Disruption of skeletal myocytes initiates superoxide release: Contribution of NAD(P)H oxidase

Generation of reactive oxygen species (ROS) as an early local reaction to muscle crush injury has frequently been predicted. However, although it is known that severe inflammatory reactions occurring after major muscle trauma originate mainly from early local incidents within the injured tissue, no detailed studies exist on the local generation of ROS in response to myocyte destruction thus far. Therefore, in this study, ROS formation after lethal mechanical damage was examined using a model of scraping injury to cultured C2C12 skeletal myocytes and superoxide detection by lucigenin chemiluminescence, nitrotetrazolium blue chloride reduction, or electron spin resonance spectroscopy. Mechanical rupture of myocytes resulted in an immediate release of superoxide from the damaged cells that could be substantially blocked by the superoxide scavengers Superoxide dismutase (51%), tiron (95%), and MAMA/NO (93%) and by hypoxia (83% inhibition). Superoxide generation was primarily confined to the myocytes' membrane fraction and 7- to 8-fold enhanced by the addition of NADH or NADPH. The NADPH-enhanced superoxide generation could largely be diminished by the NAD(P)H oxidase inhibitors diphenyleneiodonium and apocynin in cell lysates (97% and 35% inhibition, respectively) and in isolated membrane fractions (61% and 63% inhibition). We thus conclude that immediately after myocyte damage, large amounts of superoxide are formed that predominantly originate from membrane-bound electron-transferring enzymes, especially NAD(P)H oxidase. This suggests a decisive role of ROS in the pathogenesis of tissue trauma, with superoxide being an initiator of the signaling mechanism from injured myocytes to the surrounding tissue and, potentially, to the whole body.