Ser¹⁶ prevails over Thr¹⁷ phospholamban phosphorylation in the -adrenergic regulation of cardiac relaxation

Max Delbrück Center for Molecular Medicine, 13125 Berlin-Buch, Germany

	ABSTRACT

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Phospholamban is a critical regulator of sarcoplasmic reticulum Ca²⁺-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca²⁺ and cAMP pathways, we have investigated the -adrenergic-induced phosphorylation of Ser¹⁶ and Thr¹⁷ of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser¹⁶ and Thr¹⁷ with strikingly different half-maximal values (EC₅₀ = 4.5 ± 1.6 and 28.2 ± 1.4 nmol/l, respectively). The phosphorylation of Ser¹⁶ induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation (r = 0.96), whereas phosphorylation of Thr¹⁷ did not. Elevation of extracellular Ca²⁺ did not induce phosphorylation at Thr¹⁷; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr¹⁷ increased eightfold without additional effects on relaxation rate. Thr¹⁷ phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 µmol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser¹⁶ by cAMP-dependent protein kinase is the main regulator of -adrenergic-induced cardiac relaxation definitely preceding Thr¹⁷ phosphorylation and 2) the -adrenergic-mediated phosphorylation of Thr¹⁷ by Ca²⁺-calmodulin-dependent protein kinase required influx of Ca²⁺ through the L-type Ca²⁺ channel.

intact rat heart; relaxation; adenosine 3',5'-cyclic monophosphate-dependent protein kinase; calcium-calmodulin-dependent protein kinase; cross-signaling adenosine 3',5'-cyclic monophosphate/calcium

	INTRODUCTION

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PHOSPHOLAMBAN (PLB) regulates the affinity of cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase for Ca²⁺. Phosphorylation of PLB removes its inhibitory effects on SR Ca²⁺-ATPase, thereby accelerating Ca²⁺ uptake into SR vesicles to facilitate cardiac relaxation. Recently, the prominent role of PLB in the regulation of cardiac contractility was defined through genetic manipulation in a murine model (13, 21). In response to -adrenergic stimulation in vivo, PLB is phosphorylated at two adjacent amino acid residues, Ser¹⁶ and Thr¹⁷ (17, 43). In vitro studies have clearly demonstrated that Ser¹⁶ is exclusively phosphorylated by cAMP-dependent protein kinase (PKA) and Thr¹⁷ by Ca²⁺-calmodulin (CaM)-dependent protein kinase (CaM kinase II) (12, 33). Phosphorylation of each site occurs independently in vitro, although it is not clear whether the stimulatory effects of both phosphorylations on SR Ca²⁺ transport are additive (4, 12, 15).

The regulation and functional relevance of Thr¹⁷ phosphorylation to -adrenergic receptor stimulation of cardiac contractility in vivo are not clear. Phosphorylation of Ser¹⁶ and Thr¹⁷ has been demonstrated in isolated rat heart challenged with isoproterenol for 3 min (27, 41). Phosphate incorporation at Ser¹⁶ is faster than at Thr¹⁷ in guinea pig hearts exposed to 0.1 µmol/l isoproterenol (43). However, recent studies addressing -adrenergic-induced site-specific phosphorylation of PLB did not measure the time dependence of this process (27, 37, 41).

It has further been shown by several groups that increases in intracellular Ca²⁺ that bypass the cAMP signaling pathway are unable to stimulate PLB phosphorylation at Ser¹⁶ or Thr¹⁷ (19, 41, 43). Thus it was suggested that elevation of cAMP and Ser¹⁶ phosphorylation are prerequisites for the further, CaM kinase II-dependent phosphorylation of PLB (19, 43). This assumption has been elegantly verified by the use of transgenic mice overexpressing a PLB mutant in which Ser¹⁶ was replaced by Ala (22).

Recent findings have also postulated a membrane-linked CaM kinase II activity (2) that might be regulated by membrane voltage and Ca²⁺ influx in cardiomyocytes (44). Although data on a spatial CaM kinase II signaling system that may catalyze phosphorylation of PLB at Thr¹⁷ are not available, it is remarkable that L-type Ca²⁺ channel blockade affects this site of phosphorylation (27). The close apposition of the sarcolemmal L-type Ca²⁺ channel and the Ca²⁺ release channel of the SR (5, 16) favors the local increase of Ca²⁺ concentration ([Ca²⁺]). This increase may be accompanied by a local activation of CaM kinase II and may additionally accelerate Ca²⁺ channel inactivation (32). No findings concerning the cross signaling between the two Ca²⁺ flux mechanisms during -adrenergic stimulation have been previously reported with respect to site-specific PLB phosphorylation. More recently, a CaM kinase II-dependent acceleration of relaxation by PLB phosphorylation was questioned (11, 18), and it is unknown whether a Ca²⁺ release channel-sensitive CaM kinase II system linked to phosphorylation of PLB at Thr¹⁷ exists in the myocardium. The aims of the present study are to 1) elucidate differences between phosphorylation of PLB at Ser¹⁶ and Thr¹⁷ after -adrenergic stimulation, 2) evaluate the role of Ca²⁺ fluxes through sarcolemmal L-type Ca²⁺ channel and Ca²⁺ release channel of the SR for phosphorylation of PLB at Thr¹⁷, and 3) correlate phosphorylation of PLB at Ser¹⁶ and Thr¹⁷ to the lusitropic responses of the myocardium.

