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J. Biol. Chem., Vol. 275, Issue 49, 38938-38943, December 8, 2000

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A Single Site (Ser¹⁶) Phosphorylation in Phospholamban Is Sufficient in Mediating Its Maximal Cardiac Responses to -Agonists^*

Guoxiang Chu, James W. Lester, Karen B. Young, Wusheng Luo, Jing Zhai, and Evangelia G. Kranias

From the Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575

Received for publication, May 12, 2000, and in revised form, September 13, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phospholamban (PLB) can be phosphorylated at Ser¹⁶ by cyclic AMP-dependent protein kinase and at Thr¹⁷ by Ca²⁺-calmodulin-dependent protein kinase during -agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr¹⁷ phosphorylation and attenuation of the -agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Cardiac myocyte basal mechanics and Ca²⁺ kinetics were similar among the three groups. Isoproterenol stimulation was associated with phosphorylation of both Ser¹⁶ and Thr¹⁷ in wild-type PLB and Ser¹⁶ phosphorylation in T17A mutant PLB, whereas there was no detectable phosphorylation of S16A mutant PLB. Phosphorylation of Ser¹⁶ alone in T17A mutant PLB resulted in responses of the mechanical and Ca²⁺ kinetic parameters to isoproterenol similar to those in wild-type myocytes, which exhibited dual-site PLB phosphorylation. However, those parameters were significantly attenuated in the S16A mutant myocytes. Thus, Ser¹⁶ in PLB can be phosphorylated independently of Thr¹⁷ in vivo, and phosphorylation of Ser¹⁶ is sufficient for mediating the maximal cardiac responses to -adrenergic stimulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phospholamban (PLB)¹ is a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLB is an inhibitor of the affinity of SERCA2 for Ca²⁺, and phosphorylation of PLB during -adrenergic stimulation relieves its inhibitory effects on SERCA2 (1, 2). The physiological importance of PLB has been elucidated through the generation of genetically engineered mouse models with alterations in cardiac PLB expression levels (3, 4). Ablation of PLB was associated with significantly enhanced Ca²⁺ affinity of SERCA2 and myocardial performance (3, 5, 6). The elevated basal contractile parameters could be minimally stimulated by -agonists (3, 7), whereas there were no alterations in the -receptor signaling pathway or the phosphorylation states of other major cardiac phosphoproteins (8). On the other hand, overexpression of PLB was associated with significant depression of contractile parameters, which could be reversed upon phosphorylation of PLB during -agonist stimulation (4). These results indicate that PLB is a key regulator of cardiac function and a prominent mediator of the -adrenergic effects in the myocardium.

In vitro studies have shown that PLB can be phosphorylated on Ser¹⁰ by protein kinase C, Ser¹⁶ by cAMP-dependent protein kinase (PKA), and Thr¹⁷ by Ca²⁺-calmodulin-dependent protein kinase (CaMKII) (1, 9, 10). Each phosphorylation is associated with stimulation of the apparent affinity of SERCA2 for Ca²⁺. In vivo studies have shown that only Ser¹⁶ and Thr¹⁷ are phosphorylated in cardiac myocytes or perfused hearts (11, 12), whereas phosphorylation of PLB by protein kinase C has not been detected in vivo. Phosphorylation of PLB by PKA and CaMKII occurs during -agonist exposure, although the relative contribution of each phosphorylation to the cardiac stimulatory effects is not presently clear. Each phosphorylation appears to occur independently of the other (13-16). Some studies have reported additive effects of PKA and CaMKII phosphorylation of PLB on SR Ca²⁺ transport (13, 14, 17, 18), whereas others (16, 19) have proposed that maximal stimulation of the Ca²⁺ pump occurs by phosphorylation at a single site, and additional phosphorylation of the other site does not further stimulate the pump activity.

