Activity-Dependent Regulation of Mitochondrial Superoxide Flashes in Skeletal Muscle

Abstract

Reactive oxygen species (ROS) are a class of molecules that oxidize diverse cellular proteins/lipids and are generated mainly in mitochondria by the electron transport chain (ETC). We previously discovered a novel biosensor for superoxide, circularly permutated YFP (cpYFP). Using mitochondrial-targeted cpYFP (mt-cpYFP), localized bursts of superoxide in individual or clusters of mitochondria, termed “superoxide flashes,” are observed in quiescent cells across a wide range of cell types [Wang, W. et al., Cell 134:279-290, 2008]. Here, we examined the properties of superoxide flashes in flexor digitorum brevis muscle fibers from newly generated transgenic mice expressing skeletal muscle-specific mt-cpYFP. A new flash detection and analysis software (“Flash Collector”) was developed to enable automated quantification of flash frequency, amplitude, kinetics, and area. Results demonstrate that skeletal muscle fibers exhibit higher basal mitochondrial superoxide flash frequency, but similar amplitude, kinetics, and area as those observed in cardiac myocytes. Rotenone (5υM) and oligomycin (5υM) reduced flash frequency to ∼20% of control, confirming the ETC dependence of superoxide flash generation. Inhibition of adenine nucleotide translocase by bongkrekic acid (100υM) decreased flash frequency by 50%. Incubation in Ringer's containing 10mM glucose or mitochondrial substrates did not significantly alter flash frequency, but increased flash amplitude and duration by 10-15%. Importantly, superoxide flash activity was enhanced (frequency increased from18.1±1.6 to 22.3±2.0 flashes/100s·100υm2) following five consecutive brief tetani (500ms, 100Hz, 0.2 duty cycle), but was markedly suppressed following a prolonged fatiguing stimulation (40 tetani; frequency reduced from 17.6±2.2 to 7.7±1.6). These results demonstrate skeletal muscle activity regulates mitochondrial superoxide flash production. In adition, muscle-specific mt-cpYFP transgenic mice will be a powerful tool for assessing the physiological role of superoxide flash activity and how this activity is altered and contributes to skeletal muscle aging and disease.