Molecular Mechanisms of Stress-induced Reactive Oxygen Species Formation in Skeletal Muscle

Reactive oxygen species (ROS) play an important role in many biological systems. Skeletal muscles have been shown to generate considerable ROS in resting and in contracting conditions. In this study, I tested the hypothesis that increased ROS production in skeletal muscles is also associated with exposure to two other conditions of stress which are common in normal skeletal muscle during exercise, namely heat stress and hypoxia.

There is no previous direct evidence that ROS are produced during these stresses, particularly in skeletal muscle, but they may play important roles in normal contractile and cell signaling responses. Two assays for superoxide (O₂^•-) formation were used in rodent diaphragm, the cytochrome c assay for extracellular O₂^•- release and the hydroethidine oxidation for intracellular O₂^•- formation. The results demonstrate the following: 1) Markedly increased intra- and extracellular ROS formation was observed, particularly O₂^•-, at temperatures known to be physiologically relevant to exercise (i.e. 42°C). 2) The process of O₂^•-, release (extracellular formation) is not directly related to mitochondria, NADPH oxidase, or anion channels, though these are normally believed to be involved in either O₂^•- generation or the exit pathway of O₂^•- through membranes. 3) Upstream pathways of arachidonic acid (AA) metabolism, both phospholipase A₂ and nitric oxide synthase are associated with O₂^•- release. 4) Downstream pathways of AA metabolism are also involved. Though blockage of either cyclooxygenase or cytochrome P450-dependent enzymes does not cause any inhibition of O₂^•- release, blockage of lipoxygenase (LOX) results in near elimination of the signal. This suggests that O₂^•- release is dependent on AA metabolism through the LOX pathway. However, confocal measurements of intracellular O₂^•- formation suggest that intracellular ROS are produced by a separate mechanism. 5) Tissue fluorometry techniques, using a fluorescence probe sensitive to hydrogen peroxide, showed that acute hypoxia also induces ROS formation in skeletal muscle.

These findings provide insight into understanding the potential role of ROS in physiologic and pathophysiologic responses to heat exposure and acute hypoxia in skeletal muscle.