Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion

Gabriel Loor, Jyothisri Kondapalli, Hirotaro Iwase, Navdeep S. Chandel, Gregory B. Waypa, Robert D. Guzy, Terry L. Vanden Hoek, Paul T. Schumacker

Research output: Contribution to journalArticlepeer-review

171 Scopus citations


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 O2 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-XL 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-XL, 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.

Original languageEnglish (US)
Pages (from-to)1382-1394
Number of pages13
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Issue number7
StatePublished - Jul 2011
Externally publishedYes

Bibliographical note

Funding Information:
The authors thank Bhumika Sharma for general technical assistance, Danijela Dokic for assistance with immunofluorescence studies, and Juan Li for the isolation of the cardiomyocytes. We also thank Dr. Douglas R. Green for providing the HeLa cell line with stable expression of cytochrome c-GFP, and Drs. J.X. Bai and A.I. Cederbaum for providing adenoviruses expressing catalase and mitochondrial catalase. This work was supported by HL35440, HL32646, HL66315, HL079650 and the American Heart Association , Midwest Affiliates.


  • Apoptosis
  • Cardiomyocyte
  • Permeability transition
  • Reactive oxygen species
  • RoGFP
  • Superoxide dismutase


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