Tabitha Kabayo

Real-time measurement of nitric oxide in single mature mouse skeletal muscle fibres during contractions

Nitric oxide (NO) is thought to play multiple roles in skeletal muscle including regulation of some adaptations to contractile activity, but appropriate methods for the analysis of intracellular NO activity are lacking. In this study we have examined the intracellular generation of NO in isolated single mature mouse skeletal muscle fibres at rest and following a period of contractile activity. Muscle fibres were isolated from the flexor digitorum brevis muscle of mice and intracellular NO production was visualized in real‐time using the fluorescent NO probe 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF‐FM DA). Some leakage of DAF‐FM was apparent from fibres loaded with the probe, but they retained sufficient probe to respond to changes in intracellular NO following addition of the NO donor 3‐(2‐hydroxy‐1‐methyl‐2‐nitrosohydrazino)‐N‐methyl‐1‐propanamine (NOC‐7) up to 30 min after loading. Electrically stimulated contractions in isolated fibres increased the rate of change in DAF‐FM fluorescence by ∼48% compared to non‐stimulated fibres (P < 0.05) and the rate of change in DAF‐FM fluorescence in the stimulated fibres returned to control values by 5 min after contractions. Treatment of isolated fibres with the NO synthase inhibitors NG‐nitro‐l‐arginine methyl ester hydrochloride (l‐NAME) or NG‐monomethyl‐l‐arginine (l‐NMMA) reduced the increase in DAF‐FM fluorescence observed in response to contractions of untreated fibres. Treatment of fibres with the cell‐permeable superoxide scavenger 4,5‐dihydroxy‐1,3‐benzenedisulphonic acid (Tiron) also reduced the increase in fluorescence observed during contractions suggesting that superoxide, or more probably peroxynitrite, contributes to the fluorescence observed. Thus this technique can be used to examine NO generation in quiescent and contracting skeletal muscle fibres in real time, although peroxynitrite and other reactive nitrogen species may potentially contribute to the fluorescence values observed.

Role of superoxide-nitric oxide interactions in the accelerated age-related loss of muscle mass in mice lacking Cu, Zn superoxide dismutase

Mice lacking Cu,Zn superoxide dismutase (SOD1) show accelerated, age‐related loss of muscle mass. Lack of SOD1 may lead to increased superoxide, reduced nitric oxide (NO), and increased peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild‐type (WT) and Sod1−/− mice were loaded with NO‐sensitive (4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate, DAF‐FM) and superoxide‐sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for SOD enzymes, nitric oxide synthases (NOS), and 3‐nitrotyrosine (3‐NT) content. A lack of SOD1 did not increase superoxide availability at rest because no increase in ethidium or 2‐hydroxyethidium (2‐HE) formation from DHE was seen in fibers from Sod1−/− mice compared with those from WT mice. Fibers from Sod1−/− mice had decreased NO availability (decreased DAF‐FM fluorescence), increased 3‐NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1−/− mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1−/− mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF‐FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of SOD1 in Sod1−/− mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.

Skeletal muscle contractions induce acute changes in cytosolic superoxide, but slower responses in mitochondrial superoxide and cellular hydrogen peroxide.

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.

Effect of passive stretch on intracellular nitric oxide and superoxide activities in single skeletal muscle fibres: Influence of ageing

Skeletal muscle is repeatedly exposed to passive stretches due to the activation of antagonist muscles and to external forces. Stretch has multiple effects on muscle mass and function, but the initiating mechanisms and intracellular signals that modulate those processes are not well understood. Mechanical stretch applied to some cell types induces production of reactive oxygen species (ROS) and nitric oxide that modulate various cellular signalling pathways. The aim of this study was to assess whether intracellular activities of ROS and nitric oxide were modulated by passive stretches applied to single mature muscle fibres isolated from young and old mice. We developed a novel approach to apply passive stretch to single mature fibres from the flexor digitorum brevis muscle in culture and to monitor the activities of ROS and nitric oxide in situ by fluorescence microscopy. Passive stretch applied to single skeletal muscle fibres from young mice induced an increase in dihydroethidium oxidation (reflecting intracellular superoxide) with no increase in intracellular DAF-FM oxidation (reflecting nitric oxide activity) or CM-DCFH oxidation. In contrast, in fibres isolated from muscles of old mice passive stretch was found to induce an increase in intracellular nitric oxide activities with no change in DHE oxidation.