	MATERIALS AND METHODS

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Heart Perfusion

The animal experiments were performed in accordance with the recommendations of the Declaration of Helsinki and the internationally accepted principles concerning care and use of laboratory animals. Hearts from anesthetized (pentobarbital sodium, 25 mg/kg ip) and heparinized (500 U/kg) male Wistar rats (250-300 g body wt) were used for all experiments. Hearts were excised and immediately perfused in the Langendorff mode in an electronic apparatus (Hugo Sachs Electronik, March-Hugstetten, Germany). The modified Krebs-Henseleit perfusion medium was gassed with 95% O₂-5% CO₂ (pH 7.4, 37°C) and contained (in mmol/l) 118 NaCl, 4.7 KCl, 2.1 MgCl₂, 1.5 CaCl₂, 0.23 NaH₂PO₄, 24.7 NaHCO₃, 0.06 EDTA, and 11.1 glucose. Before each drug application the hearts were perfused for 10 min without stimulation, then stabilized for 20 min with a pacing rate of 340 beats/min and a perfusion pressure of 60 mmHg. A latex balloon, slightly larger than the ventricle cavity and without measurable pressure, was placed in the left ventricle. The balloon volume was initially adjusted to 14-16 mmHg. Left ventricular pressure (mmHg) and the maximal rate of left ventricular pressure development (+dP/dt, mmHg/s) and relaxation (dP/dt, mmHg/s) were measured via a Statham P23 DP pressure transducer at a 4-s intervals throughout the experiment (software from Hugo Sachs Electronik). The half-maximal relaxation time (_1/2, ms) was estimated from fast-speed recordings. Drugs were applied via an infusion pump (model 22, Harvard Apparatus).

Drug Application

Isoproterenol. Hearts were perfused with isoproterenol (Sigma Chemical, Deisenhofen, Germany) for 2 min unless otherwise indicated.

Perfusate [Ca²⁺] and A-23187. After the control perfusion with 1.5 mmol/l Ca²⁺, the perfusate [Ca²⁺] was switched to 0.5-6.0 mmol/l for 5 min. In a subset of experiments, 5 nmol/l isoproterenol was administered during the last 2 min of perfusion. In the case of the Ca²⁺ ionophore A-23187 (CalBiochem-Novabiochem, Bad Soden, Germany), hearts were preperfused with A-23187 (10 µmol/l) for 3 min before isoproterenol (5 nmol/l) addition.

Verapamil and nifedipine. Hearts were perfused for 10 min with 1 µmol/l verapamil (Sigma Chemical) or 1 µmol/l nifedipine (CalBiochem-Novabiochem), then perfused with 1 µmol/l isoproterenol for 2 min.

Ryanodine. Hearts were perfused for 5 min with 0.05, 1.0, and 10 µmol/l ryanodine (CalBiochem-Novabiochem), then with 1 µmol/l isoproterenol for 2 min.

8-Bromo-cAMP, forskolin, and 3-isobutyl-1-methylxanthine. Hearts were perfused with 8-bromo-cAMP (8-BrcAMP; Sigma Chemical; 0.5 and 2 mmol/l), forskolin (Sigma Chemical; 1 and 5 µmol/l), or 3-isobutyl-1-methylxanthine (IBMX; Sigma Chemical; 100 µmol/l) for 3 min. Drugs were dissolved according to the manufacturer's instructions. The ventricular myocardium was freeze-clamped after the experimental protocol with a Wollenberger clamp precooled in liquid nitrogen and stored at 80°C until further use.

