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1 Cardiothoracic Research Laboratory, Emory University, Atlanta, GA, USA
* To whom correspondence should be addressed. E-mail: zzhao{at}emory.edu.
We have shown that intermittent interruption of immediate re-flow at reperfusion (i.e., postconditioning) reduces infarct size in in vivo models after ischemia. Cardioprotection of postconditioning has been associated with attenuation of neutrophil-related events. However, it is unknown whether postconditioning before reoxygenation after hypoxia in cultured cardiomyocytes in the absence of neutrophils confers protection. This study tested the hypothesis that prevention of cardiomyocyte damage by hypoxic postconditioning (Post-con) is associated with a reduction in generation of reactive oxygen species (ROS) and intracellular Ca2+ overload. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned after the 3 h index hypoxia by three cycles of 5 min of reoxygenation and 5 min re-hypoxia applied before 6 h of reoxygenation. Relative to Sham control and hypoxia alone, the generation of ROS [increased lucigenin-enhanced chemiluminescence, SOD-inhibitable cytochrome c reduction and generation of hydrogen peroxide (H2O2)] was significantly augmented after immediate reoxygenation, as was the production of malondialdehyde (MDA), a product of lipid peroxidation. Concomitant with these changes, intracellular calcium ([Ca2+]i) and mitochondrial calcium ([Ca2+]m) detected by fluorescent Fluo-4 and X-rhod-1 AM staining, respectively, were elevated. Cell viability assessed by propidium iodide (PI) staining was decreased, consistent with increased levels of lactate dehydrogenase (LDH) after reoxygenation. Post-con at the onset of reoxygenation reduced ROS generation and MDA concentration in media and attenuated cardiomyocyte death assessed by PI and LDH. Post-con was associated with a decrease in [Ca2+]i and [Ca2+]m. These data suggest that hypoxic postconditioning reduces reoxygenation-induced injury in cardiomyocytes, potentially mediated by an attenuation of ROS generation, lipid peroxidation, and intracellular and mitochondrial Ca2+ overload.
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