Novel Gene-Environment Regulatory Circuit in Chamber-Specific Growth of Perinatal Heart

项目摘要。 本提案描述了一项为期5年的NIH/R01早期调查员(ESI)申请。的总目标是 我的实验室是为了阐明基因-环境调节和细胞间信号在围产期心脏中的作用。 心脏的关键窗口--胎儿向新生儿过渡期间的心房室成熟和应激反应 生长，特别是在先天性心脏缺陷(CHDS)的情况下。 建议的研究是基于我以前的工作和初步数据，展示了一部小说 涉及Wnt11信号和低氧调节小室特异性生长的调节电路被发现 围产期心脏转录组的全基因组分析。利用靶向沉默Wnt11基因表达 和体内全身低氧诱导，我进一步证实了WNT11对心肌细胞(CMC)的调节作用。 增殖可能通过调节Rb1的活性在正常和低氧过渡以及在青紫期 CHDS。为了从机制上确定Wnt11是否影响特定于小室的生长，我生成了 小鼠模型功能的获得和功能的丧失。三苯氧胺诱导WNT11-CKO小鼠模型的建立 并且通过利用携带有花丝的WNT11的预先存在的线来建立WNT11的心脏特异性消融 (WNT11FLOX/FLOX)和一条αMHC-MercreMer系列。使用这种新颖的小鼠模型，我获得了强有力的证据 支持Wnt11/Rb1信号是脑室正常成熟所必需的。重要的是 缺氧对这一调节环路的破坏在新生儿右心室(RV)比左心室(LV)更强烈 这可能会导致法洛四联症(TOF)婴儿的右室畸形。要实现 CMC特定的WNT11的功能增益，我还生成了CMC特定的WNT11的AAV9载体 过度表达(Wnt11-OE)。这些研究得到了AHA职业发展奖和 NIH/R56(高优先级短期项目奖，或“桥梁奖”)。 在这项提议中，我计划利用我们的新型小鼠模型来建立分子机制 介导Wnt11/Rb1调控小室特异性CMC增殖反应，并探讨其机制 这一新电路在TOF中的病理影响。在目标1中，我将确定Wnt11/Rb1信号在 应用Wnt11-CKO小鼠模型和AAV9介导的新生RV与LV的发育和缺氧反应 WNT11-OE，联合低氧暴露。在AIM2中，我将剖析中介WNT11的信令级联 体外培养新生大鼠心室肌细胞(NRVMs)的功能，并建立与完整的生物学相关性 活体内新生儿心脏。在目标3中，我将研究低氧和低氧之间相互作用的临床相关性。 Wnt11/Rb1在TOF发病机制中的作用 水平。实现提议的目标将建立一个新的监管环路，可能导致特定于商会的 新生儿先天性心脏病的治疗。加州大学洛杉矶分校为实现所提出的目标提供了理想的环境。

PROJECT ABSTRACT . This proposal describes a 5-year NIH/R01 early stage investigator (ESI) application. The overall goal of my laboratory is to elucidate the roles of gene-environment regulation and intercellular signaling in perinatal heart chamber maturation and responses to stress during fetal to neonatal transition, a critical window for cardiac growth, particularly, in the context of congenital heart defects (CHDs). The proposed studies are based on my previous work and preliminary data demonstrating a novel regulatory circuit involving Wnt11 signaling and hypoxia in regulating chamber-specific growth uncovered by genome-wide analysis of perinatal cardiac transcriptome. Using targeted silencing of Wnt11 gene expression and systemic hypoxia induction in vivo, I have further established that Wnt11 regulates cardiomyocyte (CMC) proliferation likely through modulating Rb1 activity during normal and hypoxic transition as well as in cyanotic CHDs. In order to, mechanistically, determine whether Wnt11 affects chamber-specific growth, I have generated both gain of function and loss of function mouse models. The Wnt11-cKO mouse model with Tamoxifen-induced and cardiac-specific ablation of Wnt11 was established by utilizing a preexisting line carrying floxed Wnt11 (Wnt11Flox/Flox) and an αMHC-MerCreMer line. Using this novel mouse model, I obtained strong evidence supporting that the Wnt11/Rb1 signaling is required for normal maturation of ventricular chambers. Importantly, this regulatory loop is disrupted by hypoxia more robustly in right ventricle (RV) than left ventricle (LV) in neonatal hearts and potentially leads to RV abnormalities in infants with cyanotic tetralogy of fallot (TOF). To achieve CMC specific gain of function for Wnt11, I also generated an AAV9 vector for CMC specific Wnt11 overexpression (Wnt11-OE). These studies were supported by an AHA career development award and an NIH/R56 (the High Priority Short-Term Project Award, or “Bridge Award’’). In this proposal, I plan to leverage our novel mouse models to establish the molecular mechanisms mediating Wnt11/Rb1 control of chamber-specific CMC proliferation in response to hypoxia, and to explore the pathological impact of this novel circuit in TOF. In AIM 1, I will determine the role of Wnt11/Rb1 signaling in neonatal RV vs LV development and hypoxia response using Wnt11-cKO mouse model and AAV9-mediated Wnt11-OE, in combination with hypoxia exposure. In AIM2, I will dissect the signaling cascade mediating Wnt11 function in neonatal rat ventricular myocytes (NRVMs) in vitro, and establish the biological relevance in the intact neonatal heart in vivo. In AIM 3, I will examine the clinical relevance of the interplay between hypoxia and Wnt11/Rb1 in TOF pathogenesis after birth by characterizing a well-defined TOF cohort at the transcriptomic level. Accomplishing the proposed aims will establish a novel regulatory loop that may lead to chamber-specific therapies for newborns with CHDs. UCLA provides an ideal environment to achieve the proposed aims.