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Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease.

ABSTRACT: Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin-D-mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3 ± 2.0 flashes/1000 ?m²·100 s before and after 5 tetani) and markedly decreased (to 7.7 ± 1.6 flashes/1000 ?m²·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature-dependent increase in mSOF frequency (11.9 ± 0.8 and 19.8 ± 2.6 flashes/1000 ?m²·100 s at 23°C and 37°C) was observed in fibers from RYR1(Y522S/WT) mice, a mouse model of malignant hyperthermia and heat-induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress

SUBMITTER: Wei L

PROVIDER: S-EPMC3157685 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease.

Wei Lan L Salahura Gheorghe G Boncompagni Simona S Kasischke Karl A KA Protasi Feliciano F Sheu Shey-Shing SS Dirksen Robert T RT

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20110606 9

Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine ...[more]

PMID: 21646399

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Project description:Mitochondrial flashes (mitoflashes) are stochastic events in the mitochondrial matrix detected by mitochondrial-targeted cpYFP (mt-cpYFP). Mitoflashes are quantal bursts of reactive oxygen species (ROS) production accompanied by modest matrix alkalinization and depolarization of the mitochondrial membrane potential. Mitoflashes are fundamental events present in a wide range of cell types. To date, the precise mechanisms for mitoflash generation and termination remain elusive. Transient opening of the mitochondrial membrane permeability transition pore (mPTP) during a mitoflash is proposed to account for the mitochondrial membrane potential depolarization. Here, we set out to compare the tissue-specific effects of cyclophilin D (CypD)-deficiency and mitochondrial substrates on mitoflash activity in skeletal and cardiac muscle. In contrast to previous reports, we found that CypD knockout did not alter the mitoflash frequency or other mitoflash properties in acutely isolated cardiac myocytes, skeletal muscle fibers, or isolated mitochondria from skeletal muscle and the heart. However, in skeletal muscle fibers, CypD deficiency resulted in a parallel increase in both activity-dependent mitochondrial Ca2+ uptake and activity-dependent mitoflash activity. Increases in both mitochondrial Ca2+ uptake and mitoflash activity following electrical stimulation were abolished by inhibition of mitochondrial Ca2+ uptake. We also found that mitoflash frequency and amplitude differ greatly between intact skeletal muscle fibers and cardiac myocytes, but that this difference is absent in isolated mitochondria. We propose that this difference may be due, in part, to differences in substrate availability in intact skeletal muscle fibers (primarily glycolytic) and cardiac myocytes (largely oxidative). Overall, we find that CypD does not contribute significantly in mitoflash biogenesis under basal conditions in skeletal and cardiac muscle, but does regulate mitoflash events during muscle activity. In addition, tissue-dependent differences in mitoflash frequency are strongly regulated by mitochondrial substrate availability.

Project description:Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.

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Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease.

Publications

Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease.

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