Abstract 4362717: Combinatorial Transcription Factor Gene Transfer Accelerates Direct Reprogramming of Ventricular Myocytes to Pacemaker Phenotype

Background: We previously demonstrated that re-expression of the embryonic transcription factor TBX18 is sufficient to reprogram ventricular myocytes (VMs) into sinoatrial node (SAN)-like induced pacemaker myocytes (iPMs). Here, we examined the timeline of this reprogramming and tested whether combined gene transfer of TBX18, SHOX2, and TBX3—transcription factors critical to SAN development—would lead to superior reprogramming. Methods: Neonatal rat VMs were isolated, cultured as monolayers, and transduced with adenoviral vectors expressing (1) GFP (control), (2) TBX18 alone, or (3) a combination of TBX18, SHOX2, and TBX3 (TST group). Spontaneous field potentials were recorded via multi-electrode arrays (MEA) on days 1, 3, 5, 7, 9, and 12. Single-cell RNA-seq (scRNA-seq) was performed on days 3, 6, and 14. Result: MEA data revealed that by d5, the TBX18 group exhibited significantly elevated beat rates compared to control (89.4±49.9 vs. 62.5±30.6 bpm, n =8, p <1e−50). Notably, TST group showed markedly faster pacing rates as early as d3 (181.6±89.5 bpm, p<1e-50), a difference that persisted throughout d12 (71.3±27.9 bpm, n=8, p<1e-50), indicating rapid onset and sustained efficacy of TST-mediated reprogramming. ScRNA-seq analysis revealed that reprogramming by TBX18 involved activation of signaling pathways such as Wnt and TGF-β, though significant changes in the expression of related molecules did not emerge until d7. In contrast, the TST group showed early enrichment (by d3) of differentially expressed genes (DEGs) involved in canonical Wnt signaling (GO:0060828, p =3.9e−4), cell-substrate adhesion (GO:0010810, p =2.1e−3), and inhibition of stem cell differentiation (GO:2000737, p =5.6e−3).Compared to VMs, the TST group exhibited early and robust expression of SAN-specific genes such as Hcn4 (log2FC=0.27, p =1.6e−22), Gjc1 (log2FC=0.27, p =1.4e−35), and Col1a1 (log2FC=1.25, p =4.1e−27), while downregulating ventricular-enriched genes including Gja1 (log2FC=1.48, p <1e−50), Ryr2 (log2FC=0.64, p <1e−50), and Slc8a1 (log2FC=0.18, p <1e−50). These findings were validated by qPCR and immunostaining. Conclusion: The results indicate that gene transfer of three transcription factors that figure prominently in the native SAN accelerates pacemaker cell reprogramming, accompanied by significantly higher spontaneous beat rates compared to TBX18 alone. This combinatorial approach enables faster onset and more sustained gene therapy for biological cardiac pacing.