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 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.
Vittone
Leticia
L
Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, La Plata, Argentina.
Mundiña-Weilenmann
Cecilia
C
Said
Matilde
M
Ferrero
Paola
P
Mattiazzi
Alicia
A
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002395:Catecholamines / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006321:Heart / Q000502:physiology*; D007511:Ischemia / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D015427:Reperfusion Injury; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D002395:Catecholamines; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D011943:Receptors, Adrenergic, beta; D012694:Serine; D019831:Sodium-Calcium Exchanger; D013890:Thiourea; D013912:Threonine; D000099211:Phospholamban; C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate