Mitochondrial oxidant stress triggers cell death in simulated ischemia–reperfusion

• Published in: Biochimica et biophysica acta 2011-07, Vol.1813 (7), p.1382-1394
• Main Authors: Loor, Gabriel, Kondapalli, Jyothisri, Iwase, Hirotaro, Chandel, Navdeep S., Waypa, Gregory B., Guzy, Robert D., Vanden Hoek, Terry L., Schumacker, Paul T.
• Format: Article
• Language: English
• Subjects: Animals; Antioxidants - pharmacology; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein - genetics; bcl-2-Associated X Protein - genetics; Cardiomyocyte; Cell Hypoxia; Cells, Cultured; Chick Embryo; Cytochromes c - metabolism; Fluoresceins; Gene Knockout Techniques; Membrane Potential, Mitochondrial; Mice; Mice, Transgenic; Mitochondria, Heart - metabolism; Mitochondria, Heart - ultrastructure; Mitochondrial Membrane Transport Proteins - metabolism; Myocardial Reperfusion Injury - metabolism; Myocardial Reperfusion Injury - pathology; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - pathology; Oxidative Stress; Permeability transition; Propidium - metabolism; Reactive oxygen species; Reactive Oxygen Species - metabolism; roGFP; Superoxide dismutase; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2010.12.008

To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia–reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O 2 during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-X L in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-X L, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection. ► Cardiomyocytes exhibit mitochondrial oxidant stress during simulated ischemia. ► Antioxidants or Mn‐SOD over‐expression attenuate cell death during reperfusion. ► Ischemic triggering of mitochondrial permeability transition requires oxidant stress. ► Knockout of Bax/Bak or cytochrome c fails to protect against cell death