Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants

• Published in: American journal of physiology. Heart and circulatory physiology Vol. 285; no. 3; pp. H1198 - H1205
• Main Authors: Said, M, Vittone, L, Mundina-Weilenmann, C, Ferrero, P, Kranias, E. G, Mattiazzi, A
• Format: Journal Article
• Language: English
• Published: United States 01.09.2003
• Subjects: Amino Acid Substitution; Animals; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Male; Mice; Mice, Inbred BALB C; Mice, Transgenic; Myocardial Contraction - physiology; Myocardial Reperfusion Injury - metabolism; Myocardial Reperfusion Injury - physiopathology; Myocardial Stunning - metabolism; Myocardial Stunning - physiopathology; Phosphorylation; Rats; Rats, Wistar
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00209.2003

1 Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, 1900 La Plata, Argentina; and 2 Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575 Submitted 6 March 2003 ; accepted in final form 15 May 2003 Phosphorylation of phospholamban (PLB) at Ser 16 (protein kinase A site) and at Thr 17 [Ca 2 + /calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca 2 + uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser 16 phosphorylation that was dependent on -adrenergic stimulation and an ischemia and reperfusion-induced Thr 17 phosphorylation that was dependent on Ca 2 + influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 µM) or the -adrenergic blocker dl -propranolol (1 µM). KN-93 diminished the reperfusion-induced Thr 17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl -Propranolol decreased the ischemia-induced Ser 16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr 17 or Ser 16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr 17 appears to play a major role. phospholamban phosphorylation residues; phospholamban mutants; ischemia-reperfusion Address for reprint requests and other correspondence: A. Mattiazzi, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas 60 y 120, 1900 La Plata, Argentina (E-mail: ramattia{at}atlas.med.unlp.edu.ar ).