Transcriptional Suppression of Connexin 43 by Tbx18 Undermines Cell-Cell Electrical Coupling in Postnatal Cardiomyocytes

Abstract

During embryonic development, critical steps in cardiac lineage specification are guided by T-box transcription factors. Mesenchymal precursor cells expressing Tbx18 give rise to the heart's pacemaker, the sinoatrial node (SAN). We sought to identify targets of Tbx18 transcriptional regulation in the heart by forced adenoviral overexpression in postnatal cardiomyocytes. Monolayers of neonatal rat cardiomyocytes (NRCMs) transduced with GFP alone showed sarcolemmal, punctate Cx43 expression. In contrast, Tbx18-transduced NRCMs exhibited sparse Cx43 expression. Both the transcript and protein levels of Cx43 were greatly downregulated within 2 days of Tbx18-transduction. Injection of Tbx18 in the guinea pig heart in vivo markedly suppressed Cx43 expression similar to what was seen in Tbx18-NRCMs. The repressor activity of Tbx18 on Cx43 was highly specific: protein levels of Cx45 and Cx40, which comprise the main gap junctions in the SAN and conduction system, were unchanged by Tbx18. A reporter-based promoter assay demonstrated that Tbx18 directly represses the Cx43 promoter. Phenotypically, Tbx18-NRCMs exhibited slowed calcein dye transfer kinetics (421±54 vs. control 127±43 ms). Intracellular Ca2+-oscillations in control NRCMs monolayers were highly synchronized. In contrast, Tbx18-overexpression led to episodes of spontaneous, asynchronous Ca2+-oscillations demonstrating reduced cell-cell coupling. The decreased electrical coupling led to slow electrical propagation; conduction velocity in Tbx18-NRVMs slowed by more than 50% relative to control (4±1 vs 9±2 cm/s). Taken together, Tbx18 specifically and directly represses the transcript and protein levels of Cx43 in NRCMs in vitro and in adult ventricular myocardium in vivo. Cx43 suppression led to significant electrical uncoupling, but the preservation of other gap junction proteins (Cx45 and Cx40) permitted action potential propagation at slower velocity. Thus, Tbx18 overexpression recapitulates a key phenotypic hallmark of the SAN, namely the characteristic loose electrical coupling.