Abstract P3132: Tgfβ Signaling Triggers Nonmyocyte Gene Expression In Tbx18-induced Pacemaker Myocytes And Abbreviates Their Automaticity

Abstract

Background: We have previously demonstrated reprogramming of ventricular myocytes into induced pacemaker cells (iPMs) using an embryonic transcription factor TBX18. We have also discovered that Tgfβ pathway is significantly upregulated in TBX18-iPMs, and triggers fibrosis upon gene transfer in vitro and in vivo. Here, we sought to investigate the impact of Tgfβ signaling on iPMs’ automaticity. Methods: Neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno-TBX18 or control Adeno-GFP. Single cell RNAseq was performed at days 3, 6, and 14. Spontaneous field potentials from NRVM monolayers were recorded with microelectrode array. Results: TBX18 gene transfer strongly repressed working cardiomyocyte gene expression, e.g., Gja1, Scn5a, Pln throughout the 14-day period. TBX18-NRVMs progressed toward pacemaker cells, evidenced by increase in nodal pacemaker-related ion channel gene expression. TBX18 also triggered cytoskeletal remodeling, and activated nonmyocyte gene expression throughout the 2-week window. This was illustrated by ECM remodeling gene expression such as Acta2, Col1a1, and Postn. Treatment with A83-01, a small molecule inhibitor of Tgfβ receptors, mitigated the increase in nonmyocyte gene expression. TBX18-NRVMs exhibited spontaneously oscillating field potentials, reaching a peak pacing frequency of 196±36 bpm during week 1, which is comparable to the sinus rhythm of newborn rats. However, the pacing frequency gradually wavered after week 1. In contrast, A83-01-treated TBX18-iPMs showed peak pacing frequency beyond week 1, with the majority of their total beat counts near their sinus rhythm (63.3%) while only 14.9% of total beats were near the sinus rhythm in TBX18-iPMs without Tgfβ inhibition (P<0.001). Interestingly, A83-01 did not impact PM-related ion channel gene expression (such as Hcn4, Gjc1) nor the percentage of TBX18-iPMs (5.1% vs. 5.4%, with or without A83-01, p=0.561), suggesting that improved automaticity may be due to its impact on nonmyocytes. Conclusion: TBX18-iPMs exhibit increased Tgfβ signaling and nonmyocyte gene expression, which negatively impacts their automaticity. Inhibition of Tgfβ signaling modulates nonmyocyte gene expression and enhances automaticity of TBX18-iPMs.