Superoxide flashes in mouse skeletal muscle are produced by discrete arrays of active mitochondria operating coherently.

Reactive Oxygen Species (ROS) constitute important intracellular signaling molecules. Mitochondria are admitted sources of ROS, especially of superoxide anions through the electron transport chain. Here the mitochondria-targeted ratiometric pericam (RPmt) was used as a superoxide biosensor, by appropriate choice of the excitation wavelength. RPmt was transfected in vivo into mouse muscles. Confocal imaging of isolated muscle fibers reveals spontaneous flashes of RPmt fluorescence. Flashes correspond to increases in superoxide production, as shown by simultaneous recordings of the fluorescence from MitoSox, a mitochondrial superoxide probe. Flashes occur in all subcellular populations of mitochondria. Spatial analysis of the flashes pattern over time revealed that arrays of mitochondria work as well-defined superoxide-production-units. Increase of superoxide production at the muscle fiber level involves recruitment of supplemental units with no increase in per-unit production. Altogether, these results demonstrate that superoxide flashes in muscle fibers correspond to physiological signals linked to mitochondrial metabolism. They also suggest that superoxide, or one of its derivatives, modulates its own production at the mitochondrial level.