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Mary L. Garcia Cazarin, Natalie N. Snider, Lorrie B. Sims, Francisco H. Andrade; Greater Oxidant Production And Less Antioxidant Enzymes In Rat Extraocular Muscles. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2221.
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Mitochondria-derived reactive oxygen species (ROS) cause cellular damage in disease and aging. Antioxidant defenses include manganese superoxide dismutase (Mn-SOD, mitochondrial), copper-zinc SOD (Cu/Zn-SOD, cytoplasmic), glutathione peroxidase (GPx) and thioredoxin (Trx) (both found in cytoplasm and mitochondria). We hypothesize that the extraocular muscles (EOMs) produce more ROS and have a higher content of antioxidant enzymes than other skeletal muscles.
We used the redox sensitive probes 2'7'-dichlorofluorescein (DCFH) and dihydrorhodamine 123 (DHR 123) to measure ROS production in mitochondria isolated from rat EOMs and gastrocnemius (a leg muscle). Mitochondria (100 μg) were placed in 96-well plates and loaded with either 100 μM of DCFH-diacetate or 10 μM DHR 123 for 45 minutes. Fluorescence (480mn excitation, 520 nm emission for both probes) was measured every 20 minutes for 2 hours using a microplate reader. We used western blots to measure the content Mn-SOD in mitochondria, Cu/Zn-SOD in cytoplasm, and GPx1 and Trx in both compartments.
EOM mitochondria produced more ROS; total DCFH fluorescence (arbitrary units, AU) from EOMs after 2 hours was 1319±220 vs. 575±56 AU from gastrocnemius (means ± SEM, p<0.001). DHR 123 gave a similar result: 495±33 from EOMs vs. 355±20 AU from gastrocnemius (p<0.001). Western blot analyses showed that EOM content of Cu/Zn-SOD and GPx1 (cytoplasmic) was 67±9% and 61±12% respectively of the gastrocnemius levels (p<0.001 for both). There was no difference in the content of mitochondrial Mn-SOD or GPx1, or cytoplasmic Trx between gastrocnemius and EOMs. Mitochondrial Trx was undetectable in either muscle.
These results show that EOM mitochondria produce ROS at a faster rate than a limb skeletal muscle. In addition, EOMs have lower content of some antioxidant enzymes needed to quench the excess ROS. We propose that this combination of factors explains the increased susceptibility of EOMs to mitochondrial disorders.
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