Polyunsaturated fatty acid analogs act antiarrhythmically on the cardiac IKs channel

Abstract

Polyunsaturated fatty acids (PUFAs) affect cardiac excitability. Kv7.1 and the β-subunit KCNE1 form the cardiac IKs channel that is central for cardiac repolarization. In this study, we explore the prospects of PUFAs as IKs channel modulators. We report that PUFAs open Kv7.1 via an electrostatic mechanism. Both the polyunsaturated acyl tail and the negatively charged carboxyl head group are required for PUFAs to open Kv7.1. We further show that KCNE1 coexpression abolishes the PUFA effect on Kv7.1 by promoting PUFA protonation. PUFA analogs with a decreased pKa value, to preserve their negative charge at neutral pH, restore the sensitivity to open IKs channels. PUFA analogs with a positively charged head group inhibit IKs channels. These different PUFA analogs could be developed into drugs to treat cardiac arrhythmias. In support of this possibility, we show that PUFA analogs act antiarrhythmically in embryonic rat cardiomyocytes and in isolated perfused hearts from guinea pig.

Keywords: IKs; KCNE1; KCNQ1; Kv7.1; antiarrhythmic.

Fig. 1.

DHA shifts the voltage dependence of Kv7.1. (A) DHA (70 μM) increases current amplitude of Kv7.1 in response to a −20-mV voltage step. (B) DHA (70 μM) shifts the G(V) of Kv7.1. DHA (●), control (○). Dashed curve in B is control curve shifted −10 mV. (C) Concentration dependence of DHA effect. Mean ± SEM. ΔV_max = −15.8 mV, c_0.5 = 50 μM. n = 3–5.

Fig. 2.

A charged head group and a polyunsaturated acyl tail are necessary. (A) Induced G(V) shifts for 70 μM of indicated substance. Means ± SEM n = 3–8. (B) pH dependence of the G(V) shift caused by the application of 70 µM DHA on Kv7.1. Means ± SEM. ΔV_max = −28.0 mV, c_0.5 = 1.8 × 10⁻⁸ = pH 7.7. n = 3–5. (C) Concentration-response curves for DHA on Kv7.1 at different pHs. pH 9.0: ΔV_max = −27.7 mV, c_0.5 = 10 μM; pH 7.4, see Fig. 1C. n = 3–8. (D) Schematic illustration of PUFA modulation. PUFAs incorporate in the lipid cleft close to the channel’s VSD and electrostatically affect the S4 movement. +++, S4 gating charges.

Fig. 3.

KCNE1 abolishes PUFA effect on Kv7.1/KCNE1 at physiological pH. (A–D) DHA (70 μM) applied on Kv7.1/KCNE1 at pH 7.4 (A and B) and pH 9 (C and D). DHA (●), control (○). Bold traces = 0 mV (A) or +10 mV (C). Dashed curve in D is control curve shifted −35 mV. (E) pH dependence for 70 µM DHA. Kv7.1/KCNE1: ΔV_max = −29.0 mV, c_0.5 = 2.5 × 10⁻⁹ = pH 8.6. n = 3–6. For Kv7.1, see Fig. 2B. (F) Model: KCNE1 changes probability of protonation of negatively charged acidic (Upper) and positively charged amine (Lower) PUFA analogs, thereby changing PUFA-Kv7.1 voltage sensor (+++) interactions. (G) G(V) shift induced by 70 µM DHA or AA+. n = 3–8.

Fig. 4.

PUFA analogs are effective on Kv7.1/KCNE1. (A) DHA-Gly (70 μM) induces a negative G(V) shift of Kv7.1/KCNE1. DHA-Gly (●), control (○). Dashed line is control curve shifted −20 mV. (B) pH dependence for 70 µM DHA-Gly or 70 µM DHA applied on Kv7.1/KCNE1. DHA-Gly: ΔV_max = −38.8 mV, c_0.5 = 7.6 × 10⁻⁸ = pH 7.1. n = 3–6. For DHA, see Fig. 3E. (C) N-arachidonoyl taurine (N-AT; 70 µM) induces a negative G(V) shift of Kv7.1/KCNE1. N-AT (●), control (○). Dashed line is control curve shifted −24 mV. (D) DHA-Gly (70 µM) does not shift Kv7.1/R228Q and has a smaller effect on Kv7.1/K218C and Kv7.1/G219C. Means ± SEM; n = 4–14. (E) Normalized fluorescence during an activating test pulse to −60 mV from a holding voltage of −120 mV. (F) N-AT (70 μM) shifts the F(V) of Kv7.1/G219C*. N-AT (●), control (○).The continuous lines are Boltzmann curves (Eq. 1) fitted to experimental data. For control: V₅₀ = −41.1 mV; s = 11.7. For N-AT: V₅₀ = −61.1 mV; s = 16.3. Means ± SEM; n = 9.

Fig. 5.

Antiarrhythmic effect of PUFA analogs. (A and B) Effect of 30 µM N-AT on action potential duration and frequency in isolated embryonic rat cardiomyocytes. (C) Example of the effect of 30 µM N-AT on current amplitude in isolated embryonic rat cardiomyocytes. Gray trace is current in control solution from a voltage 20 mV more positive than the N-AT trace. (D–F) Representative examples of arrhythmia induced by application of 5 µM Chromanol 293B or 5 µM Chromanol 293B + 30 µM N-arachidonoyl taurine (N-AT) in isolated embryonic rat cardiomyocytes. (G and H) Representative example of effect of 0.03 µM E4031 and 10 µM DHA-Gly on QT interval (G) and action potential duration (H) in isolated perfused guinea pig heart. Hearts are paced at 250 beats/min. (I) Summary of effect of 0.03 µM E4031 and 10 µM DHA-Gly on QT interval and action potential duration (APD₉₀) in isolated perfused guinea pig heart. Means ± SEM; n =3. Statistical significance compared with control.