Although the production of reactive oxygen spe- cies (ROS) during muscle contractile activity has been linked to both positive and negative adaptive responses, the sites for ROS generation within working muscle are not clearly defined. We assessed cytosolic ROS production and mitochondrial re- dox potential with a targeted redox-sensitive green fluorescent protein during repetitive field stimulation of single mature myo- fibers. Cytosolic ROS production increased by 94%, an effect that was abolished by pretreatment with the reducing agent di- thiothreitol. Mitochondrial redox potential was not altered during muscle contraction. In contrast, activity-dependent ROS produc- tion was ablated by an inhibitor of NADPH oxidase. We provide the first report on dynamic ROS production from mitochondria in single living myofibers and suggest that the mitochondria are not the major source of ROS during skeletal muscle contrac- tion. Alternatively, our data support a role for NADPH oxidase- derived ROS during contractile activity. Muscle Nerve 000: 000-000, 2010 Skeletal muscle produces low levels of reactive ox- ygen species (ROS) that are required for normal contractile function, gene regulation, and regula- tion of cellular signaling. High levels of ROS, how- ever, damage cellular components and result in contractile dysfunction and fatigue (for review, see Powers and Jackson 1 ). It has long been assumed that the mitochondria are the primary source of ROS formation in skeletal muscle cells and that the increased ROS generation that occurs during contractile activity is directly related to the increased oxygen consumption associated with increased mitochondrial activity. Reassessments of the rate of ROS production by mitochondria have indicated that only 0.1-0.2% of the O2 consumed is released as reactive oxygen, 2,3 about 10% less than originally thought. Thus, mitochondria may not be the primary source of ROS during contract- ile activity. Additional sites for ROS production within skeletal muscle include the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase gp91 phox , the cytosolic phospholipase A2, and xan- thine oxidase. The role each of these sources play in increased ROS production during contractile ac- tivity and pathology has been obscured by our inability to precisely detect ROS production in spa- tially restricted regions of the cell. It is likely that multiple sites of ROS generation are active under different situations and that the effects are rela- tively localized and important for distinct cellular functions.
