The electrophysiologic effects of KCNQ1 extend beyond expression of IKs: evidence from genetic and pharmacologic block

Abstract

Aims: While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes.

Methods and results: We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small IKs was readily recorded but the pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (μM) of moxifloxacin required to prolong APD90 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300 μM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs and markedly prolonged APD90.

Conclusion: Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs.

Keywords: KCNQ1; IKr; IKs; Long QT; Repolarization reserve.

Graphical Abstract

Figure 1

Effect of pharmacological block and genetic loss of KCNQ1 on action potential durations and I_Ks. (A) Acute pharmacological block of I_Ks in control cells. A high concentration of the I_Ks-specific blocker HMR-1556 produced no change in APD₉₀ at 0.5 Hz pacing (paired recordings, P = 0.55 by paired t-test). (B) Baseline APD₉₀ at 0.5 Hz in untreated control cells (open squares, n/N = 56/11), control cells pre-treated with HMR-1556 for 24–48 h (pink squares, n/N = 36/5), and in untreated JLN cells (red circles, n/N = 59/10). (C) Representative traces of action potentials in the same control cell (at 2 Hz) during acute exposure to PKA activators (IBMX and forskolin, red trace) followed by I_Ks block with HMR-1556 (blue trace). (D) Summary APD₉₀ data during acute exposure to PKA activators followed by HMR-1556 (n/N = 14/2). (E–G) Representative traces of I_Ks in a control cell without PKA activation (E), control cell with PKA activation (F), and a JLN cell with PKA activation (G) by IBMX and forskolin. Insets show the stimulus protocol. An inverted triangle in the inset indicates where the step current of I_Ks was measured. (H) The current–voltage relationship for activation of I_Ks measured at the end of the step in JLN cells with PKA activators (red circles, n/N = 8/3), control cells without PKA activators (open squares, n/N = 27/6), and control cells with PKA activators (blue squares, n/N = 10/3). The numbers of data points are expressed as n/N, where n indicates number of recordings and N indicates number of differentiation batches. (−) not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Kruskal–Wallis test unless otherwise specified. APD₉₀, action potential duration at 90% repolarization; JLN, Jervell and Lange-Nielsen; IBMX, 3-isobutyl-1-methylxanthine; PKA, protein kinase A.

Figure 2

Effect of acute exposure to moxifloxacin in control and JLN iPSC-CMs. (A) Representative action potential traces during acute exposure to increasing concentrations of moxifloxacin (2–3 min/concentration) in a control cell (at 0.5 Hz). (B) Log-linear interpolation to determine moxifloxacin concentration that prolonged APD₉₀ by 100 ms [(MOX)_Δ100ms] in the control cell shown in (A). (C) Representative action potential traces during moxifloxacin exposure in a JLN cell. The trace at 1000 μM moxifloxacin (red) was longer than 2000 msec at 0.5 Hz pacing (#) and showed triggered activity/early afterdepolarizations, as shown in an inset in a full trace obtained at 0.1 Hz pacing. (D) Log-linear interpolation of the JLN cell data shown in (C). Note the difference in scale on the Y-axis between panels B (maximum 200 ms) and D (maximum 1000 ms). (E) [MOX]_Δ100ms (note log-scale) in control (open squares, n/N = 7/3), JLN (red circles, n/N = 11/4), and control cells pre-treated with HMR-1556 (pink squares, n/N = 8/3). (F) Absolute change in APD₉₀ (ΔAPD₉₀) with acute exposure to 30 μM moxifloxacin in control (open squares, n/N = 8/4), JLN (red circles, n/N = 13/5), and control cells pre-treated with HMR-1556 (pink squares, n/N = 8/3). (G) Acute effect of high concentration moxifloxacin at 300 μM on APD₉₀ (at 0.5 Hz). n/N = 10–11/4–5. Comparison was made using a paired t-test. (−) not significant, ***P < 0.001 by Kruskal–Wallis test otherwise specified.