Analysis of PLB Phosphorylation in Isolated Rat Hearts

Approximately 50 mg of freeze-clamped rat ventricular heart tissue were homogenized at 4°C with a homogenizer (Ultraturrax FU 5, Janke-Kunkel) at 50,000 rpm three times for 10 s each in 10 vol of buffer containing (in mmol/l) 5 histidine-HCl, pH 7.4, 10 EDTA, 50 Na₄P₂O₇, 25 NaF, 0.2 dithiothreitol, and 0.1 phenylmethylsulfonyl fluoride. The homogenates were stored at 80°C. For electrophoresis the homogenates were solubilized in sample buffer (50 mmol/l H₃PO₄, pH 6.8 adjusted with Tris, 5 mmol/l EDTA, 2% SDS, 1% mercaptoethanol, 10% glycerol, and a trace of bromphenol blue as tracking dye) and boiled at 95°C for 5 min. Twenty micrograms of homogenate protein per lane were resolved, using a 5% stacking gel, by urea SDS-PAGE with 7.5% (wt/vol) acrylamide gels (75 × 100 × 1.5 mm) according to the method of Swank and Munkres (36). The electrophoresis was carried out at 75 V. A prestained protein marker was used for molecular weight determination (Bio-Rad, Munich, Germany). Proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Serva) for 2 h at a current of 250 mA. Remaining binding sites were blocked in TBST (50 mmol/l Tris, 150 mmol/l NaCl, 0.1% Tween 20) containing 5% dried milk for 1 h and were incubated overnight at 4°C with the primary antibodies PS-16 and PT-17 (1:10,000 dilution) raised against phosphorylated PLB peptides (Phosphoprotein Research, Leeds, UK). The membranes were washed in TBST, incubated with a 1:15,000 dilution of peroxidase-conjugated goat anti-rabbit IgG (Dianova, Hamburg, Germany) for 1.5 h, and washed again in TBST. The immunoreaction was visualized with an enhanced chemiluminescent detection kit (Amersham-Buchler, Braunschweig, Germany), exposed to X-ray film, and quantified by scanning densitometry (PDI, New York, NY).

Analysis of Site-Specific PLB Phosphorylation in Rat Hearts In Situ

To investigate baseline PLB phosphorylation in vivo, Wistar rats were anesthetized, as described above, and artificially respirated. After chest and pericardium were opened, hearts were freeze-clamped in situ. The ventricles were stored at 80°C until analysis of site-specific PLB phosphorylation.

Standardization of Immunoreaction of Site-Specific PLB Phosphorylation

Peptides corresponding to PLB residues 9-19 (9RSAIRRASTIE19 = PLB11) according to Drago and Colyer (6), monophosphorylated at Ser¹⁶ (PS-PLB11) or Thr¹⁷ (PT-PLB11) or double-phosphorylated at Ser¹⁶/Thr¹⁷ (PS/PT-PLB11), were synthesized by Biotez (Berlin-Buch, Germany) without enzymatic site-specific PLB phosphorylation. The ability of the antibodies to differentiate between the two adjacent phosphorylation sites was checked by competitive immunoblotting in hearts exposed to 1 µmol/l isoproterenol. The immunoreaction of PS-16 antibody was completely abolished in the presence of 0.1 µmol/l PS-PLB11 but remained unaffected in the presence of the same concentration of PT-PLB11 or PS/PT-PLB11. The immunoreaction of PT-17 was specifically quenched by PT-PLB11 and PS/PT-PLB11 but was not influenced by the Ser¹⁶ phosphorylated peptide. Thus the antibody PT-17, characterized as above, detects both Thr¹⁷ phosphorylated PLB and the Ser¹⁶/Thr¹⁷ double-phosphorylated PLB in the heart tissue, but not Ser¹⁶ alone.

To standardize the detection of site-specific phosphorylated PLB, various amounts of protein were checked for proportional immunoresponses. There was a linear correlation between the antibody reaction and the amount of homogenate protein in the range of 5-35 µg. The resulting calibration curves for PS-16 and PT-17 were highly reproducible over all experiments, with correlation coefficients (r) of 0.98 ± 0.01 and 0.97 ± 0.01, respectively. For internal standardization, ovalbumin-coupled PLB peptides (PS-PLB11 and PT-PLB11) were routinely loaded together with tissue extracts on the gels. These standards migrate as a 50-kDa band. Because the obtained signal intensity was linear to the amount of PS-PLB11 and PT-PBL11, the amount of phosphorylated PLB could be controlled over different sets of experiments. The amount of Ser¹⁶ and Thr¹⁷ phosphorylated PLB was expressed as optical density (arbitary units; see Figs. 3 and 4) or as percentage of PLB phosphorylation obtained from hearts exposed to 1 µmol/l isoproterenol, referred to as maximum (see Figs. 2 and 5). Data sets from one experimental run are presented as optical density (OD × mm²) of immunological signals (Fig. 1, see Figs. 6 and 7).

