WNT SIGNALING IN CARDIAC CONDUCTION AND ARRHYTHMOGENESIS

 描述（由申请人提供）：房室管是胚胎心脏中的重要结构，参与瓣膜形成和协调电脉冲延迟，以确保心房和心室的顺序激活和收缩。在这里，我们试图了解经典的Wnt信号是如何通过调节离子通道基因表达和心脏传导来调节房室（AV）交界处的电编程的。这一研究领域是重要的，因为先天性和获得性心律失常，发病率和死亡率的重要原因，可能是由于改变程序的基因表达，影响离子通道基因表达和心脏传导。胚胎心肌内经典Wnt信号传导的缺失导致涉及AV连接的先天性心脏缺陷，包括三尖瓣闭锁。在这里，我们将探讨Wnt信号如何调节AV连接的形态发育的机制，以及该组织的电模式，具有以下目的：1。阐明心肌Wnt信号调节房室管形态发生的机制。使用遗传小鼠模型来执行经典Wnt信号传导的功能获得和丧失，我们将评估Wnt在调节AV连接部内的心外膜和内膜细胞的增殖、迁移和分化中的作用。我们将阐明Wnt信号是否需要出生后的AV连接的维护，以及Wnt激活是否足以诱导异位AV连接在出生后的心脏。2：鉴定促进房室交界表型的经典Wnt信号传导的直接靶标。基于我们的初步数据，Wnt在全局上调节AV连接的表型。我们将对Wnt信号的关键转录效应子β-catenin进行染色质免疫沉淀（ChIP），以及增强子诱变试验，以确定Wnt如何调节转录因子Tbx 3的表达。我们将进行RNA测序和ChIP，然后进行大规模平行DNA测序（ChIP-seq），以确定新的推定下游Wnt靶基因及其调控元件。3：阐明Wnt介导的心脏传导调节机制。我们的初步数据表明，扰动Wnt信号在发展过程中改变AV传导和表达的钠通道和间隙连接。使用ChIP-qPCR在获得和丧失功能的小鼠突变体中，我们将确定Wnt信号如何调节钠通道表达。我们将确定Wnt突变小鼠中心室传导速度的改变是否与Nav1.5和/或间隙连接蛋白表达和磷酸化的变化相关。使用光学映射技术，我们将阐明Wnt信号是否动态调节成人心脏的心脏传导。

 DESCRIPTION (provided by applicant): The atrioventricular canal is an important structure in the embryonic heart, involved in valve formation and in coordinating electrical impulse delay to ensure sequential activation and contraction of the atria and ventricles. Here we seek to understand how canonical Wnt signaling regulates the electrical programming of the atrioventricular (AV) junction through its regulation of ion channel gene expression and cardiac conduction. This area of study is significant because congenital and acquired arrhythmias, important causes of morbidity and mortality, may result from altered programs of gene expression that influence ion channel gene expression and cardiac conduction. Loss of canonical Wnt signaling within embryonic myocardium results in congenital heart defects involving the AV junction, including tricuspid valve atresia. Here we will investigate the mechanism for how Wnt signaling regulates morphologic development of the AV junction, as well as the electrical patterning of this tissue, with the following aims: 1. Delineate the mechanisms by which myocardial Wnt signaling regulates atrioventricular canal morphogenesis. Using genetic mouse models to perform gain- and loss-of-function of canonical Wnt signaling, we will assess the role of Wnt in regulating the proliferation, migration and differentiation of epicardial and endocardial cells within the AV junction. We will elucidate whether Wnt signaling is required for postnatal maintenance of the AV junction, and whether Wnt activation is sufficient to induce ectopic AV junctions in the postnatal heart. 2: Identify direct targets of canonical Wnt signaling that promote an atrioventricular junction phenotype. Based on our preliminary data, Wnt globally regulates the phenotype of the AV junction. We will perform chromatin immunoprecipitation (ChIP) for β-catenin, the key transcriptional effector of Wnt signaling, as well as enhancer mutagenesis assays, to determine how Wnt regulates the expression of the transcription factor Tbx3. We will perform RNA-sequencing and ChIP followed by massively parallel DNA sequencing (ChIP-seq) to identify novel putative downstream Wnt target genes and their regulatory elements. 3: Elucidate the mechanisms for Wnt-mediated regulation of cardiac conduction. Our preliminary data demonstrate that perturbation of Wnt signaling during development alters AV conduction and expression of sodium channels and gap junctions. Using ChIP-qPCR in gain- and loss-of-function mouse mutants, we will determine how sodium channel expression is regulated by Wnt signaling We will determine whether altered ventricular conduction velocity in Wnt mutant mice is associated with changes in Nav1.5 and/or gap junction protein expression and phosphorylation. Using optical mapping techniques, we will elucidate whether Wnt signaling dynamically regulates cardiac conduction in the adult heart.