Ca(2+)-calmodulin-dependent protein kinase II (
CaMKII) plays an important role mediating apoptosis/
necrosis during
ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with
CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr(17) site of
phospholamban, without changes in total
protein, consistent with an increase in
CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of
ryanodine receptors (
RyR2) and the amount of
RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/
RyR2 did not change. Inhibition of the reverse
Na(+)/Ca(2+)exchanger (NCX) mode (
KBR7943) diminished
phospholamban phosphorylation, reduced apoptosis/
necrosis and enhanced mechanical recovery.
CaMKII-inhibition (KN-93), significantly decreased
phospholamban phosphorylation,
infarct area,
lactate dehydrogenase release (LDH) (
necrosis), TUNEL positive nuclei,
caspase-3 activity, Bax/Bcl-2 ratio and Ca(2+)-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog,
KN-92. Blocking SR Ca(2+) loading and release (
thapsigargin/
dantrolene), mitochondrial Ca(2+) uniporter (
ruthenium red/
RU360), or
mitochondrial permeability transition pore (
cyclosporine A), significantly decreased
infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr(17) of
phospholamban at the onset of reflow and exhibited a significant decrease in
infarct size, apoptosis and
necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by
CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.