Signaling Mechanisms Regulating Cardiac Remodeling

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DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to understand signaling mechanisms that underlie both normal and abnormal heart development as well as mechanisms that can cause congenital heart disease. Indeed, 1-2% of newborns have some form of congenital heart disease making it the most common congenital birth defect. Many of these cardiovascular defects are related to the formation and remodeling of endocardial cushion tissue into the valves and membranous septa of the heart. We have documented that the retinoid X receptor alpha null (RXRalpha-/-) mouse is an excellent model system to aid our understanding of how retinoic acid signaling influences heart development. Lethality in the embryonic day (E) 13.5 RXRalpha-/- mouse is marked, in part, by elevated TGFbeta2 and enhanced apoptosis in the outflow tract. TGFbeta2 primarily signals through phosphorylation (p) of Smad2/Smad3. pSmad2 and pSmadS show a striking coincident localization with apoptosis in E13.5 cushion mesenchyme suggesting they play a key role in programmed cell death and remodeling events mediated by TGFbeta2. Preliminary Western blot data demonstrates co-treatment of isolated trypsin-dispersed heart cells with retinoic acid and TGFbeta2 for one hour resulted in a synergistic phosphorylation of Smad2/Smad3 compared to each agent alone. This novel interaction is not due to transcriptional effects of RXRa since the effects were seen after only a one-hour treatment. Conversely, in the RXRalpha-/-, immunohistochemistry shows a lack of nuclear localized pSmad2/pSmad3 in the outflow tract. These data demonstrate a novel and significant crosstalk between the retinoic acid and TGFbeta2 signaling cascades in the developing heart that has not previously been realized. Thus, while long-term transcriptional effects of retinoic acid signaling are well documented, effects of short-term activation of the pathway have not been studied. Thus, we hypothesize that retinoic acid signaling can function in a rapid response manner to modulate TGFbeta2-mediated effects during outflow tract remodeling. Using whole mouse embryo cultures, primary cell cultures, and genetic crosses we propose three aims to test this hypothesis: 1) To determine the influence of retinoic acid on TGFbeta2 signaling in the heart and to assess the role for RXRalpha in these processes. 2) To determine how retinoic acid signaling regulates TGFbeta2-induced apoptosis and remodeling in the outflow tract. 3) To determine if RXRalpha directly interacts with Smad signaling in the outflow tract.