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 ?-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.
This project addresses several issues of central importance to the understanding of congenital and acquired arrhythmias, significant causes of morbidity and mortality. This proposal tests the hypothesis that Wnt signaling globally regulates myocardial programming and patterning of the atrioventricular junction phenotype. This proposal will result in a better understanding of how arrhythmias can result from altered programs of gene expression, which ultimately influence ion channel gene expression and cardiac conduction. The results and broad conclusions will be directly relevant to understanding the genetic basis of cardiac arrhythmias and to design more effective diagnostic and therapeutic options for cardiac arrhythmias and congenital heart defects.
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