title: Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion.
abstract: 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.
authors.affiliation: Department of Surgery, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Loor
authors.forename: Jyothisri
authors.initials: J
authors.lastname: Kondapalli
authors.forename: Hirotaro
authors.initials: H
authors.lastname: Iwase
authors.forename: Navdeep S
authors.initials: NS
authors.lastname: Chandel
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Waypa
authors.forename: Robert D
authors.initials: RD
authors.lastname: Guzy
authors.forename: Terry L
authors.initials: TL
authors.lastname: Vanden Hoek
authors.forename: Paul T
authors.initials: PT
authors.lastname: Schumacker
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002642:Chick Embryo; D045304:Cytochromes c / Q000378:metabolism; D005452:Fluoresceins; D055786:Gene Knockout Techniques; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008822:Mice, Transgenic; D008929:Mitochondria, Heart / Q000378:metabolism* / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D011419:Propidium / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: D000975:Antioxidants; D005452:Fluoresceins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D011419:Propidium; D045304:Cytochromes c; D013482:Superoxide Dismutase; C007740:fluorexon