Mechanisms of outflow tract morphogenesis regulated by extracellular matrix

PROJECT SUMMARY Congenital heart disease (CHD) is the most common birth defect in humans and patients with conotruncal defects comprise 30% 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. Among this patient cohort, the mortality rate of 17% indicates a dire need for improvement in our understanding of the early developmental signals guiding these aberrations in outflow tract (OFT) morphogenesis. The purpose of this application is to uncover the molecular, cellular, and biomechanical mechanisms that account for disruptions in OFT development and underlie human conotruncal CHDs. The emerging focus on extracellular matrix (ECM) in cardiac development and disease points to this specialized, non-cellular protein network as a key player in sculpting the conotruncus. Yet, we have limited appreciation of the individual ECM complements functioning during OFT formation. We identified Fibulin (Fbln) proteins as novel regulators of the ECM essential for OFT growth and expansion. Our preliminary data demonstrate that Fblns are required to establish the proper size of the OFT. Fblns are required for accumulation of endothelial cells (ECs) through Smad3-dependent TGF-β cues. Further, Fblns stimulate smooth muscle cell (SMC) differentiation and elastin assembly to establish the OFT caliber. Importantly, tissue stiffness of this auxiliary chamber is elevated; this decreased elasticity contributes to altered flow profiles. Altogether, our preliminary data highlight that the underlying mechanisms responsible for the impaired OFT growth in the fbln loss-of-function model are multifactorial, representing a complex interplay of defects in EC accumulation, SMC differentiation, tissue stiffness, and flow-related factors. We put forth the overarching hypothesis that Fbln genes are essential to establish OFT dimensions and compliance by regulating TGF-β signals in ECs and by promoting ECM deposition by SMCs, ultimately generating shear forces that propagate further expansion. We will examine this model with the following aims: dissect the mechanisms mediated by fbln2 in EC accumulation and OFT growth (Aim 1), investigate the function of Fbln5 in regulating elastin assembly, stiffness, and OFT expansion (Aim 2), and determine shear stress and mechanobiological mechanisms operating downstream of Fbln proteins via Piezo1 in ECs and SMCs (Aim 3). Insights gained from these proposed studies will shed light on the biomechanical mechanisms responsible for compliance and elastic deformation at the arterial pole with implications for diseases involving stenosis of the conotruncal region. Furthermore, probing the cell type-specific functions of Fbln proteins will augment our ability to identify novel therapeutic targets and protocols for tissue engineering of OFT conduits and artificial valves.