Cell-Permeable Succinate Rescues Mitochondrial Respiration in Cellular Models of Amiodarone Toxicity

Figures

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  Figure 1. Amiodarone, but not sotalol, induced concentration-dependent impairment of mitochondrial respiration in intact platelets. (A) Representative trace of amiodarone-induced mitochondrial dysfunction (amiodarone-red; DMSO-grey). (B) Respiration of human platelets was measured after submaximal uncoupling with FCCP (2 µM). The concentration-dependent effect of amiodarone (red rhombus) and sotalol (green square) was measured by titrating increasing concentrations of each drug (15–240 µM) vs. the equivalent volume of vehicle (DMSO or H2O). Non-mitochondrial respiration was evaluated by the inhibition of complex I with rotenone (2 µM) and complex III with antimycin A (1 µg/mL). (C) Representative trace of sotalol effect on mitochondrial respiration (sotalol-green; H2O/solvent-grey). Data were expressed as mean ± SEM. Two-way ANOVA with Bonferroni post hoc test was performed on the antimycin-corrected data. ROX: residual oxygen consumption. DMSO: dimethyl sulfoxide. FCCP: carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. ** p < 0.01 vs. DMSO. n = 5.

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  Figure 2. Amiodarone (but not sotalol) induced a concentration-dependent inhibition of mitochondrial respiration in permeabilized platelets. (A) Representative trace of amiodarone-induced mitochondrial dysfunction (amiodarone-red; DMSO/solvent-grey). Concentration-dependent effects were assessed for 3 doses (60 µM, 120 µM and 240 µM) of amiodarone (red rhombus) and sotalol (green square), respectively. P-L control efficiency, a measure of ATP generating respiration (B), LEAK (C), E-L coupling efficiency, a measure of ATP generating respiration (D), ET capacity (E), maximum OXPHOS (F), NADH-linked OXPHOS (G) were evaluated. (H) Representative trace of sotalol effects (sotalol-green; H2O/solvent-grey). Data were expressed as mean ± SEM of the percent of control (platelets exposed to the corresponding volume of DMSO and H2O for highest concentration of drug). Two-way ANOVA with Bonferroni post hoc test was performed on the antimycin-corrected data. DMSO: dimethyl sulfoxide. ADP: adenosine diphosphate. FCCP: carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. OXPHOS: oxidative phosphorylation. LEAK: non-phosphorylating respiration. ET: electron transfer. ROX: residual oxygen consumption. *…

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  Figure 3. The amiodarone metabolite (DEA) induced a concentration-dependent inhibition of mitochondrial respiration in permeabilized platelets. (A) Representative trace of DEA exposure (DEA-black; methanol-grey). Concentration-effects for two concentrations of DEA (30 µM-circle and 60 µM-rhombus). P-L control efficiency, a measure of ATP generating respiration (B), LEAK (C), E-L coupling efficiency, a measure of ATP generating respiration (D), ET capacity (E), maximum OXPHOS (F), NADH-linked OXPHOS (G) were evaluated. Data were expressed as individual values of the percent of control (platelets exposed to the corresponding volume of methanol). Two-way ANOVA with Bonferroni post hoc test was performed on the antimycin-corrected data. DMSO: dimethyl sulfoxide. ADP: adenosine diphosphate. FCCP: carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. OXPHOS: oxidative phosphorylation. LEAK: non-phosphorylating respiration. ET: electron transfer. ROX: residual oxygen consumption. * p < 0.05 vs. control. n = 3.

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  Figure 4. Delayed toxicity and ATP-depletion by amiodarone in mononuclear cells. Temporal decline of respiration (A) and ATP levels (B) was observed in intact PBMCs following single administration of amiodarone (100 µM, red) and rotenone (2 µM, orange), added on routine (basal) respiration. (C) Oxygen consumption (red square) and (D) ATP concentration (black square) followed amiodarone titration. Amiodarone was added at 10, 15, 20, 25 and 30 min at the final concentrations of 15, 30, 60, 120 and 240 μM. Data were expressed as mean ± SEM. Two-way ANOVA with Bonferroni post hoc test was performed to evaluate time-dependent effects of amiodarone and rotenone (A,B) panels, * p < 0.05; ** p < 0.01; **** p < 0.0001 vs. rotenone. One-way ANOVA with Bonferroni post hoc test was performed to evaluate dose-dependent effects between amiodarone and rotenone (C,D) panels, # p < 0.0001 vs. 0 µM. n = 5–6.

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  Figure 5. Effects of the succinate prodrug NV118 on amiodarone-induced mitochondrial dysfunction in human platelets. (A) Representative trace of amiodarone-exposed platelets in the absence (red) or presence (blue) of NV118. (B–D) NV118 effects on amiodarone-exposed platelets (blue) were measured as compared to its vehicle (DMSO, red). As negative control platelets were exposed to only DMSO (grey). Data were expressed as mean ± SEM. One-way ANOVA with Bonferroni post hoc test was performed on the antimycin-corrected data. DMSO: dimethyl sulfoxide. FCCP: carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. LEAK: non-phosphorylating respiration. ET: electron transfer. ROX: residual oxygen consumption. ns = not significant. * p < 0.05; ** p < 0.01 vs. DMSO. n = 5.

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  Figure 6. Effects of NV118 on amiodarone-induced mitochondrial dysfunction in human HepG2 cells. (A) Respiration of HepG2 cells was measured after mild uncoupling with FCCP (4 µM). The dose-dependent effect of amiodarone (red rhombus) was measured by titrating increasing concentrations of amiodarone or vehicle (DMSO, open black circle). Non-mitochondrial respiration was evaluated by the inhibition of complex I with rotenone (2 µM) and that of complex III with antimycin A (1 µg/mL). (B–D) NV118 effects (blue) on amiodarone-treated HepG2 cells were assessed as compared to its vehicle (DMSO, red). As negative control of the experiment HepG2 were exposed only to DMSO (grey). Data were expressed as mean ± SEM. Two-way ANOVA and one-way ANOVA with Bonferroni post hoc test were performed on the antimycin-corrected data. DMSO: dimethyl sulfoxide. ET: electron transfer. ns= not significant. * p < 0.05 vs. DMSO. n = 3–4.