Effect of puerarin on action potential and sodium channel activation in human hypertrophic cardiomyocytes

Figure 1. Differentiation and purification of hiPSC-CMs (A) After culturing with CEHCDCM I medium for 48 h, the hiPSCs showed typical deformation (short arrow) and a large number of mesodermal cells migrated out from the clones (long arrow); scale bar: 300 μm. (B) Following incubation with CEHCDCM III for 24 h, the mesoderm cells continued to grow and increase in density; scale bar: 300 μm. (C) After incubation with CEHCDCM II for another 48 h, a large number of mesodermal cells differentiated into cardiomyocytes (hiPSC-CMs, with typical myocardial morphology) (short arrow) and began to beat (long arrow); scale bar: 300 μm. (D) The differentiated hiPSC-CMs were purified in CEHMPCM, and the non-cardiomyocytes began to die; scale bar: 100 μm. (E) The differentiated hiPSC-CMs were stable after incubation with CEHMMM for 24 h; scale bar: 100 μm. (F) Figure shown a schedule of cardiomyocytes (hiPSC-CMs) differentiation from hiPSCs. CEHCDCM, CEHCDCM I, CEHCDCM II, and CEHCDCM III were different differentiation mediums purchased from CELLAPY (Beijing, China). D1, D3, D5, and D7 represented day 1,3, 5, and 7, respectively.

Figure 2. Identification of the differentiated hiPSC-CMs (A) Immunofluorescence staining of hiPSC-CMs with α-actinin (green, right-up) and TNNT2 (red, left-down) antibodies. Nuclei were stained using DAPI (blue, up-left); scale bar: 25 μm. (B) Gene expression of pluripotency-related genes (OCT4 and NANOG) and myocardial specific genes (NKX2-5, MYL2, MYH6, and MYH7) was measured using RT-PCR. GAPDH expression was used as the reference gene.

Figure 3. Immunofluorescence staining of the ET-1-induced cardiomyocytes (hiPSC-CM) (A) After induction of hiPSCs with 10 nM ET-1 (middle panels) for 24 h, expression of BNP (red) and α-actinin (green) were enhanced, and the cell shape was increased compared with the non-treated cells (up panels) and Bosentan (ET-1 antagonist) treated cells (lower panels); scale bar: 40 μm. (B) The levels of proBNP and α-actinin were significantly increased in the ET-1 group compared with the control group. However, Bosentan treatment reduced the expression of proBNP and α-actinin; however, both were still expressed at lower levels compared with the control group. *: P < 0.05 compared with control group.

Figure 4. Alteration of MYH7/MYH6 ratio after ET-1 induction ET-1 induction significantly increased the ratio of MYH7/MYH6 in the hiPSC-CMs compared with the non-ET-1 treated cells (native) as measured by RT-PCR.

Figure 5. Toxic effect of serial concentrations of puerarin on cardiomyocytes (A) Cell survival (CI) was detected under the indicated treatment concentrations of puerarin from 0 to 40 h. (B) Statistical analysis of the CI data in the groups treated with different concentration of puerarin at 0, 1, 2, 4, 8, 12, and 24 h. (C) The beating rate (BR) was recorded under the indicated treatment concentrations of puerarin from 0 to 40 h. (D) Statistical analysis of the BR data in the groups treated with different concentration of puerarin at 0, 1, 2, 4, 8, 12, and 24 h. N.S: P >0.05.

Figure 6. Activity of puerarin on action potential Action potentials were recorded in the indicated groups. APA: action potential phase 0 depolarization amplitude; APD30: depolarization start to repolarization 30% time; APD50: depolarization start to repolarization 50% time; APD90: depolarization start to repolarization 90% time; dV/dt Max (Vmax): maximum depolarization velocity in phase 0; HR: number of beats per minute in myocardium; RP: resting membrane potential; Action potential: action potential map of hiPSC-CMs. *: P<0.05 compared with the control group. N.S: P >0.05.

Figure 7. Electrophysiology measurements of the sodium ion channel (A) IV curves: cell membrane potential in the indicated groups was initially maintained at −90 mV, and then gradually stepped from −120 to −100 mV in 5 mV step intervals. (B) The average of the peak currents in each group. (C) Activation curves of sodium channel: cell membrane potential in the indicated groups was initially maintained at −90 mV, and gradually stepped to −120mV for 200 ms, and then stepped from −80 to 100 mV in 5 mV step intervals to detect the activation state of the channel. (D) Inactivation curves of sodium channel: cell membrane potential in the indicated groups was initially maintained at −90 mV, and then gradually stepped from −120 to 40 mV for 1000 ms, then stepped to 0 mV for 50 ms in 5 mV step intervals to detect sodium current. (E) Recovery curves of sodium channel: cell membrane potential in the indicated group was initially maintained at −90 mV, and then gradually stepped to −10 mV for 250 ms to inactivate sodium current, then stepped to −90 mV for different durations to recover the sodium current, and then finally stepped to −10 mV for 250 ms to detect the sodium current. Groups: control (●),…

Figure 8. Typical current trace of sodium channel Cellular membrane potential was maintained at −90 mV, and then increased at 5 mV interval in the control, puerarin, ET-1, puerarin preconditioning, and puerarin popstconditionaing group, respectively. Once the cellular membrane potentials stepped to 100 mV, the current mode of INa was recorded.