Transient hyperoxic reoxygenation reduces cytochrome C oxidase activity by increasing superoxide dismutase and nitric oxide

Abstract

Oxygen therapies have been shown to be cytoprotective in a dose-dependent fashion. Previously, we have characterized the protective effects of moderate hyperoxia on cell viability of ischemic human cardiomyocytes and their mitochondrial membrane potential by transient addition of oxygenated perfluorocarbons to the cell medium. Now, we report that the activity and expression of cytochrome c oxidase (COX) after prolonged ischemia depend on the amount of oxygen delivered during reoxygenation. Transient hyperoxia during reoxygenation results in a decrease of COX activity by 62 +/- 15% and COX expression by 67 +/- 5%, when hyperoxic tensions of approximately = 300 mm Hg are reached in the cell medium. This decrease in COX expression is prevented by the inhibition of inducible nitric-oxide synthase (iNOS). Immunoblot analysis of ischemic human cardiomyocytes revealed that hyperoxic reoxygenation causes a 2-fold increase of iNOS, leading to a rise in nitric oxide production by 140 +/- 45%. Hyperoxic reoxygenation is further responsible for a 2-fold activation of hydrogen peroxide production and an increase in cytosolic superoxide dismutase expression by 35 +/- 10%. NADPH availability has no effect on the hyperoxia-induced decrease of superoxide. Overall, these results indicate that transient hyperoxic reoxygenation in optimal concentrations increases the level of nitric oxide by activation of iNOS and superoxide dismutase, thereby inducing respiration arrest in mitochondria of ischemic cardiomyocytes.

FIGURE 1.

Oxygen delivery capacity of PFC. Oxygen concentrations dissolved in PFC are a physical gas dissolution phenomenon that follows Henry's law and are therefore much lower than oxygen concentrations found in blood where oxygen is bound to hemoglobin (30) but significantly higher than the oxygen plasma level (A). Oxygenated PFC were used to induce hyperoxia in the first 10 min of reoxygenation (B).

FIGURE 2.

Ischemic cardiomyocytes decrease oxygen consumption after hyperoxia. Red fluorescence of Ru(phen₃)²⁺ is inversely proportional to the cellular oxygen concentration. Fluorescence persisted after normoxic reoxygenation (N), indicating an increased oxygen consumption in A compared with the reduced fluorochrome signal after hyperoxic reoxygenation (H) in B. Fluorescence was quantified by densitometry analysis. Data are given as percent from normoxic value and represent mean ± S.E. (error bars) *, significantly different from normoxia (p < 0.05).

FIGURE 3.

Superoxide production of hyperoxic cardiomyocytes. Superoxide (O₂^˙̄) production is independent of NADPH during hyperoxic conditions during reoxygenation. O₂^˙̄ production after normoxic reoxygenation (N) is compared with O₂^˙̄ production after hyperoxic reoxygenation (H) and after addition of NADPH at different concentrations. Data are given as percent from normoxic value and represent mean ± S.E. (error bars). *, significantly different from normoxic value (p < 0.05).

FIGURE 4.

Hyperoxic cardiomyocytes increase production of H₂O₂. H₂O₂ production is displayed as an increase in lime green cellular fluorescence after applying the hyperoxic regime (H) in B. This signal is compared with the cellular fluorescence after reoxygenation under normoxic conditions (N) in A and quantified by densitometry analysis in C. Data are given as percent from normoxic value and represent the mean ± S.E. (error bars). *, significantly different from normoxic value (p < 0.05).

FIGURE 5.

Cytosolic copper- and zinc-containing SOD content of ischemic cardiomyocytes. Already 1 h after reoxygenation, cytosolic CuSOD expression is more prominent under hyperoxic (H) in B than under normoxic reoxygenation (N) in A. Data are quantified by densitometry analysis in C and are given as percent from normoxic value. They represent the mean ± S.E. (error bars). *, significantly different from normoxic value (p < 0.05).

FIGURE 6.

NO production inhibits COX expression after hyperoxia. Already 1 h after reoxygenation, cellular NO content was assessed in A when ischemic cardiomyocytes had been reoxygenated under normoxic (N) and hyperoxic conditions (H). Hyperoxic cardiomyocytes activate iNOS after hyperoxia in B compared with normoxic reoxygenation. In C, COX expression decreases after hyperoxic conditions when analyzed by real-time PCR. After addition of iNOS inhibitor 1400W, COX expression recovers and increases in D. Data are given as percent from normoxic value and represent the mean ± S.E. (error bars). *, significantly different from normoxic value (p < 0.05).

FIGURE 7.

Pathophysiologic model of hyperoxia-mediated increase in cardiomyocyte survival after ischemia. Treatment of ischemic cardiomyocytes with hyperoxic reoxygenation results in an activation of iNOS expression (B), whereas normoxia is not sufficient to induce iNOS (A). NO production decreases mitochondrial respiration and consecutively ATP generation. This NO effect is mediated by deactivation of COX of complex VI. Attenuation of the ETC and mitochondrial ATP generation leads to a reduced utilization of H⁺ and a pH decrease. In addition, cellular SOD protects NO, and O₂^˙̄ converts to H₂O₂.