Time Course and Mechanisms of Phosphorylation of Phospholamban Residues in Ischemia-reperfused Rat Hearts. Dissociation of Phospholamban Phosphorylation Pathways

Sarcoplasmic reticulum (SR) dysfunction is one of the multiple alterations that occurs in ischemia-reperfused hearts. Because SR function is regulated by phosphorylation of phospholamban (PLB), a SR protein phosphorylated by cAMP-dependent protein kinase (PKA) at Ser16and Ca2+-calmodulin-dependent protein kinase (CaMKII) at Thr17, the phosphorylation of these residues during ischemia and reperfusion was examined in Langendorff-perfused rat hearts. Ser16phosphorylation increased significantly after 20 min of ischemia from 2.5±0.6% to 99.8±25.5% of maximal isoproterenol-induced site-specific phosphorylation and decreased to control values immediately after reperfusion. Thr17phosphorylation transiently increased at 2–5 min of ischemia and at 1 min of reperfusion (R1, 166.2±28.2%). The ischemia-induced increase in Ser16phosphorylation was significantly diminished in hearts from catecholamine-depleted animals and/or after β -blockade and abolished in the presence of the PKA-inhibitor, H-89. Thr17phosphorylation at the beginning of ischemia was blunted by nifedipine, whereas at R1 it was significantly diminished by perfusion with 0 m m Ca2+in the presence of EGTA and by the Na+/Ca2+exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit CaMKII, decreased Thr17phosphorylation at R1 and significantly prolonged half relaxation time. The results demonstrated a dissociation between the phosphorylation of PLB sites, being phosphorylation of Ser16dependent on the β -adrenergic cascade during ischemia and phosphorylation of Thr17on Ca2+influx both, at the beginning of ischemia and reperfusion. Phosphorylation of Thr17at the onset of reflow may provide the cell a mechanism to cope with Ca2+overload, transiently favoring the recovery of relaxation during early reperfusion.