Abstract 16737: Inhibition of Tgf-β1 Signaling Protect Against Cell-Cell Decoupling and Asynchronous Automaticity of Tbx18-ipms

Abstract

Background: We have previously demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes to induced pacemaker cells (TBX18-iPMs). However, the nascent automaticity appeared to wane over time, characterized by loss of syncytial pacing but preservation of single cell automaticity. Hypothesis: Based on increase in Tgfβ ligands in TBX18-iPMs and the known role of Tgfβ signaling in electrical remodeling, we hypothesized that loss of syncytial automaticity is due to electrical decoupling of the iPMs mediated by Tgfβ signaling. Methods: Adenoviral vectors expressing either human TBX18 or GFP were used for gene transfer into neonatal rat ventricular myocytes (NRVMs) or adult rat ventricular myocardium in vivo. Results: NRVM monolayers transduced with GFP were mostly quiescent, interspersed by paroxysmal contractions. In contrast, TBX18 transduced NRVM monolayers showed spontaneous and rhythmic contractions, but the syncytial pacing wavered over the next 5-7 days although numerous iPMs continued to beat asynchronously. Treatment of TBX18-iPMs with an inhibitor of Tgfβ receptors, A83-01, preserved syncytial pacing, suggesting that electrical remodeling cues from Tgfβ signaling led to loss of iPM-iPM electrical coupling. We examined the molecules that are integral to the function of gap junction in the myocardium, namely Cx43, N-cadherin and β-catenin. Cx43 and N-cadherin were downregulated in TBX18-iPMs compared to control by 77±16% and 43±11% (p<0.01, n=6), respectively. Total β-catenin protein level was unaltered, but its distribution decreased at the sarcolemmal and increased in the nuclei of TBX18-iPMs compared to control. Inhibition of Tgfβ with A83-01 restored Cx43 protein level in TBX18-iPMs (2.2-folds higher) compared to untreated TBX18-iPMs. Similarly, adult rats that received intramyocardial injection of TBX18 showed increased Cx43 expression at the boundary between the iPMs and the host myocardium when the animals were treated with systemic delivery of A83-01 for one week compared to TBX18 animals treated with DMSO. Conclusions: TBX18 activates Tgfβ signaling which suppresses cell-cell electrical coupling. Inhibition of Tgfβ signaling in TBX18-iPMs preserves gap junction components and syncytial pacing.