View larger version (49K): [in this window] [in a new window]   Fig. 1.   Time course of isoproterenol (Iso)-induced phosphorylation of phospholamban (PLB) at Ser16 (PSer16-PLB) and Thr17 (PThr17-PLB) in perfused rat heart. Isolated hearts were perfused with 5 nmol/l () or 1 µmol/l () isoproterenol, freeze-clamped, and processed for site-specific phosphorylation of PLB. A: original autoradiograms of immunoblots (each lane represents a separate isolated rat heart). B: densitometric evaluation of Western blots represented in A. Data are expressed as optical density (OD × mm2).

Other Assays

cAMP levels were analyzed in neutralized TCA tissue extracts (8) purified by column chromatography (24). Soluble and particulate fractions of PKA were prepared by low-speed centrifugation (6,000 g for 5 min) and estimated according to Murray et al. (28). The PKA activity is expressed as the activity ratio of malantide phosphorylation in the absence and presence of 2.8 µmol/l cAMP. The protein concentration was determined by the method of Lowry et al. (20) with ovalbumin as standard.

Statistics

Values are means ± SE. Statistical significance was determined by the Student's t-test for unpaired data or ANOVA when appropriate. P < 0.05 was considered significant. Linear and nonlinear regression of dose-response relationships and statistical analysis were performed using the Prism II software Graphpad.

	RESULTS

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Time Course of Isoproterenol-Induced Ser¹⁶ and Thr¹⁷ PLB Phosphorylation

Figure 1 represents immunoblots and their densitometric evaluations of the time course of -adrenergic effects on site-specific PLB phosphorylation in perfused rat heart. A significant increase of Ser¹⁶ phosphorylation occurred within 0.5 min of exposure of the hearts to 5 nmol/l isoproterenol and was completed within the 1st min. In contrast, Thr¹⁷ phosphorylation was delayed after this low dose of isoproterenol. However, no difference in the generation of Ser¹⁶ and Thr¹⁷ phosphorylated PLB was observed at 1 µmol/l isoproterenol. In the absence of -adrenergic stimulation, no Ser¹⁶ and Thr¹⁷ PLB phosphorylation (<1% of maximum) was detected in the perfused rat heart.

Dose Response of Site-Specific PLB Phosphorylation to Isoproterenol

The generation of Ser¹⁶ and Thr¹⁷ PLB phosphorylation in response to isoproterenol in the range 1 nmol/l-1 µmol/l is given in Fig. 2. Maximal Ser¹⁶ and Thr¹⁷ PLB phosphorylation induced by 1 µmol/l isoproterenol was 18.2 ± 1.7 and 3.0 ± 0.2 arbitrary units (n = 7), respectively, 2 min after drug application.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Dose response of site-specific phosphorylation of PLB in intact heart to isoproterenol. Homogenate protein (20 µg) of rat hearts, perfused with various isoproterenol concentrations for 2 min, was resolved by urea SDS-PAGE, transferred to polyvinylidine difluoride membrane, and incubated with antibodies PS-16 and PT-17. Standardized phosphorylation of PLB derived from 1 µmol/l isoproterenol-treated hearts was regarded as 100%. Values are means ± SE of 3-5 different hearts. , Phosphorylated PLB at Ser16; , phosphorylated PLB at Thr17. Inset: autoradiograms of representative immunoblots.

Although increasing concentrations of isoproterenol caused a dose-dependent Ser¹⁶ and Thr¹⁷ phosphorylation, there was a striking difference in sensitivity to the -adrenergic agonist. Isoproterenol concentrations that produced half-maximal Ser¹⁶ and Thr¹⁷ phosphorylation (EC₅₀) were calculated to be 4.5 ± 1.6 and 28.2 ± 1.4 nmol/l, respectively (P < 0.01).

The relationship between isoproterenol-induced site-specific PLB phosphorylation and changes in cardiac relaxation are shown in Fig. 3 (dP/dt) and Fig. 4 (_1/2). Only Ser¹⁶ phosphorylation correlated with the lusitropic effect of rising isoproterenol (r = 0.93). In contrast, Thr¹⁷ phosphorylation remained extremely low in hearts with lusitropic responses of dP/dt <3,500 mmHg/s equal to _1/2 of 27.8 ms. Within this interval, corresponding to an isoproterenol concentration of 10 nmol/l, there were no obvious changes in Thr¹⁷ phosphorylation. Furthermore, -adrenergic stimulation resulted in an increased positive inotropic cardiac response. The +dP/dt was enhanced from a baseline level of 1,263 ± 87 mmHg/s to 2,035 ± 250, 3,443 ± 198, and 4,469 ± 181 mmHg/s, respectively, at 1, 5 and 1,000 nmol/l isoproterenol.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Relationship between phosphorylated PLB at Ser16 (A) and Thr17 (B) and left ventricular relaxation rate (dP/dt) of individual hearts. dP/dt was recorded during perfusion of hearts with rising isoproterenol concentration. In the same hearts, phosphorylation of PLB at Ser16 and Thr17 was analyzed as described in Fig. 2 legend. Standardized phosphorylation of PLB is represented as optical density (OD × mm2).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Relationship between shortening of relaxation time and phosphorylation of PLB at Ser16 (A) and Thr17 (B) induced by different cAMP-related agents. Rats were perfused with indicated drugs and freeze-clamped for processing of site-specific phosphorylation of PLB. Standardized phosphorylation of PLB derived from 1 µmol/l isoproterenol-treated hearts was regarded as 100%. Values are means ± SE of 4-6 different rat hearts perfused under control condition (×) or with isoproterenol [5 (), 10 (), 50 (), and 1,000 () µmol/l], forskolin [1 () and 5 () µmol/l], IBMX (100 µmol/l, ), or 8-bromo-cAMP [0.5 () and 2 () mmol/l]. r, Correlation coefficient.

