Immunodetection of Phosphorylation Sites Gives New Insights into the Mechanisms Underlying Phospholamban Phosphorylation in the Intact Heart

Abstract

Phosphorylation site-specific antibodies, quantification of 32 P incorporation into phospholamban, and simultaneous measurements of mechanical activity were used in Langendorff-perfused rat hearts to provide further insights into the underlying mechanisms of phospholamban phosphorylation. Immunological detection of phospholamban phosphorylation sites showed that the isoproterenol concentration-dependent increase in phospholamban phosphorylation was due to increases in phosphorylation of both 16 and Thr 17 , respectively (3). These phosphorylations are independent of each other, and when both are operating, they appear to have an additive action (4). In the intact heart, ␤-adrenergic stimulation phosphorylates phospholamban at both sites (5), which indicates that PKA-and CaMKII-dependent pathways are also working in the functioning heart. Whether these phosphorylation mechanisms are independent of each other and additive, as described in the isolated SR membranes, remains unknown. Different attempts to phosphorylate phospholamban by CaMKII in the intact heart have systematically failed unless cAMP levels within the cell increase (6 -11). This consistent finding strongly suggests an interaction between PKA and CaMKII pathways of phospholamban phosphorylation in the intact heart. The nature of this interaction as well as the cause for the difference between the in vivo and in vitro results have never been explored.The availability of phosphorylation-site specific antibodies to phospholamban, which precisely discriminate between Ser 16 and Thr 17 phosphorylation sites (12), prompted us to reexamine the issue. Combination of this technique with the quantitative assessment of phospholamban phosphorylation by radiochemical labeling of ATP pools and simultaneous measurements of mechanical parameters allowed us to characterize the PKA and CaMKII-dependent mechanisms of phospholamban phosphorylation in the intact heart and their relative physiological roles on cardiac performance. EXPERIMENTAL PROCEDURESHeart Perfusions-Experiments were performed in isolated hearts from male Wistar rats (250 -350 g body wt) perfused according to the Langendorff technique as described previously (8). The composition of the physiological salt solution (PSS) was (in mM): 128.3 NaCl, 4.7 KCl, 1.35 CaCl 2 , 20.2 NaHCO 3 , 0.4 NaH 2 PO 4 , 1.1 MgCl 2 , 11.1 glucose, and 0.04 Na 2 EDTA. This solution was equilibrated with 95% O 2 , 5% CO 2 to give a pH of 7.4. The mechanical activity of the heart was assessed by either sewing an isometric strain gauge arch (Micro Measurements, type MA-06 -030LB-120) to the left ventricular wall or passing into the left ventricle a latex balloon connected to a pressure transducer (Namic, perceptor DT disposable transducer). The initial length of the gauge was set by stretching the segment attached by approximately 30%. The balloon was filled with aqueous solution to achieve a left ventricular

Critical Evaluation of Cardiac Ca2+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody

“…In each case interventions that activate CaMKII have been deployed, using the optimal conditions described in the literature (26,(45)(46)(47). In experiments using cardiac SR vesicles and those using isolated rat myocytes, Thr-17 phosphorylation of phospholamban has demonstrated high levels of CaMKII activity focused on SR targets.…”

“…Others have shown that an increase in electrical stimulation frequency (45), exposure to ␤-adrenergic agonists (46) and exposure to potent Ser/Thr phosphatase inhibitors (47) results in the activation of CaMKII in isolated rat ventricular myocytes. Rat myocytes treated for 5 min with each of the above intervention regimes (stimulated electrically at 0.5 Hz, unless otherwise stated) displayed enhanced CaMKII phosphorylation of phospholamban compared with control myocytes (Fig.…”

β 2 -Adrenergic cAMP Signaling Is Uncoupled From Phosphorylation of Cytoplasmic Proteins in Canine Heart

“…[22][23][24] In addition, an augmentation in TnI phosphorylation after ␤-AR subtype stimulation might contribute to the relaxant effect. 37 Another novel finding of the present study is that the Zin-induced acceleration of relaxation occurs both in vivo and in vitro without any increase in TnI or PLB phosphorylation at either Ser16 or Thr17.…”

“…PLB plays an important role in modulating SR Ca 2ϩ pump activity, [22][23][24] and its phosphorylation at Ser16 and Thr17 is mediated by PKA and Ca 2ϩ /calmodulin-dependent kinase II, respectively. 25,26 Phosphorylation site-specific PLB antibodies 27 were used in the present study to determine whether ␤-AR subtype stimulation differentially regulates PLB phosphorylation.…”

Activation of β 2 -Adrenergic Receptors Hastens Relaxation and Mediates Phosphorylation of Phospholamban, Troponin I, and C-Protein in Ventricular Myocardium From Patients With Terminal Heart Failure

“…7,44 The CaM kinase-catalyzed phosphorylation of phospholamban can contribute to increased contractility and hastened relaxation, provided dephosphorylation is negligible. 44 Type 1 phosphatase catalyzes this dephosphorylation, and the activity of type 1 phosphatase can, in turn, be inhibited by isoproterenol through PKA-catalyzed phosphorylation of protein phosphatase inhibitor-1 in guinea pig ventricular myocytes. 45 Our results show for the first time that Thr17 phosphorylation of phospholamban occurs in failing human ventricle through both ␤ 1 -and ␤ 2 -adrenergic receptors.…”