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Free radicals are produced continuously by skeletal muscle fibers. Extracellular release of reactive oxygen species (ROS) and nitric oxide (NO) derivatives has been demonstrated, but little is known about intracellular oxidant regulation. We used a fluorescent oxidant probe, 2′,7′-dichlorofluorescin (DCFH), to assess net oxidant activity in passive muscle fiber bundles isolated from mouse diaphragm and studied in vitro. We tested the following three hypotheses. 1) Net oxidant activity is decreased by muscle cooling. 2) CO2 exposure depresses intracellular oxidant activity. 3) Muscle-derived ROS and NO both contribute to overall oxidant activity. Our results indicate that DCFH oxidation was diminished by cooling muscle fibers from 37°C to 23°C ( P < 0.001). The rate of DCFH oxidation correlated positively with CO2 exposure (0–10%; P < 0.05) and negatively with concurrent changes in pH (7.0–8.5; P < 0.05). Separate exposures to anti-ROS enzymes (superoxide dismutase, 1 kU/ml; catalase, 1 kU/ml), a glutathione peroxidase mimetic (ebselen, 30 μM), NO synthase inhibitors ( Nω-nitro-l-arginine methyl ester, 1 mM; Nω-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 μM) each inhibited DCFH oxidation ( P < 0.05). Oxidation was increased by hydrogen peroxide, 100 μM, an NO donor (NOC-22, 400 μM), or the substrate for NO synthase (l-arginine, 5 mM). We conclude that net oxidant activity in resting muscle fibers is 1) decreased at subphysiological temperatures, 2) increased by CO2 exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.

We developed an in vitro preparation to investigate shape and stress distribution in the intact rat diaphragm. Our hypothesis was that the diaphragm is anisotropic with smaller compliance in transverse fiber direction than along fibers, and therefore shape change may be small. After the animals were killed (8 rats), the entire diaphragm was excised and fixed into a mold at the insertions. Oxygenated Krebs-Ringer solution was circulated under the diaphragm and perfused over its surface. A total of 20–23 small markers were sutured on the diaphragm surface. At transdiaphragmatic pressure (Pdi) of 3–15 cmH2O, curvature was smaller in transverse direction than along fibers. Using finite element analysis we computed membrane tension. At Pdi of 15 cmH2O, tension in central tendon was larger than muscle. In costal region maximum principal tension (ς1) is essentially along the fibers and ranged from 6–10 g/cm. Minimum principal tension (ς2) was 0.3–4 g/cm. In central tendon, ς1 was 10–15 g/cm, compared with 4–10 g/cm for ς2. The diaphragm was considerably stiffer in transverse fiber direction than along the fibers.

Skeletal muscle constitutively expresses both the type I (neuronal) and type III (endothelial) isoforms of nitric oxide synthase (NOS). We tested the functional importance of type III NOS using skeletal muscles with similar levels of type III NOS expression (diaphragm and soleus) from wild-type, heterozygous, and type III NOS-deficient littermate mice. Muscles were incubated at 37°C in Krebs-Ringer solution. NO accumulation in the medium was measured by chemiluminescence; force-frequency and fatigue characteristics were measured using direct electrical stimulation. Diaphragm and soleus released NO at similar rates during passive incubation; these rates increased during active contraction. NO release by type III NOS-deficient muscle was not different from that of wild-type muscle under any condition tested. Force-frequency and fatigue characteristics also were unaffected by genotype. Because type III NOS deficiency did not alter function, we conclude that NO effects previously observed in wild-type muscle are likely to be mediated by type I NOS.