Abstract
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.
Highlights
Because the HSP-FRET sensor was expressed throughout the cytosol, these results indicate that significant oxidant stress arising from H2O2 occurs in the cytosol during ischemia
Ischemic reactive oxygen species (ROS) Predispose to Reperfusion Injury—Numerous studies have shown that a burst of oxidant stress occurs during the first few minutes after reperfusion of ischemic myocardium
The results of this study show that a lesser degree of oxidant stress begins during ischemia prior to reoxygenation
Summary
We sought to assess cellular oxidant stress during ischemia using a novel redox-sensitive fluorescence resonance energy transfer (FRET) protein expressed in the cytosol of cardiomyocytes. To assess the sensitivity of HSP-FRET to oxidant stress, fluorescent images were collected every 20 s for 30 min as the cells were perfused with normoxic medium containing H2O2 or medium alone (time controls) (Fig. 1D). To assess oxidant stress during ischemia, cardiomyocytes transiently expressing HSP-FRET were subjected to base-line normoxic perfusion (20 min) followed by simulated ischemia (30 min) while fluorescent images were collected every 60 s.
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