TNFα-induced cytoprotection requires the production of free radicals within mitochondria in C2C12 myotubes

We previously reported that tumour necrosis factor alpha (TNF alpha) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNF alpha-induced reactive oxygen species (ROS). We hypothesized that TNF alpha cytoprotection occurs through the generation of ROS which originate within the mitochondria. C2C12 myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNF alpha (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNF alpha-induced ROS was examined with Mitotracker Red CM-H(2)XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFa improved cell viability compared with the simulated ischemic control (TNFa: 75 +/- 1% versus 34 1% for the control: p < 0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNF alpha-stimulated cells to 15 1% (p < 0.001 versus TNF alpha). Similar results were obtained with IPC. TNFa stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNF alpha to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNF alpha-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNF alpha. (c) 2006 Elsevier Inc. All rights reserved.