Studies on phosphorylation of canine cardiac sarcoplasmic reticulum by calmodulin-dependent protein kinase.

124

126

17phosphorylation by CaMKII and is essential to the relaxant effect of ␤-adrenergic stimulation. To determine the role of Thr 17 PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to ␤ 1 -adrenergic stimulation or electrical field stimulation (0.1-3 Hz) or both. A ␤ 1 -adrenergic agonist, norepinephrine (10 ؊9 -10 (1-3). In the beating mammalian heart, ␤-adrenergic stimulation increases both PKA-and CaMKIImediated phosphorylation of Ser 16 and Thr 17 (4 -6). More recent studies have shown that the effects of ␤-adrenergic stimulation on PLB phosphorylation are mostly attributable to ␤ 1 -but not ␤ 2 -adrenergic receptor subtype (7-10).Over the last two decades, intensive studies have been focused on the physiological significance of the dual site phosphorylation of PLB. These previous studies in perfused hearts or in vivo have provided several lines of evidence leading to the concept that Ser 16 phosphorylation is a prerequisite for phosphorylation of Thr 17 and that Ser 16 phosphorylation is largely responsible for ␤-adrenergic modulation of cardiac relaxation (4, 5, 11, 12, 14 -18 phosphorylation is the dominant molecular event responsible for accelerated cardiac relaxation. However, in vitro studies in the isolated SR membranes have consistently indicated that Ser 16 and Thr 17 can be readily and independently phosphorylated by PKA and CaMKII, respectively, and that when both are phosphorylated, there is an additive interaction (20,21). The apparent discrepancy between in vivo and in vitro PLB phosphorylation is yet to be reconciled.To resolve this paradox and to further address the relative contribution of PKA-and CaMKII-mediated PLB phosphorylation in beat-to-beat cardiac functional modulation, individually we manipulated PKA activity, using ␤-adrenergic stimulation in quiescent rat ventricular myocytes, and CaMKII activity, by electrically pacing the myocytes at different stimulation frequency (0.1-3 Hz) in the absence of ␤-adrenergic stimulation. Both stimuli were also combined to explore possible interactions between PKA-and CaMKII-mediated signaling. Under those experimental conditions, we measured PLB phosphorylation at Ser 16 and Thr 17 as well as relaxation time of cell contraction. Here, we report our surprising findings that electrical stimulation alone increases CaMKII-dependent phosphorylation of PLB at Thr 17 in a frequency-dependent manner without altering PKA-mediated Ser 16 phosphorylation, that phosphorylation of Thr 17 is markedly enhanced by ␤-adrenergic stimulation in the electrically paced but not in quiescent myocytes, and that Thr 17 phosphorylation is associated with a significant relaxant effect, regardless of ␤-adrenergic stimulation.

Section: Phospholamban (Plb)mentioning

confidence: 99%

Frequency-encoding Thr17 Phospholamban Phosphorylation Is Independent of Ser16 Phosphorylation in Cardiac Myocytes

Hagemann

Kuschel

Kuramochi

et al. 2000

124

126

“…In vitro studies have shown that Ser 16 is phosphorylated by cAMP-dependent protein kinase, whereas Thr 17 is phosphorylated by Ca 2ϩ -calmodulin-dependent protein kinase (4). Phosphorylation of each site occurs in an independent manner, although it is not presently clear whether the stimulatory effects of the two phosphorylations on SR Ca 2ϩ transport are additive (5)(6)(7)(8)(9).…”

Section: Phospholamban (Plb)mentioning

confidence: 99%

Transgenic Approaches to Define the Functional Role of Dual Site Phospholamban Phosphorylation

Luo

Chu

Sato

et al. 1998

103

Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca 2؉ -ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser 16 and Thr 17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser 16 3 Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wildtype phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32 P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser 16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser 16 is a prerequisite for Thr 17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the ␤-agonist stimulatory responses in the mammalian heart. Phospholamban (PLB)1 is a regulator of the affinity of the cardiac sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase for Ca 2ϩ . Dephosphorylated PLB is an inhibitor, and phosphorylation of PLB removes its inhibitory effects on the SR Ca 2ϩ -ATPase. Recently, the critical role of PLB in the regulation of cardiac contractility has been defined through gene transfer (1) and knockout (2) technology in the mouse. Cardiac-specific overexpression of PLB was associated with decreases in the affinity of the SR Ca 2ϩ -ATPase for Ca 2ϩ and depressed cardiac function, whereas PLB deficiency resulted in increased Ca 2ϩ affinity of the Ca 2ϩ -ATPase and enhanced myocardial performance. Furthermore, the stimulatory effects to ␤-adrenergic agonists were more pronounced in the PLB-overexpressing hearts, whereas these effects were attenuated in the PLB-knockout hearts compared with wild types (1, 2). These studies suggested that PLB plays a prominent role in the heart's responses to ␤-agonists. However, PLB is phosphorylated on both Ser 16 and Thr 17 during isoproterenol stimulation (3) and the relative contribution of each site in the altered contractile responses of the heart is not presently well known. In vitro studies have shown that Ser 16 is phosphorylated by cAMP-dependent protein kinase, whereas Thr 17 is phosphorylated by Ca 2ϩ -calmodulin-dependent protein kinase (4). Phosphorylation of each site occurs in an independent manner, although it is not presently clear whether the stimulatory effects of the two phosphorylations on SR Ca...

“…In vitro experiments indicate that PKA and CaMKII phosphorylate phospholamban at two different sites, Ser 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.…”

Section: Cardiac Sarcoplasmic Reticulum (Sr)mentioning

confidence: 99%

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

Mundiña‐Weilenmann¹,

Vittone²,

Ortale³

et al. 1996

101

141

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