hERG potassium channel blockade by the HCN channel inhibitor bradycardic agent ivabradine

Abstract

Background: Ivabradine is a specific bradycardic agent used in coronary artery disease and heart failure, lowering heart rate through inhibition of sinoatrial nodal HCN-channels. This study investigated the propensity of ivabradine to interact with KCNH2-encoded human Ether-à-go-go-Related Gene (hERG) potassium channels, which strongly influence ventricular repolarization and susceptibility to torsades de pointes arrhythmia.

Methods and results: Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. Ih ERG was inhibited with an IC50 of 2.07 μmol/L for the hERG 1a isoform and 3.31 μmol/L for coexpressed hERG 1a/1b. The voltage and time-dependent characteristics of Ih ERG block were consistent with preferential gated-state-dependent channel block. Inhibition was partially attenuated by the N588K inactivation-mutant and the S624A pore-helix mutant and was strongly reduced by the Y652A and F656A S6 helix mutants. In docking simulations to a MthK-based homology model of hERG, the 2 aromatic rings of the drug could form multiple π-π interactions with the aromatic side chains of both Y652 and F656. In monophasic action potential (MAP) recordings from guinea-pig Langendorff-perfused hearts, ivabradine delayed ventricular repolarization and produced a steepening of the MAPD90 restitution curve.

Conclusions: Ivabradine prolongs ventricular repolarization and alters electrical restitution properties at concentrations relevant to the upper therapeutic range. In absolute terms ivabradine does not discriminate between hERG and HCN channels: it inhibits Ih ERG with similar potency to that reported for native If and HCN channels, with S6 binding determinants resembling those observed for HCN4. These findings may have important implications both clinically and for future bradycardic drug design.

Keywords: HCN; HCN4; QT interval; bradycardic agent; hERG; ivabradine; repolarization.

Figure 1.

Effect of ivabradine on I_hERG and I_hERG‐1a/1b. A, Upper traces show representative I_hERG records elicited by the step protocol shown below, in Control and after the application of 3 μmol/L ivabradine (the voltage protocol was applied at 12‐s intervals). The amplitude of peak I_hERG tails at −40 mV was measured relative to current elicited by the initial brief 50‐ms step from −80 to −40 mV. B, Normalized concentration‐response relationship for ivabradine block of I_hERG tails for WT hERG 1a and hERG 1a/1b. Fractional inhibition of I_hERG tails was assessed at each of 5 ivabradine concentrations for WT 1a and 4 ivabradine concentrations for 1a/1b hERG (n≥5 at each concentration for each expression condition). C, Voltage dependence of ivabradine block (black dotted line) and voltage‐dependent activation relations for I_hERG in Control (black continuous line) and in the presence of 3 μmol/L ivabradine (gray line). The activation relations were simulated by calculating activation variables at 2‐mV intervals using equation 4 in Data S1 and the activation parameters yielded by fitting experimental data (Figure S1). D, Upper traces show representative records of I_hERG elicited by the action potential protocol shown below, in Control and after the application of 3 μmol/L ivabradine. hERG indicates human Ether‐à‐go‐go‐Related Gene; I_hERG, hERG current; WT, wild‐type.

Figure 2.

Effect of ivabradine on ventricular monophasic action potential (MAP). A and B, Representative MAPs during Control (dashed line) and 0.2 μmol/L ivabradine (solid line) from Apex (A) and Base (B) of the guinea‐pig isolated Langendorff‐perfused heart. C and D, MAP duration (MAPD) at 50% repolarization and 90% repolarization during Control and 0.2 μmol/L ivabradine (solid bars) from apex (C) and base (D) (n=7; *P<0.05, paired t test).

Figure 3.

Effect of ivabradine on ventricular electrical restitution. A, Representative example of MAPD‐restitution curves at the left ventricular base of an isolated perfused heart during control and at each ivabradine concentration studied (0.1, 0.2, 0.3, 0.4, and 0.5 μmol/L). B and C, Mean data for maximum slope of the restitution curve (RT max) at each ivabradine concentration examined (n=6) at the left ventricular apex (B) and base (C) (**P<0.01 vs Control; *P<0.05 vs Control; repeated measure single‐factor ANOVA with Bonferroni post hoc test). D and E, Mean data for S2‐delay (duration between the pacing stimulus and S₂‐activation) at each ivabradine concentration examined at the left ventricular apex (D) and base (E) (n=7). MAPD indicates monophasic action potential duration.

Figure 4.

Time dependence of human Ether‐à‐go‐go‐Related Gene current (I_hERG) inhibition by ivabradine. A, Representative traces of I_hERG in Control (upper panel) and in the presence of 3 μmol/L ivabradine (lower panel) elicited by the “envelope of tails” protocol shown at the bottom of the lower panel. B, Time dependence of normalized tail I_hERG in Control (black) and in the presence of 3 μmol/L ivabradine (gray) (n=5). Data at each time point were normalized to the maximum tail current elicited by the protocol in Control. Lines connect successive points in each plot. C, Time dependence of fractional block of I_hERG by 3 μmol/L ivabradine, fitted with a mono‐exponential function (dotted line, equation 5, Data S1) (n=5) to yield τ value in the text.