Several in vivo studies have shown that Ser¹⁶ phosphorylation or dephosphorylation precedes Thr¹⁷ phosphorylation or dephosphorylation during exposure or removal of -agonist stimulation, respectively (7, 11, 12, 20-22). Furthermore, increases in intracellular Ca²⁺ to levels higher than those obtained by isoproterenol stimulation, elicited by changes in the perfusate Ca²⁺ or agents that cause inotropic stimulation, failed to result in phosphorylation of PLB (23). These results led to the suggestion that Thr¹⁷ phosphorylation has as a prerequisite phosphorylation of Ser¹⁶ in vivo. However, other studies, using phosphorylation site-specific antibodies for PLB (24, 25), indicated that increases in the Ca²⁺ supply (3.85 mM) to the heart, accompanied by inhibition of protein phosphatase-1 or acidosis (pH 6.8), may facilitate phosphorylation of Thr¹⁷ and acceleration of the contraction and relaxation rates. Thus, the findings on the physiological significance of Ser¹⁶ and Thr¹⁷ phosphorylation appear controversial. A major limitation in previous studies has been the use of preparations expressing wild-type PLB with both phosphorylation sites intact, which presents difficulties in discriminating the independent and/or relative contribution of each phosphorylation site to cardiac function. This limitation was recently overcome by the availability of a PLB knockout mouse, which, in combination with transgenesis, provides a unique system to delineate the physiological role of each of the phosphorylation sites, Ser¹⁶ and Thr¹⁷, in PLB. In a previous study, a PLB mutant, in which Ser¹⁶ was replaced by Ala, was expressed in the hearts of the null background, and characterization studies indicated the lack of Thr¹⁷ phosphorylation and attenuation of the -agonist stimulatory effects in Langendorff-perfused hearts (7). It was suggested that phosphorylation of Ser¹⁶ may be a prerequisite for Thr¹⁷ phosphorylation during -agonist stimulation. Although that report demonstrated the importance of Ser¹⁶ phosphorylation in PLB, the contribution of this site relative to Thr¹⁷ phosphorylation in the stimulatory effects of -agonists in vivo is not known. Thus, the present study was designed to determine the role of Ser¹⁶ phosphorylation in vivo. A PLB mutant in which Thr¹⁷ was replaced by Ala was expressed in the hearts of the null background, and the following questions were addressed: 1) Can phosphorylation of Ser¹⁶ in PLB occur independently of Thr¹⁷ in vivo? 2) What is the relative contribution of Ser¹⁶ phosphorylation to the maximal stimulatory effects of -agonists? and 3) Are the stimulatory effects of Ser¹⁶ and Thr¹⁷ phosphorylation additive? Our findings indicate that phosphorylation of Ser¹⁶ in PLB can occur independently of Thr¹⁷ in vivo, and this single-site phosphorylation is sufficient in mediating the maximal mechanical and Ca²⁺-kinetic stimulatory effects of isoproterenol in cardiac myocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PLB Phosphorylation Site-specific Mutagenesis-- PLB phosphorylation site-specific mutation of Thr¹⁷ to Ala (ACTGCT) was introduced into the PLB cDNA by polymerase chain reaction (PCR) methodology as described previously (7, 26). Briefly, a 0.9-kb SalI fragment of PLB cDNA and SV40 poly(A) signal sequence was subcloned into a pBluescript SKII() vector (Stratagene), with T3 and T7 primer sites flanking the insert. PCR mutagenesis was performed by two consecutive PCR reactions using two sets of primers. The first PCR amplification was designed to generate a desired mutant PLB minor product using the subclone plasmid DNA as a template, along with a 3'-end T7 primer and a 5'-end mutant primer (5'-CTATCAGGAGAGCCTCCGCTATTGAAATGCC-3') corresponding to nucleotides 32-62 of the PLB coding sequence. An aliquot of the first PCR product and the T3 and T7 primers were used for the second PCR to amplify the full-length insert containing the desired mutation in PLB cDNA. The final amplified product was excised and purified for sequence analysis and subsequently re-subcloned into the SalI site of PLB overexpression vector pIBI 31, which has been successfully used to generate wild-type PLB-overexpressing transgenic mice in our laboratory (4).

