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 O-2(-) 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 O-2(-) scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of O-2(-), which is then converted by SOD to H2O2, and subsequent Akt activation by H2O2 attenuates apoptosis in ischemia-reperfusion. (C) 2010 Elsevier Inc. All rights reserved.