Fig 2: Shenfu injection (SFI) promotes the differentiation of BMSCs into pacemaker-like cells. (a) Cell Counting Kit-8 was utilized to detect the effect of SFI on the viability of BMSCs. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 µL/mL. (b) BMSCs were clamped at -70 mV and stimulated with hyperpolarizing voltages ranging from -70 to -150 mV for 3 s in 10 mV steps to record cellular If currents. (c) Whole-cell patch-clamp electrophysiology was conducted and used for the purpose of recording the If current on BMSCs with different treatments. (d) SFI treatment could induce the expression of HCN4 channel in BMSCs. *P < 0.05 vs. LV-NC, #P < 0.05 vs. SFI-H under ZD7288. Data represented mean ± SEM, n = 6. SFI-L: Shenfu injection-low dose (10 µL/mL); SFI-M: Shenfu injection-medium dose (20 µL/mL); SFI-H: Shenfu injection-high dose (40 µL/mL).

Fig 3: Expression of HCN4 in BMSCs treated with Shenfu injection (SFI). (a) Western blot was conducted to examine the HCN4 expression in SFI-treated BMSCs at protein levels. (b) Real-time PCR was used to detect HCN4 mRNA expression. Data are expressed as the mean ± SEM, n = 3 - 6, *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV-NC. (c) Representative transmission electron microscopy (TEM) images of the ultrastructure derived from BMSCs. Scale bar = 2 µm. (d) ELISA was performed to detect the expression of cAMP. Data are expressed as the mean ± SEM, n = 6, *P < 0.05, **P < 0.01 vs. LV-NC. SFI-L: Shenfu injection-low dose (10 µL/mL); SFI-M: Shenfu injection-medium dose (20 µL/mL); SFI-H: Shenfu injection-high dose (40 µL/mL).