L-Type Calcium Channel Inhibition Contributes to the Proarrhythmic Effects of Aconitine in Human Cardiomyocytes

Fig 1. Effects of ACO on action potentials of hiPSC-CMs. (A) Representative AP waveforms recorded in a hiPSC-CM with 120 seconds (s) exposure of ACO at 0.3 and 3.0 μM and 180s and 300s exposure of ACO at 3.0 μM. Increased beating frequencies and decreased APD and APA were observed with short-term (120s) ACO treatment. Prolonged treatment of ACO at 3.0 μM led to frequent DADs and rapid irregular VF-like AP changes. (B),Representative AP waveforms recorded in a hiPSC-CM exposed to 0.3 μM nifedipine for 60s ~ 240s. Increased beating frequencies and decreased APD occurred at 60s while DADs emerged at 180s. APs were suppressed at 240s. The arrows indicate DADs.

Fig 2. Effects of ACO on [Ca2+]i transients in hiPSC-CMs. (A) Representative traces of the [Ca2+]i transients recorded in a hiPSC-CM at baseline and with subsequent exposure of ACO and ACO plus Bay K-8644. (B, C, and D) Bar-graphs show the changes in [Ca2+]i transient frequency, duration and amplitude in hiPSC-CMs. †p < 0.01, vs. baseline. ‡ p < 0.001, vs. ACO. n = 7.

Fig 3. Effects of ACO on sodium currents in hiPSC-CMs. (Aa) A representative time course and corresponding traces of the peak INa recorded in a hiPSC-CM showing the effect of ACO and TTX. (Ab) The I-V curve showing peak INa density in hiPSC-CMs. n = 7. (Ac) The SS-inactivation and -activation curves. n = 7. (Ba) A representative time course and the corresponding traces of late INa (INaL) recorded in a hiPSC-CM showing the effects of ACO and TTX. (Bb) A bar-graph showing the average densities of the INaL (at 200ms) recorded in hiPSC-CMs treated with 3 μM ACO. n = 7.

Fig 4. Effects of ACO on ICa,L in hiPSC-CMs. (A) Representative traces of the voltage-gated LTCC current ICa,L recorded from a hiPSC-CM at baseline followed by exposure of 1 μM ACO and addition of 1 μM nifedipine. (B) Representative traces and I-V curves recorded from hiPSC-CMs exposed to 1 μM ACO followed by 1 μM Bay K-8644. *p < 0.05, †p < 0.01, ‡ p < 0.001, ACO vs. baseline or Bay K-8644 vs ACO, n = 8. (C) The voltage-dependent activation curve of ICa,L recorded in hiPSC-CMs exposed to 1 μM of ACO. G/Gmax: normalized peak conductance. *p < 0.05, †p < 0.01, vs. baseline. n = 5. (D) The SS-inactivation curve of ICa,L. *p < 0.05, †p < 0.01, ‡ p < 0.001, vs. baseline. n = 5. (E) Recovery from inactivation of ICa,L. n = 5. Insert shows the 2-pulse protocol. (F) The IC50 of ACO on ICa,L (n = 8).

Fig 5. Effects of ACO on INa, IKr and ICa,L in heterologous expression systems. Nav1.5-HEK293, hERG-HEK293 and Cav1.2-CHO cells were exposed to 0.1~100 μM ACO. (A) The concentration-dependent inhibition curve of INa and representative traces recorded. (B) The concentration-dependent inhibition curve of IKr (peak tail: the peak of the tail current) and representative traces. (C) The concentration-dependent inhibition curve of ICa,L and representative traces.

Fig 6. Effects of ACO on the field potentials in hiPSC-CMs. (A) Representative traces of the filed potentials (FD) recorded with a cluster of hiPSC-CMs at baseline, exposed to various concentrations of ACO and after washout. (B) A bar-graph demonstrates the average beating frequency (BF), Ca2+ waves and FPDc recorded at baseline, with 0.03, 0.1, 0.3, 1.0 and 3.0 μM of ACO treatments and after washout. *p< 0.05, ‡p< 0.001; vs. baseline. n = 4.

Fig 7. Effects of ACO on sodium currents in Guinea-pig ventricular myocytes. (A) Representative traces of the peak INa recorded in a Guinea-pig ventricular myocyte prior to and after 3 μM ACO. (B) The I-V curves showing the current densities of peak INa. n = 5. (C) The SS-inactivation and -activation curves. n = 5. (D) The I-V curves of the voltage-gated INaL densities. n = 5. (E) Representative traces of voltage-dependent INaL recorded in a Guinea-pig ventricular myocyte prior to and post 3μM ACO. *p < 0.05, †p < 0.01, vs. baseline.