Figure 3

The effects of moxifloxacin and dofetilide on action potential duration, I_Ks, and KCNH2 and KCNQ1 transcripts in control and JLN iPSC-CMs. (A) The effect of chronic (24–48 h) exposure to moxifloxacin and dofetilide on APD₉₀ in control cells (at 0.5 Hz). DOF: dofetilide at 5 nM (black squares, n/N = 20/4); MOX: moxifloxacin at 300 μM (blue squares, n/N = 27/4). The dotted line and gray band indicate the mean ± SE for control cells from Figure 1B. (B) I_Ks  I–V relationship showed a dose-dependent effect of moxifloxacin in control cells isolated from identical differentiation batches: Untreated cells (open squares, n = 4); pre-treated with 30 μM moxifloxacin (light blue squares, n = 4); pre-treated with 300 μM moxifloxacin (blue squares, n = 5). (C) I_Ks  I–V relationship in control cells pre-treated with moxifloxacin 300 μM (MOX, blue squares, n/N = 13/5) or dofetilide 5 nM (DOF, black squares, n/N = 16/4). Comparisons were made with control cells without treatment (control, open squares, n/N = 27/6). (D) I_Ks amplitude measured at +40 mV in untreated control cells (open squares, n/N = 27/6), pre-treated with DOF (black squares, n/N = 16/4), and with MOX (blue squares, n/N = 13/5). (E) The effect of chronic exposure to moxifloxacin 300 μM on APD₉₀ in JLN cells (at 0.5 Hz) (red-filled blue circles, n/N = 23/3). The statistical comparison shown used Mann–Whitney U test between JLN cells and those pre-treated with moxifloxacin. (F) KCNH2 and KCNQ1 transcript levels during pre-treatment with I_Kr blockers (n = 5–7). (−) not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by Kruskal–Wallis test otherwise specified.

Figure 4

The effect of KCNQ1 knock-down on I_Ks and action potential duration. (A) The efficiency of gene-specific knock-down of KCNQ1 assessed by qPCR. Raw ΔCT values are plotted as relative expression normalized to TNNT2 expression. Mann–Whitney U test was employed. (B, C) I–V relationship (B) and I_Ks densities at +40 mV (C) in control cells: untreated, pre-treated with moxifloxacin 300 μM, pre-treated with non-targeting siRNA (Negative siRNA), siRNA targeting both exon1 and exon6 of KCNQ1 (KCNQ1-KD), and KCNQ1-KD cells pre-treated with moxifloxacin 300 μM. (D, E) The effect of KCNQ1 knock-down (KCNQ1-KD) and the I_Kr blocker moxifloxacin on APD₉₀ (at 0.5 Hz). KCNQ1-KD cells exhibited significant prolongation of APD at baseline (P < 0.0001 vs. untreated control cells and P = 0.31 vs. untreated JLN cells) (D). Furthermore, pre-treatment with moxifloxacin (300 μM) resulted in further prolongation of APD in KCNQ1-KD cells (P < 0.0001 vs. pre-treated control cells and P = 0.0007 vs. pre-treated JLN cells) (E). (−) not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Kruskal–Wallis test otherwise specified.

Figure 5

Effect of the common KCNQ1 variant G643S on action potential duration and I_Ks with I_Kr blocker challenge. (A) APD₉₀ in control and G643S cells at baseline (open squared and open triangles, respectively) and after chronic exposure to moxifloxacin (300 μM, blue squares and blue triangles, respectively) (at 0.5 Hz). ****P < 0.0001 by Kruskal–Wallis test. (B) The I–V relationship of baseline I_Ks in control (n/N = 27/6, open squares) and G643S cells (n/N = 15/3, open triangles). (C) The I–V relationship of I_Ks in response to acute PKA activation by IBMX and forskolin in control (n/N = 10/3, yellow squares) and G643S cells (n/N = 8/2, yellow triangles). (D) The I–V relationship of I_Ks in response to chronic treatment with moxifloxacin (300 μM) for 24–48 h in control (n/N = 13/5, blue squares) and G643S cells (n/N = 11/3, blue triangles). (−) not significant, *P < 0.05, **P < 0.01 by Mann–Whitney U test otherwise specified.