Generation of Transgenic Mice Expressing T17A Mutant PLB in the Null Background-- The -myosin heavy chain (-MHC) promoter was used to direct cardiac-specific expression of the mutant PLB in the null background. The mutant mice expressing T17A PLB in the PLB knockout mouse heart (KO+T17A) were generated in the same manner as transgenic mice expressing wild-type (KO+WT) or S16A mutant PLB (KO+S16A) in the null background (7). Briefly, the entire expression construct was composed of the -MHC promoter (5.5 kb), the PLB coding region with the T17A mutation (0.65 kb), and the SV40 poly(A) signal sequence (0.25 kb). The KpnI-HindIII fragment of vector pIBI 31 containing the entire expression construct was released and purified for pronuclear microinjection. The founder mice harboring the PLB mutant transgene were identified by PCR analysis using primers corresponding to the -MHC promoter (primer 1, 5'-CACATAGAAGCCTAGCCCACAC-3') and the PLB-encoding sequence (primer 2, 5'-GATTCTGACGTGCTTGCTGAGG-3') with a resultant PCR product of 150 bp. Transgenic mice with the desired mutation in the null background were identified using PCR methodology and confirmed by Southern blot analysis of genomic DNA isolated from tail biopsies. The transgene expression, driven by the cardiac-specific -MHC promoter, was determined by Northern analysis of total RNA from the transgenic mouse hearts. A ~0.5-kb random-primed labeled PLB cDNA was used as a probe for hybridization.

Quantitative Immunoblotting of PLB and SERCA2-- Hearts from transgenic mice were homogenized in buffer (pH 7.0) containing imidazole (10 mM), sucrose (300 mM), dithiothreitol (1 mM), sodium metabisulfite (1 mM), and phenylmethylsulfonyl fluoride (0.3 mM). The cardiac homogenates were incubated with equal volume of loading buffer (20% glycerol, 2% -mercaptoethanol, 4% SDS, 0.001% bromphenol blue, and 130 mM Tris-Cl, pH 6.8), subjected to 13% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (27), and blotted onto nitrocellulose membranes (Schleicher & Schuell). The membranes were then reacted with a mouse monoclonal antibody to phospholamban or SR Ca²⁺-ATPase (Affinity Bioreagents Inc., Golden, CO). After washing out the unbound antibody with Tris-buffered saline (10 mM Tris-HCl and 150 mM NaCl, pH 7.8), the blots were incubated with an alkaline phosphatase-conjugated anti-mouse secondary antibody (1:1000; Cappel Division of Organon Teknika). The phospholamban and SR Ca²⁺-ATPase protein bands were visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates for the alkaline phosphatase reaction, and the signals were analyzed by laser densitometry using ImageQuant software.

In Vitro Phosphorylation of PLB-- In vitro phosphorylation of PLB was performed using cardiac homogenates from mutant mice, as described previously (7). PKA phosphorylation of the cardiac homogenates (60 µg) was carried out at 30 °C in 30 µl of reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 10 mM MgCl₂, 5 mM NaF, 0.5 mM EGTA, 0.1 mM ATP, 20 µCi of [-³²P]ATP, and 45 units of the PKA catalytic subunit. For endogenous CaMKII phosphorylation of the cardiac homogenates, 0.5 mM CaCl₂, 2 µM calmodulin, and 1 µM protein kinase inhibitor peptide 5-24 amide (Sigma) were added to the above reaction mixture. Reactions were terminated with 30 µl of SDS sample buffer after a 2-min (PKA) or 5-min (CaMKII) incubation, which was associated with optimal phosphate incorporation in PLB. Thirty µg of protein was subjected to 15% SDS-PAGE and autoradiography.

Isolation of Mouse Left Ventricular Myocytes-- Isolation of mouse left ventricular myocytes was carried out as described previously (27, 28). Briefly, mouse hearts were excised from anesthetized (pentobarbital sodium, 70 mg/kg, i.p.) adult mice, mounted in a Langendorff perfusion apparatus, and perfused with Ca²⁺-free Tyrode solution at 37 °C for 3 min. The normal Tyrode solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose, and 5 mM HEPES, pH 7.4. Perfusion was then switched to the same solution containing 75 units/ml type 1 collagenase (Worthington), and perfusion continued until the heart became flaccid (~10-15 min). The left ventricular tissue was excised, minced, pipette-dissociated, and filtered through a 240-µm screen. The cell suspension was then sequentially washed in 25, 100, and 200 µM Ca²⁺-Tyrode and resuspended in 1 mM Ca²⁺-Tyrode for further analysis.