Isoproterenol-Associated Alterations in cAMP Level and Activation of PKA

Exposure of rat hearts to 1 µmol/l isoproterenol resulted in a 3.7-fold rise in cAMP (predrug value = 4.0 ± 0.3 pmol/mg protein, n = 8). This was accompanied by an activation of soluble PKA (expressed as activity ratio = cAMP/+cAMP) from 0.08 ± 0.01 to 0.55 ± 0.04 (P < 0.01). The particulate PKA activity rose from 0.21 ± 0.05 to 0.36 ± 0.02 (P < 0.05). The correlation coefficients between isoproterenol-induced Ser¹⁶ phosphorylation and increases in cAMP and PKA were 0.71 and 0.72, respectively.

-Adrenoceptor-Independent Stimulation of PLB Phosphorylation

Inasmuch as site-specific phosphorylation of PLB at Ser¹⁶ and Thr¹⁷ differs significantly after -adrenergic stimulation, we reestimated the effects of 8-BrcAMP, forskolin, and IBMX on Ser¹⁶ and Thr¹⁷ phosphorylation. In our hands, all drugs elicited significant increases in relaxation rate (Table 1) and shortening of relaxation time (Fig. 4), which was associated to a variable extent with PLB phosphorylation. The increase of +dP/dt obtained with 8-BrcAMP, forskolin, and IBMX was 2,633 ± 135, 3,565 ± 386, and 3,444 ± 176 mmHg/s, respectively. Ser¹⁶ was the predominantly phosphorylated amino acid residue under all conditions studied (Fig. 4). No significant Thr¹⁷ phosphorylation was detectable in the presence of 2 mmol/l 8-BrcAMP and 100 µmol/l IBMX. However, 5 µmol/l forskolin resulted in Ser¹⁶ as well as Thr¹⁷ phosphorylation.

View this table: [in this window] [in a new window]   Table 1.   Effect of cAMP-elevating interventions on +dP/dt, dP/dt, and site-specific phosphorylation of PLB

Effect of Extracellular Ca²⁺ and A-23187 on PLB Phosphorylation

To examine the influence of extracellular [Ca²⁺] on the generation of Ser¹⁶ and Thr¹⁷ phosphorylated PLB, hearts were exposed to 0.5, 1.5, 4.5, and 6.0 mmol/l Ca²⁺ in the absence and presence of a submaximal concentration (5 nmol/l) of isoproterenol. In the absence of -adrenergic stimulation, no PLB phosphorylation was detectable (data not shown). Whereas isoproterenol induces PLB phosphorylation at Ser¹⁶ independently of [Ca²⁺], Thr¹⁷ phosphorylation correlates with rising extracellular [Ca²⁺] (Fig. 5). At 6 mmol/l perfusate Ca²⁺ and 5 nmol/l isoproterenol, the contractile parameter +dP/dt was significantly higher than at 1.5 mmol/l Ca²⁺ (4,752 ± 190 vs. 3,443 ± 198 mmHg/s). Reducing [Ca²⁺] from 1.5 mmol/l to 1.0 and 0.5 mmol/l prolonged _1/2 from 39.1 ± 0.6 ms to 46.0 ± 2.0 and 52.5 ± 2.5 ms, respectively, whereas higher [Ca²⁺] (4.5 and 6.0 mmol/l) were without effects on _1/2. Isoproterenol (5 nmol/l) hastened _1/2 to 35.0 ± 1.3, 30.5 ± 0.5, and 28.3 ± 1.7 ms, respectively, at 0.5, 1.0, and 1.5 mmol/l Ca²⁺. Under these conditions, Ser¹⁶ PLB phosphorylation prevailed in comparison to Thr¹⁷. However, at high [Ca²⁺] (4.5 and 6.0 mmol/l), _1/2 was not affected (28.3 ± 1.7 vs. 27.5 ± 0.1 and 27.5 ± 1.4 ms, respectively) although Thr¹⁷ PLB phosphorylation was severalfold increased. Additionally, Thr¹⁷ phosphorylation was not observed in the presence of the Ca²⁺ ionophore A-23187 in the absence or presence of -adrenergic stimulation (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Dependence of phosphorylation of PLB at Ser16 (A) and Thr17 (B) on isoproterenol-stimulated rat heart on perfusate Ca2+. After 30 min of perfusion at 1.5 mmol/l, Ca2+ concentration was changed to 0.5, 1.5, 4.5, or 6.0 mmol/l for 3 min, then 5 nmol/l isoproterenol for another 2 min. Samples were analyzed as described in Fig. 2 legend. Values were calculated to standardized phosphorylation of PLB obtained at 1 µmol/l isoproterenol and 1.5 mmol/l Ca2+. Values are means ± SE of 3-6 different hearts. * P < 0.05 vs. 6.0 mmol/l Ca2+.

