Increased mitochondrial matrix-directed superoxide production by fatty acid hydroperoxides in skeletal muscle mitochondria - PubMed (original) (raw)
Increased mitochondrial matrix-directed superoxide production by fatty acid hydroperoxides in skeletal muscle mitochondria
Arunabh Bhattacharya et al. Free Radic Biol Med. 2011.
Abstract
Previous studies have shown that muscle atrophy is associated with mitochondrial dysfunction and an increased rate of mitochondrial reactive oxygen species production. We recently demonstrated that fatty acid hydroperoxides (FA-OOHs) are significantly elevated in mitochondria isolated from atrophied muscles. The purpose of this study was to determine whether FA-OOHs can alter skeletal muscle mitochondrial function. We found that FA-OOHs (at low-micromolar concentrations) induce mitochondrial dysfunction assessed by a decrease in the rate of ATP production, oxygen consumption, and activity of respiratory chain complexes I and III. Using methods to distinguish superoxide release toward the matrix and toward the intermembrane space, we demonstrate that FA-OOHs significantly elevate oxidative stress in the mitochondrial matrix (and not the intermembrane space), with complex I as the major site of superoxide production (most probably from a site upstream of the ubiquinone binding site but downstream from the flavin binding site-the iron sulfur clusters). Our results are the first to indicate that FA-OOHs are important modulators of mitochondrial function and oxidative stress in skeletal muscle mitochondria and may play an important role in muscle atrophies that are associated with increased generation of FA-OOHs, e.g., denervation-induced muscle atrophy.
2010. Published by Elsevier Inc.
Figures
Figure 1. FA-OOH decreases skeletal muscle mitochondrial function
Oxygen consumption as a measure of mitochondrial function was determined in actively phosphorylating (State 3) and resting (State 4) mitochondria respiring on 5 mM glutamate/malate (G/M). The respiratory control ratio (RCR) was determined as the ratio of State 3 to State 4 (A). The rate of mitochondrial ATP production was measured using a luciferase based assay as described under ‘Experimental Procedures’ in mitochondria respiring on (B) G/M (5 mM) and (C) succinate (5 mM) + rotenone (0.5 μM) (S/R). Activities of NADH-ubiquinone oxidoreductase (complex I, D) and Ubiquinol–cytochrome c oxidoreductase (complex III, E) were measured as described under ‘Experimental Procedures’. FA-OH and FA-OOH were added at a final concentration of 0.75 μM. Results shown represent means ± S.E.M. for 5-6 individual mitochondrial preparations. Statistical significance was assessed by one-way ANOVA with Newman Keul’s multiple comparison test (#p < 0.01, *p < 0.05 vs untreated mitochondria).
Figure 2. FA-OOH increases the rate of mitochondrial ROS production during forward electron transfer
Amplex red was used to measure H2O2 production in the presence of respiratory substrates specific for complex I and complex II and complex I inhibitor, rotenone - (A) glutamate/malate (G/M), 5 mM; (B) succinate (5 mM) + rotenone (0.5 μM) (S/R) and (C) succinate, 5 mM. FA-OH or FA-OOH was added at the concentrations indicated. The amplex red assay was performed in the absence/presence of 2 mM ADP to determine the effect of FA-OH or FA-OOH (0.75 μM) on ROS production in resting vs phosphorylating mitochondria in the presence of (D) G/M (5 mM) and (E) succinate (5 mM) + rotenone (0.5 μM). The results represent means ± S.E.M. for 6-7 individual mitochondrial preparations. Statistical significance was assessed by one-way ANOVA with Newman Keul’s multiple comparison test (*p < 0.0001 vs. untreated mitochondria (State 4), #p < 0.001 vs. untreated mitochondria (State 3).
Figure 3. FA-OOH does not affect extra-mitochondrial superoxide release during forward electron transfer
Amplex red was used to measure H2O2 production in the presence of substrates specific for complex I or II and complex I inhibitor, rotenone - (A) glutamate/malate, 5 mM and (B) succinate, 5 mM + rotenone, 0.5 μM. The amplex red buffer was prepared with ± 30 Units/mL of CuZnSOD. FA-OH or FA-OOH was added at a final concentration of 0.75 μM. Statistical significance was assessed by one-way ANOVA with Newman Keul’s multiple comparison test (* p < 0.0001 vs. untreated mitochondria). The results shown represent means ± S.E.M. for 5-6 individual mitochondrial preparations.
Figure 4. FA-OOH inhibits aconitase activity during forward electron transfer
Muscle mitochondria (0.5 mg of protein/ml; final concentration 0.1 mg/ml) were incubated with EGTA-free incubation buffer (with or without isocitrate dehydrogenase), and aconitase activity was measured via fluorescence at 355 nm (excitation) and 460 nm (emission). The difference in the fluorescence reading in the presence/absence of isocitrate dehydrogenase was taken as a measure of aconitase activity. FA-OH or FA-OOH was added at a final concentration of 0.75 μM per assay. The assays were performed with respiratory substrates specific for complex I or complex II and complex I inhibitor, rotenone - (A) glutamate/malate (G/M, 5 mM); (B) succinate (S, 5 mM) + rotenone (R, 0.5 uM) and (C) succinate (Succ, 5 mM). Statistical significance was assessed by one-way ANOVA with Newman Keul’s multiple comparison test (* p < 0.05; #p < 0.001; $ p < 0.0001 vs. untreated mitochondria). The results shown represent means ± S.E.M. for 8-10 individual mitochondrial preparations.
Figure 5. FA-OOH increases the rate of mitochondrial ROS production and inhibits aconitase activity in rotenone inhibited mitochondria
(A) Amplex red assay to measure H2O2 production and (B) aconitase assay were performed in the presence of 5 mM glutamate/malate (G/M) + 0.5 μM rotenone (R). FA-OH or FA-OOH was added at a final concentration of 0.75 μM. The effect of DPI (final concentration 25 μM) on the rate of mitochondrial ROS production was measured using amplex red in the presence of 5 mM G/M + 0.5 μM R. Statistical significance was assessed by one-way ANOVA with Newman Keul’s multiple comparison test (* p < 0.0001; $p < 0.001 vs. untreated mitochondria The results shown represent means ± S.E.M. for 7-8 (amplex red assay) and 8-10 (aconitase activity) individual mitochondrial preparations.
Figure 6. The addition of vitamin E does not inhibit FA-OOH induced increase in the rate of mitochondrial ROS production
Skeletal muscle mitochondria were incubated with FA-OH or FA-OOH (final concentration 0.75 μM) for 10 mins followed by the addition of 25 μM vitamin E (vit E). The rate of mitochondria H2O2 production was measured using amplex red in the presence of substrates specific for complex I or II and complex I inhibitor, rotenone - (A) glutamate/malate, 5 mM and (B) succinate, 5 mM + rotenone, 0.5 μM. Statistical significance was assessed by one-way ANOVA with Newman Keul’s multiple comparison test (* p < 0.0001 vs. untreated mitochondria). The results shown represent means ± S.E.M. for 3-4 individual mitochondrial preparations.
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