Selective targeting of gain-of-function KCNQ1 mutations predisposing to atrial fibrillation

Abstract

Background: Atrial fibrillation is the most common sustained cardiac arrhythmia in adults. We hypothesized that gain-of-function KCNQ1 mutations previously associated with familial atrial fibrillation have distinct pharmacological properties that may enable targeted inhibition.

Methods and results: Wild-type (WT) KCNQ1 or the familial atrial fibrillation mutation KCNQ1-S140G was heterologously coexpressed with KCNE1 to enable electrophysiological recording of the slow delayed rectifier current (IKs) and investigation of pharmacological effects of the IKs selective blocker HMR-1556. Coexpression of KCNQ1-S140G with KCNE1 generated potassium currents (S140G-IKs) that exhibited greater sensitivity to HMR-1556 than WT-IKs. Enhanced HMR-1556 sensitivity was also observed for another gain-of-function atrial fibrillation mutation, KCNQ1-V141M. Heteromeric expression of KCNE1 with both KCNQ1-WT and KCNQ1-S140G generated currents (HET-IKs) with gain-of-function features, including larger amplitude, a constitutively active component, hyperpolarized voltage dependence of activation, and extremely slow deactivation. A low concentration of HMR-1556, which had little effect on WT-IKs but was capable of inhibiting the mutant channel, reduced both instantaneous and steady state HET-IKs to levels that were not significantly different from WT-IKs and attenuated use-dependent accumulation of the current. In cultured adult rabbit left atrial myocytes, expression of S140G-IKs shortened action potential duration compared with WT-IKs. Application of HMR-1556 mitigated S140G-IKs-induced action potential duration shortening and did not alter action potential duration in cells expressing WT-IKs.

Conclusions: The enhanced sensitivity of KCNQ1 gain-of-function mutations for HMR-1556 suggests the possibility of selective therapeutic targeting, and, therefore, our data illustrate a potential proof of principle for genotype-specific treatment of this heritable arrhythmia.

Keywords: antiarrhythmic drugs; arrhythmias, cardiac; atrial fibrillation; genetics; potassium channels.

Figure 1

S140G-I_Ks and HET-I_Ks exhibit enhanced sensitivity to HMR-1556. A, B and C, Representative current recordings from cells expressing WT-I_Ks (A), S140G-I_Ks (B), or HET-I_Ks (C). Recordings illustrated in A, B and C were obtained using the activation protocol described in the Methods. D, E, and F, Average current densities (current normalized to cell capacitance) elicited by a 2 s voltage step to +40 mV followed by a 10 s interpulse during application of vehicle or various concentration of HMR-1556 from cells expressing WT-I_Ks (D), S140G-I_Ks (E), or HET-I_Ks (F). Current density traces in D, E, and F are averages from 9–11 cells.

Figure 2

HMR-1556 concentration-response curves for WT-I_Ks (○), S140G-I_Ks (△), and HET-I_Ks (●). Solid lines represents fits of the averaged data to either monophasic (WT-I_Ks) or biphasic (S140G-I_Ks and HET-I_Ks) Hill function (see Supplemental Material). IC₅₀ values and Hill coefficients are provided in the text.

Figure 3

V141M-I_Ks exhibits enhanced sensitivity to HMR-1556. A, Representative current densities (current normalized to cell capacitance) recorded from cells expressing WT-I_Ks that were elicited by a 2 s voltage step to +40 mV followed by a 10 s interpulse during application of vehicle or various concentration of HMR-1556. B, HMR-1556 concentration-response curves for V141M-I_Ks (▲) and WT-I_Ks (○). Solid lines represents fits of the averaged data (9–11 cells) to a biphasic Hill function (see Supplemental Material). For V141M-I_Ks, the high affinity state had an IC₅₀ of 0.72 nM and Hill coefficient of 0.6; the low affinity state had an IC₅₀ of 204 nM and Hill coefficient of 1.7.

Figure 4

Selective inhibition of HET-I_Ks by HMR-1556. A and B, Effects of vehicle on whole-cell currents during an activation voltage clamp protocol (middle panel) and during repetitive stimulation (right panel) from cells expressing WT-I_Ks (A) or HET-I_Ks (B). C and D, Effect of HMR-1556 (20 nM) on whole-cell currents during activation (middle panel) and repetitive stimulation (right panel) protocols from cells expressing WT-I_Ks (C) or HET-I_Ks (D). In A–D, traces in each row are from the same cell.

Figure 5

Selective inhibition of HET-I_Ks by HMR-1556. A, Voltage dependence of instantaneous current density for vehicle-treated WT-I_Ks (○, n = 10), vehicle-treated HET-I_Ks (●, n = 11), HMR-1556 (20 nM) treated WT-I_Ks (□, n = 10), and HMR-1556 (20 nM) treated HET-I_Ks (■, n = 9). Differences between vehicle-treated HET-I_Ks and other groups were significant at the p<0.001 level for voltages between −20 and +60 mV. B, Voltage dependence of steady-state current density for vehicle or HEM-1556 treated WT-I_Ks or HET-I_Ks (symbols defined in A). Differences between vehicle-treated HET-I_Ks and other groups were significant at the p<0.02 level for voltages between −40 and +60 mV. C, Use dependence of instantaneous current density for vehicle or HEM-1556 treated WT-I_Ks or HET-I_Ks (symbols defined in A). Differences between vehicle-treated HET-I_Ks and other groups were significant at the p<0.001 level at all tested potentials. D, Use dependence of steady-state current density for vehicle or HEM-1556 treated WT-I_Ks or HET-I_Ks (symbols defined in A). Differences between vehicle-treated HET-I_Ks and other groups were significant at the p<0.02 level at all tested potentials. In A–D, there were no significant differences (p=0.09–0.74) among vehicle-treated WT-I_Ks, HMR-1556 treated WT-I_Ks, and HMR-1556 treated HET-I_Ks at any voltage. E, Ratios of instantaneous current density to steady-state current density. Differences between vehicle-treated WT-I_Ks (open black bar) or HET-I_Ks (solid black bar) was significant at p<0.001, whereas there was no significant difference (p=0.06) between HMR-1556 treated WT-I_Ks (open red bar) and HET-I_Ks (solid red bar).

Figure 6

HMR-1556 mitigates atrial action potential shortening by S140G-I_Ks. Representative averages of 10 sequential action potentials from cultured rabbit left atrial myocytes expressing either WT-I_Ks (black line, n=6) or S140G-I_Ks (blue line, n=6). A, Action potentials elicited after application of vehicle. B, Action potentials elicited after application of 1 μM HMR-1556. APD₉₀ values are provided in the text.