Altering CO2 during reperfusion of ischemic cardiomyocytes modifies mitochondrial oxidant injury

Objective: Acute changes in tissue CO2 and pH during reperfusion of the ischemic heart may affect ischemia/reperfusion injury. We tested whether gradual vs. acute decreases in CO2 after cardiomyocyte ischemia affect reperfusion oxidants and injury. Design: Comparative laboratory investigation. Setting: Institutional laboratory. Subjects: Embryonic chick cardiomyocytes. Interventions: Microscope fields of approximately 500 chick cardiomyocytes were monitored throughout 1 hr of simulated ischemia (Po-2 of 3-5 torr, Pco(2) of 144 torr, pH 6.8), followed by 3 hrs of reperfusion (Po-2 of 149 torr, Pco(2) of 36 torr, pH 7.4), and compared with cells reperfused with relative hypercarbia (Pco(2) Of 71 torr, pH 6.8) or hypocarbia (Pco(2) of 7 torr, pH 7.9). Measurements and Main Results: The measured outcomes included cell viability (via propidium iodide) and oxidant generation (reactive oxygen species via 2',7'-dichlorofluorescin oxidation and nitric oxide [NO] via 4,5-diaminofluorescein diacetate oxidation). Compared with normocarbic reperfusion, hypercarbia significantly reduced cell death from 54.8% +/- 4.0% to 26.3% +/- 2.8% (p < .001), significantly decreased reperfusion reactive oxygen species (p < .05), and increased NO at a later phase of reperfusion (p < .01). The NO synthase inhibitor N-nitro-L-arginine methyl ester (200 mu M) reversed this oxidant attenuation (p < .05), NO increase (p < .05), and the cardioprotection conferred by hypercarbic reperfusion (increasing death to 54.3% +/- 6.0% [p < .05]). Conversely, hypocarbic reperfusion increased cell death to 80.4% +/- 4.5% (p < .01). It also increased reactive oxygen species by almost two-fold (p = .052), without affecting the NO level thereafter. Increased reactive oxygen species was attenuated by the mitochondrial complex III inhibitor stigmatellin (20 nM) when given at reperfusion (p < .05). Cell death also decreased from 85.9% +/- 4.5% to 52.2% +/- 6.5% (p < .01). The nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin (300 mu M) had no effect on reperfusion reactive oxygen species. Conclusions: Altering CO2 content during reperfusion can significantly affect myocardial postresuscitation injury, in part by modifying mitochondrial oxidants and NO synthase-induced NO production.