Modulation of hERG potassium channel gating normalizes action potential duration prolonged by dysfunctional KCNQ1 potassium channel

Abstract

Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, ∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as I(Ks) and I(Kr). The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I(Kr) by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if I(Ks) is reduced. Using a unique specific chemical activator for I(Kr) that has a primary effect of causing a right shift of V(1/2) for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an I(Kr) chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent.

Fig. 1.

Comparison between simulated and recorded ventricular-like action potentials from iPSC-derived cardiomyocytes from a healthy control and patient with LQT1. (A) qRT-PCR analysis of HERG1a, HERG1b, and KCNQ1 in cardiomyocyte clusters from control (bar with light shading) and LQT1 (open bar) iPSCs at 4-mo maturation compared with human adult (AH, solid bar) and fetal (FH, bar with dark shading) heart. Expression values are relative to those of AH, normalized to GAPDH, and presented as mean ± SD, n = 3. (B) Representative traces of the ventricular-like AP recorded from cardiomyocytes derived from either a healthy control or a patient with LQT1 with R190Q mutation as indicated. The experimentally recorded traces are shown on the Left, whereas the simulated ventricular-like APs based on a modified ten Tusscher model are shown on the Right.

Fig. 2.

Simulation study of different compound effects on iPSC-derived cardiomyocytes from a healthy control and a patient with LQT1. (A) Simulated AP from a healthy control without compound treatment (I_NaL = 0.055 nS/pF) and a presumed patient with LQT3 without compound treatment (I_NaL = 0.33 nS/pF) or with I_NaL blocker treatment (I_NaL = 0.070 nS/pF). (B) Simulated action potential from a healthy control without compound treatment (I_NaL = 0.055 nS/pF, scale factor of G_Ks = 1), a patient with LQT1 without compound treatment (I_NaL = 0.055 nS/pF, scale factor of G_Ks = 0.3), a healthy control with I_NaL blocker treatment (I_NaL = 0.014 nS/pF, scale factor of G_Ks = 1), or a patient with LQT1 with I_NaL blocker treatment (I_NaL = 0.014 nS/pF, G_Ks =0.3). (C) Simulated APs from a healthy control (scale factor of G_Ks = 1, x_r2 V_1/2 = −87 mV) and a healthy control with a progressive shift in x_r2 V_1/2 = −70 mV, −40 mV, −10 mV, respectively, to show different doses of compound effect on inactivation V_1/2. (D) Simulated APs from a patient with LQT1 without compound treatment (scale factor of G_Ks = 0.3, x_r2 V_1/2 = −87 mV) and a patient with LQT1 with progressive shift in x_r2 V_1/2 = −70 mV, −40 mV, or −10 mV, respectively. (E) Simulated AP from a patient with LQT1 without compound treatment (scale factor of tx_r1 = 1) and a patient with LQT1 with progressive decrease in I_Kr decay. Scale factor of tx_r1 = 2 or 3, respectively, to mimic different doses of compound effect on deactivation.

Fig. 3.

ML-T531 effects on expressed hERG and KCNQ1/KCNE1 channels. (A) ML-T531, when tested at 10 μM, significantly potentiates hERG steady-state currents and tail currents in CHO cells. The holding potential was −80 mV. The steady-state currents were examined from −80 mV to +80 mV in 20-mV increments, whereas tail currents were elicited at −50 mV. (B) Steady-state current density of hERG either in the absence or in the presence of 10 μM ML-T531, plotted against the voltage. (C) ML-T531, when tested at 10 μM, has no significant effect on hERG activation V_1/2. Tail currents were elicited at −50 mV and the G-V curve was fitted by the Boltzmann equation. (D) ML-T531, when tested at 10 μM, slows hERG deactivation rates. Tail current deactivation was examined from −130 mV to −80 mV and was fitted by a standard double-exponential equation. (E) Inactivation of hERG in the presence and the absence of 10 μM ML-T531 was measured at different voltages using the indicated voltage protocol.

Fig. 4.

ML-T531 potentiates native potassium currents in iPSC-derived cardiomyocytes. (A) A pair of representative traces from a single differentiated cardiomyocyte in the absence and the presence of 10 μM ML-T531 as indicated. The holding potential was −60 mV. Steady-state currents were examined from −10 mV to +50 mV in 20-mV increments, whereas tail currents were elicited at −30 mV. Except for I_Kr and I_Ks, other major ionic components including I_Na, I_K1, I_to, and I_CaL were pharmacologically suppressed under the recording conditions (Experimental Procedures). (B) Summary of the compound effects on steady-state current (Upper) and tail current (Lower) at the indicated voltages (n = 11). (*P < 0.05; **P < 0.001).

Fig. 5.

ML-T531 normalizes the disease phenotype of cardiomycytes derived from a patient with LQT1. (A) A train of action potentials recorded from cardiomycytes derived from a patient with LQT1. The time window of ML-T531 application is indicated. Representative action potentials from indicated areas were expanded to appreciate the difference in APD. (Lower) Parameters for individual action potentials are shown in heatmap format. Data are z-score normalized to the first 20 potentials in the train, with color intensity representing the number of SDs of change from this baseline. The plotted parameters are as indicated on the left. (B) Comparison of APD₉₀ recorded from healthy control cells and cells from a patient with LQT1. (C) Overlay of representative action potential traces of cardiomyocytes from a patient with LQT1 at baseline (black), in the presence of 10 μM ML-T531(red), and after washout (blue). (D) ML-T531 effects on action potential durations (APD₉₀) of cardiomyocytes derived from a patient with LQT1 at the indicated concentrations (n = 3 or more). (E) Dose–response curve of ML-T531 on shortening APD₉₀ in LQT1 cardiomyocytes. The curve was fitted by a Hill equation with a Hill coefficient of 2.0.