Bone morphogenetic protein 4 promotes the differentiation of Tbx18-positive epicardial progenitor cells to pacemaker-like cells

Clarifying the mechanisms via which pacemaker- like cells are generated is critical for identifying novel targets for arrhythmia-associated disorders and constructing pacemakers with the ability to adapt to physiological requirements. T-box 18 (Tbx18)⁺ epicardial progenitor cells (EPCs) have the potential to differentiate into pacemaker cells. Although bone morphogenetic protein 4 (Bmp4) is likely to contribute, its role and regulatory mechanisms in the differentiation of Tbx18⁺ EPCs into pacemaker-like cells have remained to be fully elucidated. In the present study, the association between Bmp4, GATA binding protein 4 (Gata4) and hyperpolarization- activated cyclic nucleotide gated Potassium Channel 4 (Hcn4) to regulate NK2 homeobox 5 (Nkx2.5), which is known to be required for the differentiation of Tbx18⁺ EPCs into pacemaker-like cells, was assessed. Tbx18⁺ EPCs were isolated from Tbx18:Cre/Rosa26R^{enhanced yellow fluorescence protein (EYFP)} murine embryos at embryonic day 11.5 and divided into the following four treatment groups: Control, Bmp4, Bmp4+LDN193189 (a Bmp inhibitor) and LDN193189. In vitro Bmp4 promoted the expression of Hcn4 in Tbx18⁺ EPCs via lineage tracing of Tbx18:Cre/Rosa26R^EYFP mice, which was likely due to upregulation of Gata4 expression. Gata4 knockdown experiments were then performed using the following five treatment groups: Control, control small interfering RNA (siRNA), Bmp4, Bmp4+siRNA targeting Gata4 (siGata4) and siGata4 group. Knockdown of Gata4 caused a downregulation of Hcn4 and an upregulation of Nkx2.5, but had no effect on Bmp4 expression. In conclusion, it was indicated that in Tbx18⁺ EPCs, the expression of Nkx2.5 was regulated by Bmp4 via Gata4. Taken together, these results provide important information on regulatory networks of pacemaker cell differentiation and may serve as a basis for further studies.