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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ageing is associated with several physiological declines to both the cardiovascular (e.g. reduced aerobic capacity) and musculoskeletal system (muscle function and mass). Ageing may also impair the adaptive response of skeletal muscle mitochondria and redox-regulated stress responses to an acute exercise bout, at least in mice and rodents. This is a functionally important phenomenon, since (1) aberrant mitochondrial and redox homeostasis are implicated in the pathophysiology of musculoskeletal ageing and (2) the response to repeated exercise bouts promotes exercise adaptations and some of these adaptations (e.g. improved aerobic capacity and exercise-induced mitochondrial remodelling) offset age-related physiological decline. Exercise-induced mitochondrial remodelling is mediated by upstream signalling events that converge on downstream transcriptional co-factors and factors that orchestrate a co-ordinated nuclear and mitochondrial transcriptional response associated with mitochondrial remodelling. Recent translational human investigations have demonstrated similar exercise-induced mitochondrial signalling responses in older compared with younger skeletal muscle, regardless of training status. This is consistent with data indicating normative mitochondrial remodelling responses to long-term exercise training in the elderly. Thus, human ageing is not accompanied by diminished mitochondrial plasticity to acute and chronic exercise stimuli, at least for the signalling pathways measured to date. Exercise-induced increases in reactive oxygen and nitrogen species promote an acute redox-regulated stress response that manifests as increased heat shock protein and antioxidant enzyme content. In accordance with previous reports in rodents and mice, it appears that sedentary ageing is associated with a severely attenuated exercise-induced redox stress response that might be related to an absent redox signal. In this regard, regular exercise training affords some protection but does not completely override age-related defects. Despite some failed redox-regulated stress responses, it seems mitochondrial responses to exercise training are intact in skeletal muscle with age and this might underpin the protective effect of exercise training on age-related musculoskeletal decline. Whilst further investigation is required, recent data suggest that it is never too late to begin exercise training and that lifelong training provides protection against several age-related declines at both the molecular (e.g. reduced mitochondrial function) and whole-body level (e.g. aerobic capacity).
    Biogerontology 12/2014; DOI:10.1007/s10522-014-9546-8 · 3.01 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Oxidative stress is recognized as one of the primary processes underlying the initiation and progression of atherosclerotic vascular disease. Under physiological conditions, the balance between reactive oxygen species (ROS) generation and ROS scavenging is tightly controlled. As part of normal cellular metabolism, regulated oxidative stress is responsible for a variety of cellular responses. Excess generation of ROS that could not be compensated by antioxidant system has been suggested to be responsible for a number of pathological conditions. Due to their short biological half-lives, direct measurement of ROS is not available and surrogate measures are commonly used. Plasma lipoproteins, by virtue of their close interactions with endothelial cells in the vasculature and the susceptibility of their surface lipids to oxidative modification, are perfect biological sensors of oxidative stress in the arterial wall. In particular, with each consumed meal, triglyceride-rich lipoproteins, secreted by the intestine into the circulation, are responsible for the delivery of 20-40 grams of fat to the peripheral tissues. This flux of dietary lipids is accompanied by concomitant increases in glucose, insulin and other meal-associated metabolites. The contribution of postprandial lipemia to the pathogenesis of atherosclerosis has been previously suggested by several lines of investigation. We have extended this hypothesis by demonstrating the acute generation of oxidative epitopes on plasma lipoproteins as well as transient changes in the oxidative susceptibility of plasma lipoproteins.
    International Journal of Molecular Sciences 01/2014; 16(1):401-419. DOI:10.3390/ijms16010401 · 2.46 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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. DOI:10.1371/journal.pone.0096378 · 3.53 Impact Factor

Full-text (2 Sources)

Available from
May 30, 2014