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

围产期心脏室特异性生长的新型基因环境调节回路

• 批准号: 10471992
• 负责人: Marlin Touma
• 金额: $ 39万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471992
• 关键词: Ablation; Address; Affect; Attenuated; Award; Biological; Birth; Calcineurin; Cardiac; Cardiac Myocytes; Cardiac development; Common Ventricle; Congenital Heart Defects; Critical Congenital Heart Defects; Data; Development; DiGeorge Syndrome; Environment; Environmental Risk Factor; FZD4 gene; Gene Expression; Genes; Genetic; Genetic Models; Goals; Growth; Growth and Development function; Heart; Howard Temin Award; Human; Hypoxia; In Vitro; Infant; K-Series Research Career Programs; Knockout Mice; Knowledge; Laboratories; Lead; Left ventricular structure; LoxP-flanked allele; Mediating; Mediator of activation protein; Molecular; Mus; Muscle Cells; Mutation; Neonatal; Newborn Infant; Outcome Study; Oxygen; Pathogenesis; Pathologic; Pathology; Pathway Analysis; Patients; Perinatal; Perinatal Hypoxia; Physiology; Process; Rattus; Regulation; Research Personnel; Right ventricular structure; Role; Sampling; Signal Pathway; Signal Transduction; Specimen; Stress; Subgroup; Tamoxifen; Testing; Tetralogy of Fallot; Time; United States National Institutes of Health; Ventricular; WNT Signaling Pathway; WNT11 gene; Work; base; cardiogenesis; clinically relevant; cohort; congenital heart disorder; differential expression; experimental study; expression vector; fetal; gain of function; genome-wide analysis; in vivo; loss of function; mouse model; neonatal mice; novel; overexpression; perinatal development; perinatal period; postnatal; receptor; response; transcriptome; transcriptome sequencing; transcriptomics; vector

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.

项目摘要。 该提案描述了为期5年的NIH/R 01早期研究者（ESI）申请。的总目标 本实验室主要研究基因环境调控和细胞间信号转导在围产期心脏中的作用 在胎儿到新生儿过渡期间，心腔成熟和对应激的反应是心脏 生长，特别是在先天性心脏病（CHD）的背景下。 建议的研究是基于我以前的工作和初步数据证明一种新的 在调节室特异性生长中涉及Wnt 11信号传导和缺氧的调节回路 围产期心脏转录组的全基因组分析。使用Wnt 11基因表达的靶向沉默 和体内全身缺氧诱导，我进一步确定Wnt 11调节心肌细胞（CMC） 增殖可能通过调节Rb 1活性在正常和缺氧过渡以及在紫绀 冠心病为了从机制上确定Wnt 11是否影响室特异性生长，我生成了 功能获得和功能丧失小鼠模型。具有他莫昔芬诱导的Wnt 11-cKO小鼠模型 并通过利用预先存在的携带floxed Wnt 11的线建立了Wnt 11的心脏特异性消融 （Wnt 11 Flox/Flox）和αMHC-MerCreMer线。利用这种新颖的小鼠模型，我获得了强有力的证据， 支持Wnt 11/Rb 1信号传导是心室腔正常成熟所必需的。重要的是， 新生儿右心室（RV）的缺氧比左心室（LV）的缺氧更能破坏这一调节回路。 心脏和潜在的导致右心室异常发绀法洛四联症（TOF）的婴儿。实现 由于Wnt 11的CMC特异性功能增益，我还产生了CMC特异性Wnt 11的AAV 9载体 过表达（Wnt 11-OE）。这些研究得到了AHA职业发展奖和 NIH/R56（高优先短期项目奖，或“桥梁奖”）。 在这个建议中，我计划利用我们的新型小鼠模型来建立分子机制， 介导Wnt 11/Rb 1调控室特异性CMC对缺氧的反应性增殖，并探讨其在细胞增殖中的作用。 这种新型电路在TOF中的病理影响。在AIM 1中，我将确定Wnt 11/Rb 1信号在 使用Wnt 11-cKO小鼠模型和AAV 9介导的小鼠模型的新生RV与LV发育和缺氧反应 Wnt 11-OE，与缺氧暴露组合。在AIM 2中，我将剖析介导Wnt 11的信号级联 功能的新生大鼠心室肌细胞（NRVMs）在体外，并建立在完整的生物学相关性， 新生儿体内心脏。在AIM 3中，我将研究缺氧和缺氧之间相互作用的临床相关性。 Wnt 11/Rb 1在出生后TOF发病机制中的作用，通过在转录组学上表征明确定义的TOF队列 水平实现拟议的目标将建立一个新的监管循环，这可能会导致特定于腔室的 治疗新生儿心脏病加州大学洛杉矶分校提供了一个理想的环境，以实现拟议的目标。