CaMKII and reactive oxygen species contribute to early reperfusion arrhythmias, but oxidation of CaMKIIδ at methionines 281/282 is not a determining factor

Background: Available evidence suggest that Ca 2+ /calmodulin-dependent protein kinase type II (CaMKII) and reactive oxygen species (ROS) are important in early ischemia-reperfusion arrhythmias (IRA). Since ROS can activate CaMKII by oxidation of two methionines at positions 281/282, oxidized-CaMKII (Ox-CaMKII) has been proposed to be important for IRA. However, direct evidence for this is missing. Methods: We exposed Langendorff-perfused hearts and ventricular cardiomyocytes from C57BL/6 mice to global and simulated ischemia, respectively, and recorded arrhythmic events during early reperfusion. Hearts were collected for immunoblotting of key phosphoproteins. We evaluated the effects of beta-adrenoceptor stimulation, inhibition of CaMKII, and reduced ROS levels with isoprenaline, KN93/AIP and N-acetylcysteine (NAC), respectively. We further tested the importance of Ox-CaMKII by using hearts and cardiomyocytes from mice with CaMKII resistant to oxidation of methionines 281 and 282 (MMVV). Results: Hearts treated with KN93, AIP or NAC had lower incidence of early IRA, and NAC-treated cardiomyocytes had lower incidence of arrhythmogenic events. However, hearts from MMVV mice had a similar incidence of early IRA to wild type mice (WT), and MMVV and WT cardiomyocytes had a similar frequency of Ca 2+ waves and Ca 2+ sparks. Immunoblotting confirmed high levels of oxidation in early reperfusion, but revealed no significant differences in the phosphorylation levels of Ca 2+ -handling proteins in MMVV and WT hearts. Conclusions: Although CaMKII and ROS both contribute to early IRA, hearts from mice with CaMKII resistant to oxidation at methionines 281/282 were not protected from such arrhythmias, suggesting that oxidation at these sites is not a determining factor.