Oxidant Stress during Simulated Ischemia Primes Cardiomyocytes for Cell Death during Reperfusion

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.Previous studies have examined the cellular consequences of tissue ischemia, but the molecular mechanisms that regulate cell death in this syndrome are still not fully understood. When blood flow to a tissue is interrupted, the extent of injury is influenced by the severity and duration of the ischemic challenge such that longer periods of ischemia are associated with more extensive cell death (1, 2). Restoration of blood flow relieves the ischemic stress, although morphological evidence of tissue injury appears only after the oxygen supply has been restored (3). This has led to the widely accepted notion that reperfusion is responsible for the cellular damage in ischemia-reperfusion injury (4). However, the events during ischemia that prime the cell for death during reperfusion are not fully understood.Oxidant stress arising from excessive production of reactive oxygen species (ROS) 3 has long been associated with ischemia-reperfusion injury (4, 5). In this regard, animals with genetic impairment of antioxidant systems exhibit increased susceptibility to myocardial ischemia-reperfusion injury (6); antioxidants administered during ischemia-reperfusion confer protection against subsequent cell death (7-9); and overexpression of antioxidant enzymes confers protection against ischemia-reperfusion injury (10, 11). Multiple studies have demonstrated that a burst of oxidant stress is generated upon reperfusion of ischemic cardiomyocytes (5, 12, 13). Int...

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