Retinoic Acid Plays Critical Roles in the Late Development of the Heart

Abstract

Vitamin A, via its active metabolite retinoic acid (RA), actively participates in many biological processes including cardiogenesis. Yet RA was found to be highly teratogenic as both excess and deficiency of this critical morphogen result in congenital heart defects. Studies presented here aims to gain insight in mechanisms regulating RA metabolism during embryogenesis and enrich our knowledge of the critical roles of RA during late heart development. Previously, our lab reported that a short-chain dehydrogenase/reductase, i.e. DHRS3, is required for preventing excessive accumulation of RA and thus safeguarding mouse embryogenesis during mid-gestation. The current study expanded the investigation and studied the physiological importance of DHRS3 in RA metabolism at multiple embryonic stages. Consistent with the elevated RA synthesis and signaling at E14.5, genetic ablation of Dhrs3 results in an expansion of RA signaling at E10.5 and E12.5 globally; as well as in the fetal hearts at E10.5, 12.5 and 13.5. Dhrs3-null mutants display a spectrum of congenital defects, including defects in the heart, skeleton and cranial nerves, which collectively result in mid-gestational lethality in Dhrs3-/- embryos. Reduction of maternal intake of vitamin A successfully rescued the Dhrs3-/- fetuses and allows them to survive into full-size adults with normal growth rate. These data jointly demonstrated the indispensability of DHRS3 in reducing the accumulation of RA in various developmental stages and in the formation of fetal organs. With the advancement of knowledge in RA metabolism gained in the first part of this dissertation, the critical roles of RA in cardiogenesis was further explored in the current work. During late cardiogenic stages, the major source of RA is the epicardium. Epicardium contributes greatly to the formation of coronary vessels and the myocardium via 1) giving rise to migratory epicardial cells that differentiate into perivascular cells, and 2) secreting cardiogenic factors. This dissertation employed multiple in vivo and in vitro models to determine the influence of RA on epicardial behaviors and the subsequent epicardial-regulated cardiogenic events. In vitro studies demonstrated that inhibition of RA synthesis in epicardial cell disrupted cytoskeletal reorganization and preserved epithelial characteristics, which resulted in a reduction of migration of epicardial cells. On the contrary, addition of RAR agonist to activate RA signaling strongly induced remodeling of cytoskeleton represented by the formation of stress fibers as well as filopodia marked by polymerized F-actin. RA signaling also abolished the membranous distribution of epithelial markers and induces expression of numerous metalloproteases to pave the way for cell migration. Data from in vivo models showed consistent observation in the regulation of epicardial migration by RA: excess RA in Dhrs3-/- embryos enhances the intramyocardial invasion of epicardial cells whereas deficiency of RA largely retained epicardial cells in the intact epicardium on the surface of the myocardium. To further understand the molecular mechanisms underlying the regulation of epicardial EMT by RA, we employed transcriptomic analysis, which in combination with further molecular assays clearly demonstrated that RhoA pathway is activated by RA and proves to be critical for RA-induced morphological changes in epicardial cells. Furthermore, potentially as a consequence of altered epicardial behavior and functions in response to aberrant RA signaling, both deficiency and excess RA led to compromised coronary vessel formation and hypoplastic ventricular myocardium in vivo. Vascular hierarchy was severely impaired and density of intramyocardial vessels was drastically diminished by altered RA signaling. Lack of proper recruitment and differentiation of vascular smooth muscle cells further acerbated the malformations in coronary vessels in response to abnormal RA signaling. These observations provided novel evidence of the teratogenic nature of RA and collectively demonstrated that RA signaling is not only actively involved but also critically important in the late heart development, which may shed light on the discovery of methods in preventing congenital heart diseases as well as the identification of novel targets for treating cardiovascular diseases.