Ca(2+)-
calmodulin kinase II (
CaMKII) activation is deleterious in cardiac
ischemia/reperfusion (I/R). Moreover, inhibition of
CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents
CaMKII-induced I/R damage. However, the downstream targets of
CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of
CaMKII at the SR level,
phospholamban (PLN) and
ryanodine receptors (
RyR2), in
CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary
CaMKII sites, S2814 on cardiac
RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of
RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated
serine 2814 phosphorylation site on
RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced
infarct size and decreased apoptosis. Conversely, knock-in mice, in which
CaMKII site of
RyR2 is constitutively activated (S2814D), significantly increased
infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional
myocardial ischemia,
infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on
RyR2 in
CaMKII-dependent I/R cardiac damage. In contrast, they showed that
CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.