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

Lugar: London, New York; Año: 2002 vol. 34 p. 39 - 50

0022-2828

J Mol Cell Cardiol. 2002 Jan;34(1):39-50. 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. Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Medicas, Universidad Nacional de La Plata, 60 y 120, La Plata, Argentina. 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 Ser(16)and Ca(2+)-calmodulin-dependent protein kinase (CaMKII) at Thr(17), the phosphorylation of these residues during ischemia and reperfusion was examined in Langendorff-perfused rat hearts. Ser(16)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. Thr(17)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 Ser(16)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. Thr(17)phosphorylation at the beginning of ischemia was blunted by nifedipine, whereas at R1 it was significantly diminished by perfusion with 0 m m Ca(2+)in the presence of EGTA and by the Na(+)/Ca(2+)exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit CaMKII, decreased Thr(17)phosphorylation at R1 and significantly prolonged half relaxation time. The results demonstrated a dissociation between the phosphorylation of PLB sites, being phosphorylation of Ser(16)dependent on the beta-adrenergic cascade during ischemia and phosphorylation of Thr(17)on Ca(2+)influx both, at the beginning of ischemia and reperfusion. Phosphorylation of Thr(17)at the onset of reflow may provide the cell a mechanism to cope with Ca(2+)overload, transiently favoring the recovery of relaxation during early reperfusion.