Effect of oxidative stress and acidosis on diaphragm contractile function

Acidosis during exercise has long been associated with skeletal muscle fatigue. Recent evidence also has linked reactive oxygen species (ROS) with fatigue in skeletal muscle, including the diaphragm. We hypothesized that acidosis (designed to mimic blood pH during maximal exercise) would worsen ROS-induced depression of diaphragm contractility. The xanthine oxidase (XO) reaction in solution (0.01 U/ml) allows direct assessment of the effects of oxidant stress by ROS. Costal diaphragm fiber bundles from 24 Sprague-Dawley rats (200-250 g) were divided into four treatment groups: 1) pH 7.4, no XO (H); 2) pH 7.4 + XO (HXO); 3) pH 7.0, no XO (L); and 4) pH 7.0 + XO (LXO). Baseline twitch mechanics and force-frequency relationships (Pre) were determined in control Krebs solution (pH 7.4, no XO) before treatment. Treatment solutions were introduced, and the diaphragm underwent 2 min of contractions at 25 Hz (250 ms) at a rate of 1/s. After 10 min of recovery, the control solution was reintroduced into the bath and postcontractile function (Post) was measured. Significant reductions in twitch tension and low-frequency tetanic tension were greater in HXO and LXO compared with H, without an effect on maximal tetanic tension. One-half relaxation time was prolonged only by the combination of acidosis and oxidative stress. Addition of superoxide dismutase (50 U/ml) worsened and catalase (1,800 U/ml) attenuated XO-induced depression of diaphragm contractility. We concluded that XO induced a reduction of low-frequency tension in the fatigued diaphragm, which was mediated directly or indirectly through hydrogen peroxide and was exacerbated to a modest extent with acidosis.