Signaling Mechanisms Regulating Cardiac Remodeling

描述(申请人提供)：我们实验室的长期目标是了解心脏正常和异常发育的信号机制，以及可能导致先天性心脏病的机制。事实上，1%-2%的新生儿患有某种形式的先天性心脏病，使其成为最常见的先天性出生缺陷。这些心血管缺陷中的许多与心内膜垫组织的形成和重塑进入心脏瓣膜和膜间隔有关。我们已经证明，维甲酸X受体α零(RXRpha-/-)小鼠是帮助我们理解维甲酸信号如何影响心脏发育的一个很好的模型系统。胚胎13.5d(E)RXRα/-小鼠的致死性部分是由于TGFbeta2升高和流出道细胞凋亡增加所致。TGFbeta2主要通过Smad2/Smad3的磷酸化(P)来传递信号。PSmad2和pSmadS在E13.5缓冲间充质中的定位与凋亡有显着的一致性，提示它们在TGFbeta2介导的细胞程序性死亡和重塑事件中发挥关键作用。初步的Western印迹数据表明，与单独使用两种药物相比，维甲酸和TGFbeta2共同处理分离的胰酶分散的心肌细胞一小时，可导致Smad2/Smad3协同磷酸化。这种新的相互作用并不是由于RXRA的转录效应，因为这种效应只在处理一小时后就能看到。相反，在RXRα-/-中，免疫组织化学显示在流出道中缺乏核定位的pSmad2/pSmad3。这些数据表明，在发育中的心脏中，维甲酸和TGFbeta2信号级联之间存在着一种新颖而显著的串扰，这是以前尚未实现的。因此，虽然维甲酸信号的长期转录效应已经被很好地记录下来，但该通路的短期激活的影响还没有被研究。因此，我们假设在流出道重构过程中，维甲酸信号能够以快速反应的方式调节TGFbeta2介导的效应。利用小鼠胚胎培养、原代细胞培养和遗传杂交，我们提出了三个目的来验证这一假说：1)确定维甲酸对心脏中TGFbeta2信号的影响，并评估RXRpha在这些过程中的作用。2)研究维甲酸信号对TGFbeta2诱导的流出道细胞凋亡和重塑的调节作用。3)确定RXRpha是否与流出道中的Smad信号直接相互作用。

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.