Reactive oxygen intermediates modulate skeletal muscle contraction, but little is known about the role of nitric oxide (NO). Here we show that rat skeletal muscle expresses neuronal-type NO synthase and that activity varies among several respiratory and limb muscles. Immunohistochemistry showed prominent staining of type II (fast) fibre cell membranes with antibodies against neuronal-type NO synthase. NO synthase activity in muscles correlated with type II fibre density. Resting diaphragm muscle produced detectable NO chi, but no reactive oxygen intermediates. In contrast, actively contracting muscle generated increased levels of reactive oxygen intermediates. Contractile function was augmented by blockers of NO synthase, extracellular NO chelation, and guanylyl cyclase inhibition; it was depressed by NO donors and by increased levels of cyclic GMP. Force-frequency plots of different muscles showed an inverse correlation between NO synthase activity and force development. Our results support two physiological functions of NO in skeletal muscle. The first is to promote relaxation through the cGMP pathway. The second is to modulate increases in contraction that are dependent on reactive oxygen intermediates and which are thought to occur through reactions with regulatory thiols on the sarcoplasmic reticulum.

Atrogin1/MAFbx is an ubiquitin ligase that mediates muscle atrophy in a variety of catabolic states. We recently found that H2O2 stimulates atrogin1/MAFbx gene expression. Since the cytokine tumor necrosis factor-alpha (TNF-alpha) stimulates both reactive oxygen production and general activity of the ubiquitin conjugating pathway, we hypothesized that TNF-alpha would also increase atrogin1/MAFbx gene expression. As with H2O2, we found that TNF-alpha exposure up-regulates atrogin1/MAFbx mRNA within 2 h in C2C12 myotubes. Intraperitoneal injection of TNF-alpha increased atrogin1/MAFbx mRNA in skeletal muscle of adult mice within 4 h. Exposing myotubes to either TNF-alpha or H2O2 also produced general activation of the mitogen-activated protein kinases (MAPKs): p38, ERK1/2, and JNK. The increase in atrogin1/MAFbx gene expression induced by TNF-alpha was not altered significantly by ERK inhibitor PD98059 or the JNK inhibitor SP600125. In contrast, atrogin1/MAFbx up-regulation and the associated increase in ubiquitin conjugating activity were both blunted by p38 inhibitors, either SB203580 or curcumin. These data suggest that TNF-alpha acts via p38 to increase atrogin1/MAFbx gene expression in skeletal muscle.

We used intact single fibres from a mouse foot muscle to study the role of oxidation‐reduction in the modulation of contractile function. The oxidant hydrogen peroxide (H2O2, 100‐300 μM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. The reductant dithiothreitol (DTT, 0.5‐1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation‐reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor alpha (TNF-alpha) in a variety of chronic diseases. However, little is known about the direct effects of TNF-alpha on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF-alpha on the mouse-derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF-alpha treatment of differentiated myotubes stimulated time- and concentration-dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF-alpha concentrations (1-3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF-alpha activated binding of nuclear factor kappaB (NF-kappaB) to its targeted DNA sequence and stimulated degradation of I-kappaBalpha, an NF-kappaB inhibitory protein. TNF-alpha stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10-40 microM) blocked TNF-alpha activation of NF-kappaB. Catalase 1 kU/ml inhibited NF-kappaB activation by TNF-alpha; exogenous hydrogen peroxide 200 microM activated NF-kappaB and stimulated I-kappaBalpha degradation. These data demonstrate that TNF-alpha directly induces skeletal muscle protein loss, that NF-kappaB is rapidly activated by TNF-alpha in differentiated skeletal muscle cells, and that TNF-alpha/NF-kappaB signaling in skeletal muscle is regulated by endogenous reactive oxygen species.

We hypothesized that muscle fiber bundles produce reactive oxygen intermediates and that reactive oxidant species contribute to muscular fatigue in vitro. Fiber bundles from rat diaphragm were mounted in chambers containing Krebs-Ringer solution. In studies of intracellular oxidant kinetics, bundles were loaded with 2',7'-dichlorofluorescin, a fluorochrome that emits at 520 nm when oxidized; emissions were quantified using a fluorescence microscope. Emissions from unstimulated muscles increased over time (P < 0.001). Accumulation of fluorescence was slowed by addition of catalase (P < 0.001) or superoxide dismutase (P < 0.001) and was accelerated by repetitive muscular contraction (P < 0.05). To determine effects of reactive oxygen intermediates on fatigue, curarized bundles were stimulated to contract isometrically; force was measured. Catalase, superoxide dismutase, and dimethyl sulfoxide were screened for effects on low- and high-frequency fatigue. Antioxidants inhibited low-frequency fatigue [after 5 min of repetitive contractions, force at 30 Hz was 20% greater than control (P < 0.015)] and increased the variability of fatigue at 30 Hz (P < 0.03). Antioxidants did not alter high-frequency (200-Hz) fatigue. We conclude that 1) diaphragm fiber bundles produce reactive oxygen intermediates, including O2-. and H2O2; 2) muscular contraction increases intracellular oxidant levels; and 3) reactive oxygen intermediates promote low-frequency fatigue in this preparation.