Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion.
Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.
Robin
Emmanuel
E
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Guzy
Robert D
RD
Loor
Gabriel
G
Iwase
Hirotaro
H
Waypa
Gregory B
GB
Marks
Jeremy D
JD
Hoek
Terry L Vanden
TL
Schumacker
Paul T
PT
D000818:Animals; D002374:Catalase / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D031541:Fluorescence Resonance Energy Transfer; D006352:Heart Ventricles / Q000166:cytology; D006861:Hydrogen Peroxide / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D017202:Myocardial Ischemia / Q000378:metabolism / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology*; D018384:Oxidative Stress / Q000502:physiology*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
D013481:Superoxides; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase