Amiodarone irrecoverably impairs the function of human ether-a-go-go-related gene potassium channels

Figures

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  Fig. 1. Culture of hERG-HEK cells with AMIO overnight decreases IhERG recorded in the drug-free conditions. (A) Chemical structures of AMIO and DEA. (B) Upper panel: families of IhERG recorded using the voltage protocol shown above the currents from cells after overnight culture without (CTL) or with 0.2 μM and 5 μM AMIO. Lower panel: concentration-response relationships for overnight culture with AMIO-mediated IhERG inhibition. IhERG is plotted against AMIO concentrations, and data are fitted to the Hill equation. Numbers in brackets indicate the number of cells recorded from 3 to 6 independent experiments. Data are presented as mean ± SD.

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  Fig. 2. Culture of hERG-HEK cells with AMIO overnight did not affect hERG expression levels. (A) Representative Western blot images showing the effects of overnight culture of cells with various concentrations of AMIO on hERG channel expression (n = 5). (B) Box plots overlapping individual data showing expression levels of mature hERG channel protein in cells cultured with 5 μM AMIO overnight relative to those in the CTL condition. The shaded square inside the box represents the mean, and the whiskers represent the SD. (C) Mature hERG channel proteins (the upper, 155-kDa band) from both untreated cells and AMIO-treated cells are susceptible to extracellularly applied PK treatment. hERG-HEK cells were cultured without or with 5 μM AMIO for 12 hours. For each group, cells were untreated (CTL) or treated with PK (200 μg/mL, 20 minutes), and whole-cell lysates were then extracted for Western blot analysis.

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  Fig. 3. AMIO irrecoverably inhibits IhERG. (A) Acute application of AMIO to the bath solution inhibits IhERG in a concentration-dependent manner. Left panel: representative current traces under CTL, and with 0.2 μM or 5 μM AMIO. Right panel: concentration dependence of AMIO-induced IhERG inhibition, in which summarized relative IhERG (n = 3–7 for each data point from 3 independent experiments) is plotted against AMIO concentrations, and data are fitted to the Hill equation. (B) IhERG relative to the CTL (IhERG-Rel) during drug application and washout, plotted against time. IhERG did not recover from 5 μM AMIO-mediated inhibition, whereas it recovered partially or nearly completely from 1 μM E-4031–mediated or 5 μM verapamil-mediated inhibition. (C) IhERG recorded in drug-free condition from cells that underwent overnight culture in CTL or with 5 μM AMIO, 1 μM E-4031, or 5 μM verapamil. Box plots overlapping with individual recordings from 3 to 5 experiments are shown. The shaded square inside the box represents the mean, and the whisker represents the SD. ∗∗P < .01. One-way ANOVA with Tukey’s post hoc test comparing AMIO treatment to CTL, E-4031, or verapamil treatment.

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  Fig. 4. Effects of stimulating frequency and mutations on AMIO-mediated IhERG inhibition. (A) Development of IhERG inhibition during repetitive pulsing with the voltage protocol shown above, with a pulse interval of 2 seconds in the presence of 3 μM AMIO in the bath solution. Current traces at 0-, 20-, 40-, and 60-second time points are shown. (B) Frequency-dependent inhibition of IhERG by 3 μM AMIO. Development of IhERG inhibition by 3 μM AMIO during repetitive pulsing at intervals of 0.6 seconds, 2 seconds, or 20 seconds is shown. IhERG relative to the control (IhERG-Rel) is plotted against time. (C) Concentration-response relationships of acute bath-applied AMIO for WT and mutant hERG channels. IhERG at each AMIO concentration relative to the control (IhERG-Rel) is plotted against AMIO concentrations. Each data point was summarized from 3 to 7 independent recordings. Data are presented as mean ± SD. (D) IC50 values of AMIO-induced IhERG inhibition of WT and mutant hERG channels. (E) IhERG during drug application and washout relative to the control (IhERG-Rel) for various mutant hERG channels is plotted against time. Washout of AMIO did not lead to IhERG recovery from the block in any…

