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 murine embryos at embryonic day 11.5 and divided into the following four treatment groups: Control, Bmp4, Bmp4+LDN193189 (a Bmp inhibitor) and LDN193189. Bmp4 promoted the expression of Hcn4 in Tbx18 EPCs via lineage tracing of Tbx18:Cre/Rosa26R 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.