Abstract We086: Identifying Gene Regulatory Subnetwork Drivers of Cardiomyocyte Chamber Specification

Abstract

Introduction: Fetal heart development is a highly coordinated process directed by complex gene regulatory networks (GRN). Imbalances in such networks can lead to congenital heart defects, a leading cause of morbidity and mortality in children. Many of these defects are related to the improper specification of cardiac progenitor cells to chamber-specific (i.e., atrial, or ventricular) cardiomyocytes, but the GRNs involved in this process are not well characterized. Hypothesis: We hypothesize that cardiomyocyte chamber specification depends on discordant atrial and ventricular regulatory subnetworks within heart field cardiac progenitor cells. Approach: We used CellOracle, a GRN inference software, and publicly available fetal mouse heart single cell RNA and ATAC sequencing data to infer cell type specific GRNs during cardiomyocyte differentiation. Each of the first heart field and two second heart field networks included 4,000 directed interactions and more than 600 genes. We performed a systematic in silico knockdown study with these networks to identify TFs that differentially modulate atrial and ventricular gene expression. Correlations in the downstream responses to TF perturbations were used to extract chamber specification subnetworks. Results: The most central genes within the heart field networks included several previously identified cardiac developmental TFs (Nkx2-5, Mef2c, Tbx5). Perturbations of these heart field GRNs identified 12 TFs that increased ventricular gene expression while also decreasing atrial gene expression (ventricular program), as well as 20 genes with the opposite effect (atrial program). Previous in vitro knockdown studies during iPSC-CM differentiation validated one of the ventricular (Hey2) and two of the atrial programming TF (Nr2f2, Tead2) predictions. Discordant atrial and ventricular regulatory subnetworks were identified, including one network with opposing effects on the expression of Wnt2 by ventricular (repressing) and atrial (activating) TFs. Conclusions: We identified a set of TFs organized within regulatory subnetworks that drive discord specification of ventricular vs. atrial cardiomyocyte gene expression within cardiac progenitor cells in mouse development. These predictions will be validated through in vitro and in vivo studies.