Fig 1.
Concentration-dependent effects of vandetanib on action potentials in rabbit Purkinje fiber.
(A) Representative traces of action potential recorded in vehicle control (VC) condition and presence of vandetanib at concentrations of 0.3, 1, and 3 μM. (B-F), Normalized AP parameters of rabbit Purkinje fibers in the control and presence of 0.3, 1, and 3 μM vandetanib. Data are expressed as mean ± standard error of mean (SEM) and compared by ANOVA followed by Dunnett’s test. Vmax: maximum upstroke velocity, RMP: Resting membrane potential, APA: action potential amplitude, APD50: action potential duration at 50% repolarization, APD90: action potential duration at 90% repolarization. *p < 0.05 and **p < 0.01 compared to VC (rabbit n = 3).
Fig 2.
Effects of vandetanib on cardiac ion channel currents.
Representative traces demonstrating the dose-dependent effects of vandetanib on IhERG (A), IKs (B), IK1 (C), and INa (D) currents expressed in HEK293 cells and ICa (E) in rat ventricular myocytes, respectively. Dose-response relationship of vandetanib showing the IC50 values for hERG (1.15 ± 0.02 μM, n = 4), IKs (28.04 ± 1.81 μM, n = 4), IK1 (94.52 ± 4.57 μM, n = 3, No effect at 10 μM), INa (36.63 ± 8.61 μM, n = 4), and ICa (32.74 ± 0.64 μM, n = 3). IhERG, human ether-a-go-go-related gene (hERG) currents; IKs, slow delayed rectifier potassium currents; IK1, inward rectifier potassium currents; INa, sodium channel currents; ICa, calcium channel currents; VC, vehicle control.
Fig 3.
Effect of mutation at the S6 domain and pore region on vandetanib-induced hERG inhibition.
Representative current traces of (A) wild type (WT)-, (B) T623A-, (C) S624A-, (D) Y652A-hERG currents under extracellular normal K+ condition and (E) WT-, (F) F656A-hERG currents under extracellular high K+ (94 mM) in vehicle control (VC) condition (black lines) and after application of 3 μM vandetanib (red lines). (G) The mean and SEM of the percent inhibition rates produced by 3 μM vandetanib for WT-, T623A-, S624A-, Y652A-, WT (inward)-, and F656A-hERG currents. *p < 0.05 compared to WT by Dunnett’s test. ##p < 0.01 compared to WT (inward) by Student’s t-test.
Fig 4.
Schematic diagrams of vandetanib bound to hERG.
Hydrophobic and hydrogen-bonding interactions are depicted as red and blue dotted lines, respectively. (A) Proposed binding mode of vandetanib in hERG open-state homology model. Vandetanib is given as the yellow stick. (Up) The four subunits of hERG are represented as ribbons in different colors. (Down) Subunits interacting with vandetanib are shown as ribbons, but the others as wires. The residues with interactions are represented as sticks. (B) 2D vandetanib interaction diagram. The residues in red and blue circles form hydrophobic and hydrogen-bonding interactions with vandetanib, respectively.
Fig 5.
Effects of vandetanib on action potential parameters of hiPSC-CMs.
(A) Typical action potential traces of hiPSC-CMs in the absence (vehicle control, VC) and presence of 0.3, 1, and 3 μM vandetanib. (B) Normalized AP parameters of hiPSC-CMs in VC and in the presence of 0.3, 1, and 3 μM vandetanib. Data are expressed as mean ± SEM and compared by ANOVA followed by Dunnett’s test. Vmax, maximum upstroke velocity; APD90 or APD50, action potential duration at 90 or 50% repolarization; APA, action potential amplitude; MDP, maximal diastolic potential. *p < 0.05 and **p < 0.01 compared to VC (n = 6).
Fig 6.
Effects of vandetanib on IhERG and INa currents in hiPSC-CMs.
(A) Representative traces demonstrating the dose-dependent effects of vandetanib on IhERG currents. E-4031: IhERG specific blocker (B) Dose–response relationship showing an IC50 value for IhERG in hiPSC-CMs (mean ± SEM, n = 3). (C) Representative traces showing the dose-dependent effects of vandetanib on INa currents. (D) Dose–response relationship demonstrating an IC50 value for INa in hiPSC-CMs (mean ± SEM, n = 3). TTX (tetrodotoxin): a sodium channel blocker.
Fig 7.
Comparison of tetrodotoxin (TTX)-sensitive INa current density and sensitivity in hiPSC-CMs and SCN5A-encoded HEK293 cells.
(A) Representative I-V traces of INa in hiPSC-CMs under control (left) and 3 μM TTX (middle). I-V relationships of TTX-sensitive currents in hiPSC-CMs (right, mean ± SEM, n = 3) (B) Dose–response relationship showing an IC50 value for INa in hiPSC-CMs (mean ± SEM, n = 6). (C) Representative I-V traces of INa in HEK293 under control (left) and 10 μM TTX (middle). I-V relationships of TTX-sensitive currents in heterogeneous HEK293 cells (right, mean ± SEM, n = 3). (D) Dose–response relationship showing an IC50 value for INa in hiPSC-CMs (mean ± SEM, n = 4).
Fig 8.
Concentration-dependent effects of class I antiarrhythmic agents, quinidine, lidocaine, and flecainide, on INa currents expressed in hiPSC-CMs or HEK293 cells.
(A) Representative current traces for quinidine (class IA, left)-, lidocaine (class IB, middle)-, and flecainide (class IC, right)-induced INa current inhibitions in hiPSC-CMs. (B) Dose-dependent inhibition of INa currents by quinidine (left), lidocaine (middle), and flecainide in SCN5A-encoded HEK293 cells. Dose-response curves are inserted (each n = 3).