Measurements of Cell Shortening and Ca2+ Transients-- Cell shortening and Ca²⁺ transients were simultaneously measured from the same cardiomyocytes. To obtain intracellular Ca²⁺ signals, cells were incubated with the acetoxymethyl ester form of fura-2 (fura-2/AM; 2 µM) for 30 min at room temperature and resuspended in 1.0 mM Ca²⁺-Tyrode solution. The myocyte suspension was placed in a Plexiglas chamber, which was positioned on the stage of an inverted epifluorescence microscope (Nikon Diaphot 200), and perfused with normal Tyrode solution at room temperature (22 °C-23 °C). Myocyte contraction was field-stimulated by a Grass S5 stimulator (0.5 Hz, square waves), and contractions were videotaped and digitized on a computer. A video edge motion detector (Crescent Electronics) was used to measure myocyte length and cell shortening, from which the shortening fraction and maximal rates of shortening and re-lengthening (±dL/dt) were calculated. For Ca²⁺ signal measurements, the cells were alternately excited at 340 and 380 nm by a Delta Scan dual-beam spectrophotofluorometer (Photon Technology International). Ca²⁺ transients were recorded as the 340/380 nm ratio of the resulting 510 nm emissions. The baseline, amplitude, and time for 80% decay of the Ca²⁺ signal were analyzed using software from Photon Technology International.

Immunodetection of Site-specific Phosphorylation of PLB in Vivo-- To detect the phosphorylation of PLB in mouse cardiomyocytes, ventricular myocytes were isolated from transgenic mice as described above. Myocytes obtained from two mouse hearts with the same genotype were combined and suspended in 1 ml of the normal Tyrode solution containing 1.0 mM Ca²⁺. Aliquots (100 µl) of the myocyte suspension were then incubated with a final concentration of 0.1 µM isoproterenol for 5 min. The reaction was stopped by adding SDS-stop solution containing 1 mM dithiothreitol, 30 mM Tris-HCl, 3 mM EDTA, 6% SDS, 15% glycerol, and a trace of bromphenol blue, pH 7.8. An aliquot of samples containing 50 µg of myocyte protein, as determined by the Bio-Rad protein assay, was applied to each well. For immunodetection of site-specific phosphorylation of PLB, myocyte preparations were subjected to 15% SDS-PAGE and immunoblotted with PLB polyclonal antibodies that specifically recognize either PLB phosphorylated at Ser¹⁶ (1:10,000) or PLB phosphorylated at Thr¹⁷ (1:5,000) (PhosphoProtein Research). The immunoreactivity was visualized by alkaline phosphatase-conjugated anti-rabbit secondary antibodies in conjunction with an ECL chemiluminescence detection system (Amersham Pharmacia Biotech).

Statistical Analysis-- Data are expressed as mean ± S.E. Statistical analysis was performed using Student's t test for unpaired observations and analysis of variance for multiple comparisons. Values of p < 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cardiac-specific Expression of T17A Mutant PLB in the Null Background-- Mutation of ACT to GCT (Thr¹⁷ to Ala) was introduced into the coding region of the mouse PLB cDNA by site-directed PCR mutagenesis, as described previously (7, 26). The mutation and transgene construct integrity were confirmed by DNA sequencing before submission for pronuclear microinjection. The -MHC promoter was used to direct expression of the PLB mutant in the cardiac compartment of the PLB knockout mouse. Three germ lines hosting the PLB transgene were identified using PCR analysis of genomic DNA isolated from tail biopsies. Southern blot hybridization of genomic DNA with ³²P-labeled PLB cDNA revealed that the PLB transgene migrated at ~3 kb, whereas the endogenous PLB gene migrated at ~7.0 kb (Fig. 1A). The genomic DNA also served as a template to amplify the transgene, using a set of primers flanking the PLB cDNA. The resultant PCR product was sequenced, and the presence of the desired mutation of ACT to GCT in PLB was confirmed. Expression of the transgene, which migrated at 1 kb, was detected by Northern blot analysis of total RNA prepared from transgenic hearts (Fig. 1B). This 1.0-kb transcript was not present in PLB knockout or wild-type hearts. The wild-type control hearts showed the presence of the endogenous PLB transcripts, which migrated at 2.8 and 0.7 kb (Fig. 1B).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   A, Southern blot analysis of PLB. Genomic DNA was isolated from mouse tail biopsies and digested with EcoRI and BamHI. The digested DNA was electrophoresed and transferred to a nitrocellulose membrane, which was hybridized with a 32P-labeled PLB cDNA (0.5 kb) probe. B, Northern blot analysis of PLB. Total RNA was isolated from mouse hearts, separated by gel electrophoresis, and transferred to a nitrocellulose membrane, which was hybridized with a 32P-labeled PLB cDNA probe. Lanes 1, wild-type control; lanes 2, PLB knockout; lanes 3, T17A mutant PLB in the null background.