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  Fig. 5. Recovery of IhERG from AMIO-mediated inhibition relies on newly trafficked mature channels. (A) Time-dependent recovery of IhERG in hERG-HEK cells after various treatments. IhERG from AMIO-pretreated cells, PK-treated CTL cells, AMIO-pretreated cells further treated with PK, and DGX-treated cells displayed similar recovery time courses. Presence of BFA (10 μM) during cell cultures abolished IhERG recovery in AMIO-pretreated cells, PK-treated CTL cells, and DGX-treated cells. Data were summarized from 5 to 16 cells in 3 independent experiments for each treatment. (B) Western blots of hERG protein expressions in various treatment conditions described in (A). (C) Quantified relative densities of the hERG upper bands (155-kDa) in groups presented in (B). Values at each time point were normalized to each group’s respective CTL and plotted against time (n = 3–6 experiments). (D) The AMIO-pretreated upper band of hERG was not associated, but the newly trafficked upper band was associated with the hERG current. Upper panel: Western blot images of hERG expression in CTL cells, cells after overnight culture with 5 μM AMIO, AMIO-pretreated cells further cultured with BFA and bafilomycin for…

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  Fig. 6. Effects of AMIO on the degradation of mature hERG proteins. (A) Western blot images showing hERG expression from CTL or AMIO-pretreated (AMIO-Pret) conditions cultured in medium with BFA (10 μM) without (middle panel) or with 5 μM AMIO (lower panel) for various periods. (B) Relative densities of the hERG upper bands of cells under various conditions collected at various periods of culture. The densities of the hERG upper bands at various time points were normalized to their respective initial (0-hour) value and plotted against time (n = 4). Data are presented as mean ± SD. ∗∗P < .01. Two-way ANOVA with Tukey’s post hoc test comparing AMIO-Pret to CTL and AMIO-Pret + AMIO groups at each concentration.

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  Fig. 7. DEA, the major metabolite of AMIO, also irrecoverably inhibits IhERG. (A) Effects of overnight culture with DEA on IhERG recorded in the drug-free condition. Upper panel: families of IhERG recorded in drug-free conditions from CTL cells as well as cells that underwent overnight culture with 5 μM DEA. Lower panel: concentration-response relationships of overnight DEA-treatment mediated IhERG inhibition. IhERG is plotted against DEA concentrations, and the data are fitted to the Hill equation. Summarized IhERG was from 4 to 17 cells for each concentration in 6 independent experiments. (B) Effects of acute application of DEA to the bath solution on IhERG recorded in the incremented concentration in the same cells. Upper panel: superimposed IhERG traces from the same cell in the CTL condition and after adding 1 μM and 5 μM DEA to the bath solution. Lower panel: concentration dependence of acute DEA-induced IhERG inhibition. IhERG at each concentration relative to the CTL (IhERG-Rel) is plotted against DEA concentrations, and data are fitted to the Hill equation. Summarized IhERG-Rel was from 15 to 22 cells for each concentration in 5 independent experiments. (C) DEA-mediated IhERG…

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  Fig. 8. Acute effects of AMIO and DEA on the currents of hERG channels heterologously expressed in H9c2 cells. (A) IhERG traces recorded from the same cell in the CTL condition and after acute bath application of 0.3 μM or 10 μM AMIO. (B) Concentration-response relationships of acute AMIO-induced IhERG inhibition. IhERG-Rel is plotted against AMIO concentrations, and data are fitted to the Hill equation. Summarized IhERG-Rel was from 4 to 6 cells for each concentration in 3 independent experiments. (C) IhERG during AMIO application and washout relative to the CTL current (IhERG-Rel) plotted against time (n = 4). (D) IhERG traces recorded from the same cell in the CTL condition and after acute bath application of 1 μM and 10 μM DEA. (E) Concentration-response relationships of acute DEA-induced IhERG inhibition. IhERG-Rel is plotted against DEA concentrations, and data are fitted to the Hill equation. Summarized IhERG-Rel was from 3 to 4 cells for each concentration. (F) IhERG during DEA application and washout relative to the CTL current (IhERG-Rel) plotted against time (n = 3). Data are presented as mean ± SD.