Tbx18 Drives Nodallike Self- assembly Of Pacemaker Myocytes And Non-myocytes

Background: We have demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes (CMs) to induced pacemaker cells. Cell-cell interactions and cell-extracellular matrix (ECM) are important regulators of microtissue organization and assembly. Here, we sought to gain a finer understanding of self-organization and assembly of pacemaker microtissue with myocytes and non-myocytes driven by TBX18. Methods: 2D monolayer of neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno- GFP and Adeno-TBX18 in vitro model. AAV9-TBX18 was delivered via tail vein injection of Hcn4 (+/eGFP) transgenic mice to track the induced pacemaker myocytes. Result: TBX18-NRVMs showed more compact nodal-like aggregates with a higher density of CMs and surrounded by nonmyocytes. The number of alpha smooth muscle actin (αSMA) + myofibroblasts producing specific ECM and facilitating cell motility was increased by 190% in TBX18-NRVMs as well. Systemic delivery of AAV9-TBX18 via tail vein of mice created aggregation of de novo Hcn4+ induced pacemaker myocytes (iPMs) surrounded by αSMA+ myofibroblasts in vivo. Then, we treated pre-plated neonatal rat ventricular cells (NRVCs, including CMs and nonmyocytes) to explore the potential role of cell-cell communication and cell-ECM interaction in the formation of nodal-like aggregates through scRNA-seq. The data indicated that TBX18+ CM (28.6% % of all CMs) and TBX18-negative CMs (71.4% of all CMs) presented highly consistent transcriptomic profiles at day 3. No significant difference in Hcn4 transcript levels and the ratio of iPMs (defined as Hcn4+, Gja1 low , Nkx2.5 low , Tnni3 high , Actn2 high CM) was observed between them, as well as other pacemaker genes, suggesting cross-communication between them. Despite of TBX18+ fibroblasts (FBs) accounting for only 5% of all FBs, a higher proportion of activated myofibroblasts (39.3% vs. 25.4%) and lower proportion of quiescent FBs were observed in TBX18-NRVCs compared to control. Enrichment analysis revealed that TBX18-CMs lost ventricular specific electrical coupling and sarcomere organization (down-regulated Gja1, Cdh2, Irx3 ), but reconstructed hemidesmosome assembly and cell-ECM interaction enriched in sinoatrial node myocytes (Upregulated Lamc1, Itgb1, Plec, Des, Actn1, and Acta2 ) and activated Tgfβ signaling. Furthermore, treatment with gap junction inhibitor palmitoleic acid as well as Tgfβ receptor inhibitor A83-01 significantly suppressed the activation of quiescent fibroblasts to αSMA+ myofibroblasts and the formation of nodal-like aggregation of TBX18-NRVMs. Conclusion: TBX18 drives self-organization and nodal-like assembly of pacemaker microtissue with myocytes and non-myocytes through cell-cell communication of gap junction and Tgfβ signal.