T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system

Abstract

Genome-wide association studies have identified noncoding variants nearTBX3that are associated with PR interval and QRS duration, suggesting that subtle changes inTBX3expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygousTbx3mutant (Tbx3+/−) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes inTbx3+/−mutants. Notably,Tbx3+/−atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs nearRyr2,an up-regulated chamber-enriched gene, and inCacna1g,a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governsCacna1gexpression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in theTBX3locus.