To determine the PLB protein expression levels, cardiac homogenates from transgenic mice were prepared and subjected to quantitative immunoblotting. The protein levels of PLB were 0.7-fold in one line and 2.0-fold in the other two transgenic lines, compared with control hearts. The line expressing 0.7-fold PLB, compared with nontransgenic wild-types, was propagated for additional characterization studies. Transgenic lines expressing similar (0.7-fold) levels of wild-type or S16A mutant PLB in the hearts of the null background (7) were also processed in parallel. To verify incorporation of the re-expressed PLB in the SR membrane, quantitative immunoblotting of PLB and SERCA2 was performed, using SR-enriched membranes prepared from our mouse models (Fig. 2A). Consistent with findings obtained in cardiac homogenates, the SR protein levels of T17A mutant PLB reinserted in the null background were ~70% of those of the non-transgenic wild types (Fig. 2B). Similar levels of wild-type or S16A mutant PLB were also observed in SR-enriched membranes, as described previously (7). No significant alterations in SERCA2 protein levels were detected among the SR-enriched membranes prepared from PLB knockout hearts with either wild-type, S16A mutant, or T17A mutant PLB reinserted in the null background (Fig. 2A).

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Quantitative immunoblotting of PLB and SERCA2 in transgenic hearts. A, representative Western blots of PLB and SERCA2 using cardiac SR-enriched membrane preparations from transgenic hearts. B, PLB levels in transgenic hearts relative to the level of PLB in wild-type hearts, which was set as 100%. KO+WT, wild-type PLB reintroduced in the PLB knockout hearts; KO+T17A, T17A mutant PLB reintroduced in the PLB knockout hearts.

In Vitro Phosphorylation of PLB-- To determine whether Ser¹⁶ can be phosphorylated in vitro upon mutation of Thr¹⁷ to Ala in PLB, cardiac homogenates from the T17A mutant PLB mice were incubated with the protein kinase A catalytic subunit (PKA) under optimal phosphorylation conditions and subjected to SDS-PAGE and autoradiography. The degree of ³²P incorporation in PLB was similar in cardiac homogenates with either wild-type or T17A mutant PLB reinserted in the null background (Fig. 3A). The Ca²⁺-calmodulin-dependent phosphorylation of mutant PLB was also assessed in vitro. In the presence of Ca²⁺ and calmodulin, phosphorylation of wild-type PLB was detected, and, as expected, no ³²P incorporation in T17A mutant PLB was observed (Fig. 3B). Because endogenous CaMKII was used in the in vitro phosphorylation assays, quantitative immunoblotting was performed to determine the levels of this enzyme in the different models. There were no significant alterations of CaMKII between wild-type, S16A mutant, T17A mutant, and knockout hearts (Fig. 3C). Furthermore, phosphorylation of the T17A mutant PLB by protein kinase A catalytic subunit was completely abolished by a PKA inhibitor peptide (Fig. 4A). Similarly, Ca²⁺-calmodulin-dependent phosphorylation of the S16A mutant PLB was inhibited by a CaMKII inhibitor (Fig. 4B). These data indicate: 1) the specificity of the PLB phosphorylation pathways, and 2) the independence of the two phosphorylation sites in PLB in vitro.

