title: TNFalpha-induced cytoprotection requires the production of free radicals within mitochondria in C2C12 myotubes.
abstract: We previously reported that tumour necrosis factor alpha (TNFalpha) 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 TNFalpha-induced reactive oxygen species (ROS). We hypothesized that TNFalpha cytoprotection occurs through the generation of ROS which originate within the mitochondria. C(2)C(12) myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFalpha (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 TNFalpha-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 TNFalpha improved cell viability compared with the simulated ischemic control (TNFalpha: 75 +/- 1% versus 34 +/- 1% for the control: p<0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFalpha-stimulated cells to 15 +/- 1% (p<0.001 versus TNFalpha). Similar results were obtained with IPC. TNFalpha stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFalpha to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFalpha-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFalpha.
authors.affiliation: Hatter Institute for Cardiology Research, University of Cape Town, South Africa. lydia@capeheart.uct.ac.za
authors.forename: Lydia
authors.initials: L
authors.lastname: Lacerda
authors.forename: Robert M
authors.initials: RM
authors.lastname: Smith
authors.forename: Lionel
authors.initials: L
authors.lastname: Opie
authors.forename: Sandrine
authors.initials: S
authors.lastname: Lecour
mesh_terms: D015687:Cell Hypoxia / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019610:Cytoprotection / Q000187:drug effects*; D005434:Flow Cytometry / Q000379:methods; D005452:Fluoresceins / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D019194:Ischemic Preconditioning; D008564:Membrane Potentials / Q000187:drug effects; D008931:Mitochondria, Muscle / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D005452:Fluoresceins; C110658:N-(2-mercaptoproprionyl)-glycine; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D014409:Tumor Necrosis Factor-alpha; C029569:diacetyldichlorofluorescein; D005998:Glycine