Skeletal muscles produce low levels of reactive oxygen species (ROS) under resting conditions while during contractile activity the rate of ROS production increases. Low levels of ROS production may act to stimulate adaptive responses in skeletal muscle, while increased ROS dependent oxidation at sarcoplasmic reticulum, myofilaments and other EC coupling components likely lead to decrements in contractile function. Multiple sites exist for ROS production, including mitochondria and NADPH oxidase; however, the contribution of each of these and the factors that regulate the increased production of ROS during contractile activity remains to be determined. Here we use repetitive field stimulation of single FDB myofibers as a model of ROS secondary to repetitive activity. In FDB's loaded with the cytosolic, non-specific ROS probe DCFH, we have imposed intermittent (0.5 Hz) trains (150msec, 0.5msec sq. pulse @, 50Hz) of tetanic stimulation to establish a reliable in vitro model for activity dependent ROS production. In recent studies with this model, we have begun to explore site dependent generation of ROS with a redox sensitive variant of green fluorescent protein (roGFP) that is targeted to the mitochondria (mito-roGFP). Following cDNA electroporation in vivo, expression of mito-roGFP, and FDB isolation, we report evidence of the fidelity and specificity of this probe in mitochondria and the activity dependent mitochondrial redox status during our stimulation paradigm.