Figure 5.

Effect of ivabradine on hERG channel availability. A, Voltage protocol used to study hERG channel availability. B and C, Sample traces of hERG transient current in Control (B) and in the presence of 3 μmol/L ivabradine (C) elicited by the portion of the 3‐step protocol shown at the bottom of (C) (expanded from the dashed box in (A). For clarity of display, only selected test voltages are reported, while the full protocol spans from −140 to +50 mV with a 10‐mV increase at each step). D, Voltage dependence of the normalized resurgent current elicited by the third step of the 3‐step protocol in Control (black) and in the presence of 3 μmol/L ivabradine (gray) (n=6). Experimental data were fitted with equation 6 (dotted lines, Data S1) to give the V_0.5 and k values in the Results. E, Time constant of I_hERG inactivation calculated by fitting the peak transient current at +40 mV after a 2‐ms step to −120 mV with a mono‐exponential decay function (equation 7, Data S1). The application of 3 μmol/L ivabradine had no significant effect on τ_inactivation (n=6, ns P>0.05, Wilcoxon matched‐pairs signed‐rank test). hERG indicates human Ether‐à‐go‐go‐Related Gene; I_hERG, current hERG.

Figure 6.

Effect of hERG mutants on ivabradine block of I_hERG. A, Representative traces of N588K I_hERG elicited by a step protocol identical to that used to study WT I_hERG in Figure 1A in control and in the presence of 3 μmol/L ivabradine. B, Concentration response relation for ivabradine action on N588K I_HERG compared with that for WT I_hERG. Fractional inhibition was assessed for I_hERG tails at each of 4 concentrations (n≥5 at each concentration). C, Representative traces of S624A I_hERG elicited by same protocol as used to study N588K in control and in the presence of 3 μmol/L ivabradine. D, Voltage protocol with hyperpolarizing step to −120 mV used to elicit WT (upper panel) and F656A (lower panel) inward currents in high (94 mmol/L) external potassium condition is shown as an inset above Figure 6F. The dotted box frames the portion of the protocol shown on an expanded timescale at the bottom of the lower panel. Upper and lower panels each show representative traces in Control and 3 μmol/L ivabradine while the insets to both panels show peak inward currents on expanded scale for clarity of display. E, Representative traces for Y652A I_hERG elicited by same protocol as used to study N588K and S624A hERG, in Control and in the presence of 3 μmol/L ivabradine. The inset shows tail currents on an expanded timescale in order to aid visualization of the peak I_hERG tail in control and drug. F, Bar charts that summarize the effect of 3 (black bars) and 10 μmol/L (white bars) ivabradine on WT I_hERG in standard (4 mmol/L) external potassium condition elicited at −40 mV by a standard outward I_hERG protocol (n=5 for 3 μmol/L and n=6 for 10 μmol/L), on inward WT I_hERG elicited at −120 mV in high (94 mmol/L) external potassium condition (n=5 for both concentrations), on F656A inward current elicited at −120 mV in high potassium condition (n=5 for both concentrations) and on S624A and Y652A outward current elicited at −40 mV in standard external potassium condition (n≥5 at each concentration) (**P<0.01 against respective Control, ***P<0.0001 against respective Controls; for details of tests used see legend to Table). hERG indicates human Ether‐à‐go‐go‐Related Gene; I_hERG, current hERG; WT, wild‐type.

Figure 7.

Docking simulation. Lateral (A) and intracellular (B) views of a representative pose for ivabradine docked to the MthK‐based human Ether‐à‐go‐go‐Related Gene (hERG) open‐state homology model. Pore and S6 helices are represented as faint gray ribbon. S624, T623, and V625 pore helical residues are represented as green sticks, while Y652 and F656 are represented respectively as pink and blue sticks. Ivabradine is shown in yellow, while the purple spheres represent the K⁺ ions (displayed at full ionic diameter) in positions S1 and S3 of the channel selectivity filter. C, Representative GOLD low‐energy score pose for ivabradine docked to the MthK‐based model. The side chains of the aromatic residues that make π‐π (black dotted line) and cation‐π (blue dotted line) interactions with the drug molecule are represented in light blue and pink sticks. The S624 side chains are also shown as green sticks. The potassium ion in the S3 site of the selectivity filter is shown as a purple sphere. The interactions shown include the following: π‐π interaction 4.0 Å with Y652; π‐π interaction 3.9 Å with F656; π‐π interaction 3.7 Å with F656; and π‐cation interaction 3.9 Å with F656. For clarity, only the Y652 and F656 residues that make interactions with ivabradine are shown. D, Sequence alignment between hERG and HCN4, focusing on pore helical region/selectivity filter (the GFG and GYG sequences for hERG and HCN4 are shaded) and on the S6 domain for which Y652 and F656 in hERG are shaded and corresponding aromatic residues in HCN4 are also shaded.