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*

*Corresponding author for this work

Research output: Contribution to journalArticle

130 Scopus citations

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 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
Volume1813
Issue number7
DOIs
StatePublished - Jul 1 2011

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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