermore, probing the cell type-specific roles of Fbln proteins will augment our ability to identify novel therapeutic targets and protocols for tissue engineering of OFT conduits and artificial valves.