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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.

Retinoic acid plays a pivotal role during the heart development. The mammalian heart is formed from cardiac progenitors in the first and second heart fields (FHF and SHF). FHF progenitors populate the left ventricle and the atria; SHF progenitors populate the right ventricle, outflow tract and parts of the atria. Loss of retinoic acid signaling leads to depletion of FHF progenitors and compensatory increase in SHF progenitors. Here we hypothesized that mimicking the in vivo retinoic acid signaling during human pluripotent stem cell differentiation could lead to FHF cardiomyocytes.Human pluripotent stem cells were differentiated to cardiac lineage by treatment with Activin A, BMP4 and bFGF (control) for the first three days (d0-3). This lead to >70% Brachyury+ mesodermal cells, and >90% of Brachyury+ cells became cTnT+ cardiac myocytes. Retinoic acid signaling activated with exogenous RA (1μM) from d5-8 (RA protocol). This RA protocol was compared to another protocol in which d5-8 retinoic acid activation was followed by initial inhibition with a pan-retinoic acid (RiRA protocol). At d14, both RA and RiRA cardiomyocytes expressed higher transcript and protein levels of the FHF markers, NKX2.5, TBX5and HCN4, but lower levels of the SHF markers, ISL1, MEF2c, FGF8/10, compared to control. RiRA cardiomyocytes showed highest expression of ventricular markers, IRX4 and HRT2 , lowest expression of an atrial specific marker CoupTFII, which were mirrored in RA cardiomyocytes. In line with their atrial/ventricular-specific gene expression profile, RA cardiomyocytes demonstrated higher beating rates, higher maximum shortening velocity, faster conduction velocity, higher atrial specific ANP secretion and higher sensitivity to atrial-selective sodium blocker, ranolazine compared to control. In contrast, RiRA cardiomyocytes generated higher force but shorter action potential duration compared to control.Taken together, the data demonstrate temporal regulation of retinoic acid signaling can not only accurately recapitulate ventricular to atrial cardiomyocyte differentiation by delineating FHF vs. SHF lineages.