Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemia–reperfusion

We examined whether and how pretreatment with carbon monoxide (CO) prevents apoptosis of cardioblastic H9c2 cells in ischemia–reperfusion. Reperfusion (6 h) following brief ischemia (10 min) induced cytochrome c release, activation of caspase-9 and caspase-3, and apoptotic nuclear condensation. Brief CO pretreatment (10 min) or a caspase-9 inhibitor (Z-LEHD-FMK) attenuated these apoptotic changes. Ischemia–reperfusion increased phosphorylation of Akt at Ser472/473/474, and this was enhanced by CO pretreatment. A specific Akt inhibitor (API-2) blunted the anti-apoptotic effects of CO in reperfusion. In normoxic cells, CO enhanced O2- generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A) but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an O2- scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of O2-, which is then converted by SOD to H2O2, and subsequent Akt activation by H2O2 attenuates apoptosis in ischemia–reperfusion.