In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy.

Division of Metabolic and Cellular Medicine, School of Clinical Sciences, University of Liverpool, Liverpool, United Kingdom.
Antioxidants & Redox Signaling (Impact Factor: 8.2). 09/2008; 10(8):1463-74. DOI: 10.1089/ars.2007.2009
Source: PubMed

ABSTRACT Reactive oxygen species (ROS) produced by skeletal muscle stimulate adaptive responses to activity and mediate some degenerative processes. ROS activity is usually studied by measuring indirect end-points of their reactions with various biomolecules. In order to develop a method to measure the intracellular ROS generation in real-time in mature skeletal muscle fibers, these were isolated from the flexor digitorum brevis (FDB) muscle of mice and cultured on collagen-coated plates. Fibers were loaded with 5- (and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-DCFH DA) and measurements of 5- (and 6-) chloromethyl-2',7'-dichlorofluorescin (CM-DCF) fluorescence from individual fibers obtained by microscopy over 45 min. The sensitivity of this approach was demonstrated by addition of 1 microM H(2)O(2) to the extracellular medium. Contractions of isolated fibers induced by field electrical stimulation caused a significant increase in CM-DCF fluorescence that was abolished by pre-treatment of fibers with glutathione ethyl ester. Thus, CM-DCF fluorescence microscopy can detect physiologically relevant changes in intracellular ROS activity in single isolated mature skeletal muscle fibers in real-time, and contractions generated a net increase that was abolished when the intracellular glutathione content was enhanced. This technique has advantages over previous approaches because of the maturity of the fibers and the analysis of single cells, which prevent contributions from nonmuscle cells.

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    ABSTRACT: Reactive oxygen species (ROS) are important signaling molecules with regulatory functions, and in young and adult organisms, the formation of ROS is increased during skeletal muscle contractions. However, ROS can be deleterious to cells when not sufficiently counterbalanced by the antioxidant system. Aging is associated with accumulation of oxidative damage to lipids, DNA and proteins. Given the pro-oxidant effect of skeletal muscle contractions, this effect of age could be a result of excessive ROS formation. We evaluated the effect of acute exercise on changes in blood redox state across the leg of young (23±1 years) and older (66±2 years) sedentary humans by measuring the whole blood concentration of the reduced (GSH) and oxidized (GSSG) form of the antioxidant glutathione. To assess the role of physical activity, lifelong physically active older subjects (62±2 years) were included. Exercise increased the venous concentration of GSSG in an intensity-dependent manner in young sedentary subjects, suggesting an exercise-induced increase in ROS formation. In contrast, venous GSSG levels remained unaltered during exercise in the older sedentary and active groups despite a higher skeletal muscle expression of the superoxide generating enzyme NADPH oxidase. Arterial concentration of GSH and expression of antioxidant enzymes in skeletal muscle of older active subjects was found to be increased. The potential impairment in exercise-induced ROS formation may be an important mechanism underlying skeletal muscle and vascular dysfunction with sedentary aging. Lifelong physical activity up-regulates antioxidant systems which may be one of the mechanisms underlying the lack of exercise-induced increase in GSSG.
    Free Radical Biology and Medicine 05/2014; · 5.71 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 05/2014; 9(5):e96378. · 3.53 Impact Factor
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    ABSTRACT: Several redox-regulated responses to an acute exercise bout fail in aged animal skeletal muscle including the ability to up regulate the expression of antioxidant defence enzymes and Heat Shock Proteins (HSPs). These findings are generally derived from studies on sedentary rodent models and thus may be related to reduced physical activity and/or intra species differences as opposed to aging per se. The present study, therefore, aimed to determine the influence of age and training status on the expression of HSPs, antioxidant enzymes and NOS isoenzymes in quiescent and exercised human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis before and 3 days after an acute high-intensity-interval exercise bout in young trained, young untrained, old trained and old untrained subjects. Levels of HSP72, PRX5 and eNOS were significantly higher in quiescent muscle of older compared with younger subjects, irrespective of training status. 3-NT levels were elevated in muscles of the old untrained but not the old trained state, suggesting that lifelong training may reduce age-related macromolecule damage. SOD1, CAT and HSP27 levels were not significantly different between groups. HSP27 content was up-regulated in all groups studied post-exercise. HSP72 content was up-regulated to a greater extent in muscle of trained compared with untrained subjects post-exercise, irrespective of age. In contrast to every other group, old untrained subjects failed to up-regulate CAT post-exercise. Aging was associated with a failure to up-regulate SOD2 and a down-regulation of PRX5 in muscle post-exercise, irrespective of training status. In conclusion, lifelong training is unable to fully prevent the progression towards a more stressed muscular state as evidenced by increased HSP72, PRX5 and eNOS protein levels in quiescent muscle. Moreover, lifelong training preserves some (e.g. CAT) but not all (e.g. SOD2, HSP72, PRX5) of the adaptive redox-regulated responses following an acute exercise bout. Collectively, these data support many but not all of the findings from previous animal studies and suggest parallel aging effects in man and mice at rest and following exercise that are not modulated by training status in human skeletal muscle.
    Free Radical Biology and Medicine 05/2014; · 5.27 Impact Factor

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