Effect of L-Type Ca²⁺ Channel and Ca²⁺ Release Channel on PLB Phosphorylation

To assess whether the Ca²⁺-dependent phosphorylation of Thr¹⁷ PLB during -adrenergic stimulation is related to Ca²⁺ influx through sarcolemmal Ca²⁺ channels or Ca²⁺-induced Ca²⁺ release from the SR, hearts were perfused with verapamil, nifedipine, or ryanodine. Figure 6 shows that ryanodine at low concentrations (0.05 and 1.0 µmol/l) did not significantly modify isoproterenol-induced Thr¹⁷ phosphorylation, whereas in the presence of 10 µmol/l ryanodine this phosphorylation was decreased 28%. Ser¹⁶ phosphorylation remained unaffected under these conditions. As summarized in Table 2, ryanodine did not affect the rate of contraction at low concentration but decreased this parameter at 1 and 10 µmol/l to 25 and 12% of control, respectively. Relaxation time rises with increasing drug concentration up to 170% of untreated hearts. However, isoproterenol (1 µmol/l) elicited positive inotropic and lusitropic effects under these conditions (Table 2).

View larger version (63K): [in this window] [in a new window]   Fig. 6.   Influence of ryanodine on isoproterenol-induced site-specific phosphorylation of PLB in rat hearts. Hearts were pretreated without and with various concentrations of ryanodine for 5 min, then with isoproterenol (1 µmol/l) for 2 min. Tissue samples were processed for site-specific PLB phosphorylation as described in Fig. 2 legend. Each lane represents 1 separately treated heart. Bars represent means ± SE from 3 separate experiments. Densitometric evaluation of Western blots (inset) are expressed as optical density (OD × mm2) of immunological signals. * P < 0.05 vs. isoproterenol without ryanodine. Ctr, control heart.

View this table: [in this window] [in a new window]   Table 2.   Positive inotropic and lusitropic effects of isoproterenol on perfused rat hearts without and with ryanodine, nifedipine, or verapamil

The isoproterenol-induced Thr¹⁷ phosphorylation of PLB was completely abolished in the presence of 1 µmol/l verapamil or nifedipine without changes in Ser¹⁶ phosphorylation (Fig. 7). In the presence of these compounds, +dP/dt was diminished and the time of relaxation was increased (Table 2).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Influence of L-type Ca2+ channel inhibition on isoproterenol-induced phosphorylation of PLB at Ser16 (A) and Thr17 (B) in rat hearts. Left: autoradiographs of immunoblots representing effects of isoproterenol (Iso, 1 µmol/l) and L-type Ca2+ channel blockers [nifedipine (Nife, 1 µmol/l) and verapamil (Vera, 1 µmol/l)] in presence of isoproterenol on phosphorylation of PLB at Ser16 and Thr17. For details of drug perfusion protocol and for analytic details see MATERIALS AND METHODS. Right: densitometric evaluation of immunological signals represented on left. Values are means ± SE of optical density (OD × mm2) of 2 - 3 individual hearts.

Site-Specific Phosphorylation of PLB in Rat Hearts In Situ

In isolated, perfused rat hearts the level of phosphorylated PLB under control conditions was not detectable. Because the heart in situ is permanently controlled by the central nervous system, in particular by sympathetic activity, we additionally analyzed the cardiac PLB phosphorylation in anesthetized and artificially respirated rats. On average, 14.8 ± 0.4% of Ser¹⁶ PLB and 3.5 ± 0.1% of Thr¹⁷ PLB were phosphorylated compared with the PLB phosphorylation obtained from isolated hearts exposed to 1 µmol/l isoproterenol (n = 5). The level of cAMP was assayed to be 4.1 ± 0.2 pmol/mg protein, and soluble and particulate PKA activity ratios were measured to be 0.10 ± 0.01 and 0.21 ± 0.05, respectively, under this condition.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study addressed the phosphorylation of PLB at Ser¹⁶ and Thr¹⁷ in -adrenergic-stimulated intact rat hearts. We have particularly focused on the cross talk of cAMP and Ca²⁺ signaling and the contribution of different cardiac Ca²⁺ transporting systems to phosphorylation at Thr¹⁷ .

