CaMKII and reactive oxygen species contribute to early reperfusion arrhythmias, but oxidation of CaMKII8 at methionines 281/282 is not a determining factor

Background: Available evidence suggest that Ca2+/calmodulin-dependent protein kinase type II8 (CaMKII8) and reactive oxygen species (ROS) are important in early ischemia-reperfusion arrhythmias (IRA). Since ROS can activate CaMKII8 by oxidation of two methionines at positions 281/282, oxidized-CaMKII8 (Ox-CaMKII8) 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-CaMKII8 by using hearts and cardiomyocytes from mice with CaMKII8 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 Ca2+ waves and Ca2+ sparks. Immunoblotting confirmed high levels of oxidation in early reperfusion, but revealed no significant differences in the phosphorylation levels of Ca2+-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.

Automated summary

The study suggests that CaMKII8 and ROS play important roles in ischemia-reperfusion arrhythmias (IRA), but direct evidence is lacking. Inhibition of CaMKII and reduction of ROS levels can reduce the incidence of arrhythmias. However, mice with CaMKII resistant to oxidation at methionines 281/282 were not protected from arrhythmias, indicating that oxidation at these sites is not a determining factor.