Nkx2.5, Gata4, and Tbx5 activated contractile cardiomyocyte program. (A) Overexpression of Tbx3 specifically repressed the expression of Nkx2.5 (n = 3 in each group). *P < 0.05. (B) Overexpression of Nkx2.5 activated the expression of Nkx2.5, Gata4, and Tbx5 (n = 3 in each group). *P < 0.05. (C) Overexpression of Tbx5 did not significantly affect the expression of Nkx2.5, Gata4, and Tbx5 (n = 3 in each group). (D) Overexpression of Gata4 did not affect the expression of Nkx2.5, but activated the expression of Gata4 and Tbx5 (n = 3 in each group). *P < 0.05. (E) Co-expression of Nkx2.5, Gata4, and Tbx5 significantly activated expression of Gata4 and Tbx5 (n = 3 in each group). (F-I) Activation of ion channel genes by overexpression of Nkx2.5 (F), Tbx5 (G), Gata4 (H), and Nkx2.5/Tbx5/Gata4 triplet (I) (n = 3 in each group). *P < 0.05.