Specificity of PS-16 and PT-17 Antibodies

Further characterization of the commercially available phosphorylation site-specific antibodies to PLB raised questions as to the specificity of PT-17 to monophosphorylated PLB at Thr¹⁷. Only recently have the properties of antibody PT-17 been confirmed by Colyer and co-workers (personal communication). From our data we conclude that antibody PT-17 mainly reflects the generation of monophosphorylated PLB, inasmuch as neither enhancement (Fig. 5) nor inhibition of Thr¹⁷ phosphorylation (i.e., after Ca²⁺ channel blockade; Fig. 7) influenced the signal intensity for Ser¹⁶ PLB. The amount of double-phosphorylated PLB is thus rather small in comparison to the monophosphorylated species, which is consistent with the hypothesis that distinct populations of PLB are preferentially phosphorylated by PKA or CaM kinase II (9). However, Ser¹⁶/Thr¹⁷ PLB double-phosphorylated species were electrophoretically characterized in guinea pig hearts after -adrenergic stimulation (43). More specific tools are required to clarify whether the monophosphorylation of PLB at Thr¹⁷ can indeed occur.

Basal Level of PLB Phosphorylation in Hearts

In our hands, in contrast to former studies (10, 27, 34, 40, 43), the double phosphorylation of PLB at Ser¹⁶ and Thr¹⁷ is not detectable in the intact perfused rat hearts under basal conditions. These discrepancies might at least in part be attributable to different experimental conditions for cardiac tissue extraction and fractionation as well as analyses of PLB phosphorylation. To clarify these discrepancies, we analyzed PLB phosphorylation in cardiac tissue freeze-clamped in situ, reflecting more precisely the situation in the beating heart, to determine modulatory mechanisms of the central nervous system (17). Thus the steady-state level of Ser¹⁶ PLB phosphorylation, ~15% of total Ser¹⁶, should account for the sympathetic control of the myocardium.

Ser¹⁶ and Thr¹⁷ PLB Phosphorylation in Response to Isoproterenol

Here we report a time-delayed phosphorylation of PLB at Thr¹⁷ that is apparent only at submaximal doses of isoproterenol. Former studies in isolated rat and guinea pig hearts measured such phosphorylation 3-4 min after exposure to isoproterenol (27, 37) or used ³²P prelabeling and phosphoamino acid analysis after partial acid hydrolysis (43). Consistent with former results, it is shown that Ser¹⁶ precedes Thr¹⁷ phosphorylation for submaximal -adrenergic stimulation (Fig. 2). However, we were not able to detect this time delay at a high dose of the drug.

Increasing isoproterenol induces phosphorylation of PLB at Ser¹⁶ and Thr¹⁷ in a dose-dependent manner. EC₅₀ for Ser¹⁶ PLB phosphorylation is consistent with earlier studies (14). Strikingly, the present results demonstrate that phosphorylation of PLB at Thr¹⁷ is more than fivefold less sensitive to the -adrenergic agonist isoproterenol, which is in contrast to the findings described by Mundiña et al. (27), who obtained no differences in the sensitivity of the Ser¹⁶ and Thr¹⁷ phosphorylation. This may be explained, at least in part, by the use of different times of exposure to isoproterenol and different experimental conditions used to analyze PLB phosphorylation.

Recent findings have indicated that phosphorylation of Ser¹⁶ is a prerequisite for Thr¹⁷ phosphorylation, and prevention of Ser¹⁶ phosphorylation results in attenuation of the -agonist-mediated cardiac responses (22, 23). Our study accordingly demonstrates that phosphorylation of PLB at Ser¹⁶ is strongly related to the -adrenoceptor-mediated lusitropic response and, therefore, predominates in modulating SR Ca²⁺-ATPase activity. The significance of Ser¹⁶ phosphorylation in regulating intracellular Ca²⁺ is further stressed by our new data on the PLB phosphorylation status in situ, favoring the Ser¹⁶ residue for a modulation of Ca²⁺ uptake into the SR. Thr¹⁷ PLB phosphorylation is only moderately involved in this regulation, which is in line with results from a transgenic approach (22). The phosphorylation of this residue may be more relevant under pathophysiological conditions (i.e., acidosis), as recently shown (10, 42).

