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

VITTONE L.; MUNDIÑA - WEILENMANN C; SAID M.; FERRERO P; MATTIAZZI A

JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY

ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD

Lugar: Amsterdam; Año: 2002 vol. 34 p. 39 - 50

0022-2828

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 Ser16 and Ca2+-calmodulin-dependent protein kinase (CaMKII) at Thr17, the phosphorylation of these residues during ischemia and reperfusion was examined in Langendorff-perfused rat hearts. Ser16 phosphorylation 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. Thr17 phosphorylation transiently increased at 2?5 min of ischemia and at 1 min of reperfusion (R1, 166.2±28.2%). The ischemia-induced increase in Ser16 phosphorylation was significantly diminished in hearts from catecholamine-depleted animals and/or after beta-blockade and abolished in the presence of the PKA-inhibitor, H-89. Thr17 phosphorylation at the beginning of ischemia was blunted by nifedipine, whereas at R1 it was significantly diminished by perfusion with 0 m Ca2+ in the presence of EGTA and by the Na+/Ca2+ exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit CaMKII, decreased Thr17 phosphorylation at R1 and significantly prolonged half relaxation time. The results demonstrated a dissociation between the phosphorylation of PLB sites, being phosphorylation of Ser16 dependent on the -adrenergic cascade during ischemia and phosphorylation of Thr17 on Ca2+ influx both, at the beginning of ischemia and reperfusion. Phosphorylation of Thr17 at the onset of reflow may provide the cell a mechanism to cope with Ca2+ overload, transiently favoring the recovery of relaxation during early reperfusion.