Furthermore, we show that cAMP-elevating agents such as forskolin and IBMX, as well as 8-BrcAMP, accelerate relaxation and increase phosphorylation of PLB at Ser¹⁶ but only moderately affect Thr¹⁷ phosphorylation. The responses to IBMX (100 µmol/l) as well as to forskolin at the level of PLB phosphorylation are consistent with earlier data (30, 34) in which a ³²P-labeling technique was used. Thus the inhibition of SR protein phosphatase I activity by cAMP-mediated phosphorylation of inhibitor-1 (29), which may maintain phosphorylation at Thr¹⁷, as suggested recently (27), is not effective under these conditions.

Ca²⁺ Cycling Systems and Phosphorylation at Thr¹⁷

The present data confirm earlier observations that in the absence of -adrenergic stimulation an increase in extracellular Ca²⁺ does not induce phosphorylation of PLB in the intact heart (19, 27, 40). Only in the presence of isoproterenol does the phosphorylation at Thr¹⁷ occur in response to extracellular Ca²⁺. Stimulation of -adrenoceptors leads to an enhanced influx of Ca²⁺ through the L-type Ca²⁺ channel mediated by PKA-dependent phosphorylation or interaction with G proteins (7, 39). There is evidence that an increase in Ca²⁺ influx results in an activation of CaM kinase II localized closely to the sarcolemmal membranes in cardiomyocytes (44). After -adrenergic stimulation of isolated rat heart, an autophosphorylation of CaM kinase II is demonstrated (1), which converts the enzyme to an autonomous kinase (2). Interestingly, BAY K 8846, a L-type Ca²⁺ channel activator, induces phosphorylation at Thr¹⁷ in cultivated cardiomyocytes also in the absence of isoproterenol (unpublished data). Therefore, we propose that a local rise in intracellular Ca²⁺ is followed by a spatial CaM kinase II activation and phosphorylation of PLB at Thr¹⁷ in the SR adjacent to the sarcolemma. Blockade of L-type Ca²⁺ channel activity by verapamil or nifedipine interrupts this phosphorylation, whereas the PKA-catalyzed phosphorylation at Ser¹⁶ is unaffected.

In our experiments, ryanodine produces dose-dependent negative inotropic responses and an increase in the duration of contraction in the isolated heart, as shown earlier (35, 38). Additionally, the end-diastolic pressure rises with increased drug concentration, indicating an opening of the SR Ca²⁺ release channels, then a depletion of the SR Ca²⁺ by ryanodine (25, 31). We observed a significant reduction in phosphorylation at Thr¹⁷ at higher ryanodine concentration (Fig. 5). Therefore, we suggest that Ca²⁺ released from the SR participates in the activation of CaM kinase II.

The function of phosphorylation of PLB at Thr¹⁷ in vivo is not definitely clear. Inasmuch as KN-93, a CaM kinase II inhibitor, was shown to reduce the SR Ca²⁺ uptake and the decay in cytosolic Ca²⁺ concentration in intact cardiomyocytes, these data have been related to PLB phosphorylation (3, 26), although the phosphorylation status was not elucidated. More recent studies demonstrate a CaM kinase II-mediated acceleration of cardiac relaxation in PLB knockout mice (18) to suggest other targets for CaM kinase II (45).

In summary, the PKA-dependent phosphorylation of PLB at Ser¹⁶ is preferentially involved in the acceleration of cardiac relaxation after -adrenergic stimulation or other interventions that elevate cAMP levels and predominates over phosphorylation at Thr¹⁷ in rat heart. Phosphorylation of PLB at Thr¹⁷ required Ca²⁺ influx through the L-type Ca²⁺ channel in the presence of -adrenergic receptor occupation as well as increased PKA activity. In addition, the Ca²⁺-triggered Ca²⁺ release is involved in the regulation of CaM kinase II activation. PLB phosphorylated by CaM kinase II probably reflects a distinct part of the SR that is less involved in the acute and transiently occurring -adrenergic augmentation of cardiac relaxation.

	ACKNOWLEDGEMENTS

The authors thank Inge Beyerdörfer and Donathe Vetter for excellent technical assistance, Drs. Roland Willenbrock and Martin Philipp (Franz Volhard Clinic, Humboldt University, Berlin) for expert help in the performance of in situ studies, and Dr. H. B. Smith for critical reading of the manuscript.

	FOOTNOTES

This work was supported in part by the Deutsche Forschungsgemeinschaft. M. Kuschel and P. Hempel were supported by grants from Sonnenfeld-Stiftung, Berlin.

Present address of M. Kuschel: Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Bartel, Max Delbrück Center for Molecular Medicine, Robert Rössle Str. 10, 13125 Berlin-Buch, Germany (E-mail: egkrause{at}mdc-berlin.de).

Received 15 April 1998; accepted in final form 12 January 1999.

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