View larger version (66K): [in this window] [in a new window]   Fig. 3.   In vitro phosphorylation of PLB. Cardiac homogenates from mice with wild-type or T17A mutant PLB in the null background were phosphorylated by either the catalytic subunit of cAMP-dependent protein kinase (A, PKA) or the endogenous Ca2+-calmodulin-dependent protein kinase (B, CaMKII). Reactions were terminated by SDS sample buffer, boiled, and subjected to SDS-PAGE and autoradiography. C, quantitative immunoblotting of CaMKII. 10 µg of cardiac homogenate was incubated with an equal volume of loading buffer (20% glycerol, 2% -mercaptoethanol, 4% SDS, 0.001% bromphenol blue, and 130 mM Tris-HCl, pH 6.8), subjected to 4-20% gradient SDS-polyacrylamide gel electrophoresis, and blotted onto nitrocellulose membranes. The membranes were then reacted with a mouse monoclonal antibody to CaMKII (Transduction Laboratories, Lexington, KY), and the primary antibody binding was detected by ECL (Amersham Pharmacia Biotech). KO+WT, wild-type PLB reintroduced in the PLB knockout hearts; KO+S16A, S16A mutant PLB reintroduced in the PLB knockout hearts; KO+T17A, T17A mutant PLB reintroduced in the PLB knockout hearts; KO, PLB knockout hearts.

View larger version (89K): [in this window] [in a new window]   Fig. 4.   In vitro phosphorylation of PLB. Cardiac homogenates from mice with wild-type or mutant PLB reintroduced in the null background were phosphorylated by either the catalytic subunit of cAMP-dependent protein kinase (A, PKA) or the endogenous Ca2+-calmodulin-dependent protein kinase (B, CaMKII). Reactions were performed in the absence () or presence (+) of 1 µM PKA inhibitor peptide (Upstate Biotechnology, catalogue number 12-151) or 1 µM CaMKII inhibitor (Upstate Biotechnology, catalogue number 14-361), terminated by SDS sample buffer, boiled, and subjected to SDS-PAGE and autoradiography. KO+WT, wild-type PLB reintroduced in the PLB knockout hearts; KO+S16A, S16A mutant PLB reintroduced in the PLB knockout hearts; KO+T17A, T17A mutant PLB reintroduced in the PLB knockout hearts; KO, PLB knockout hearts.

Cardiomyocyte Mechanics and Ca2+ Transients-- To examine the effect of T17A mutant PLB on cardiac contractile parameters, left ventricular myocytes were loaded with fura-2 and paced at 0.5 Hz, and simultaneous measurements of cell shortening and Ca²⁺ transients were obtained. Myocytes from PLB knockout hearts exhibited significantly enhanced contractile properties and markedly accelerated Ca²⁺ transient kinetics compared with wild types (data not shown), consistent with previous reports (5, 28-30). To assess the functional significance of Thr¹⁷ phosphorylation in PLB, myocyte mechanics and Ca²⁺ kinetics were examined in parallel, using transgenic hearts expressing similar levels of wild-type PLB reinserted in the null background. Furthermore, myocytes from hearts expressing similar levels of S16A mutant PLB in the null background were studied in parallel to determine the role of dual-site PLB phosphorylation. The shortening fraction, maximal rates of shortening and re-lengthening (Fig. 5), Ca²⁺ amplitude, and rate of Ca²⁺ transient decline (Fig. 6) were similar among myocytes expressing wild-type, S16A, or T17A mutant PLB in the null background. Thus, the mutant S16A or T17A form of PLB inhibited myocyte mechanics and Ca²⁺ transients to a similar degree as wild-type PLB under basal conditions. The findings at the myocyte level of the S16A mutant PLB were similar to previous findings in Langendorff-perfused hearts (7).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Contractile parameters of isolated ventricular myocytes loaded with fura-2 and paced at 0.5 Hz in the absence or presence of isoproterenol (0.1 µM). A, shortening fraction; B, maximal rates of shortening (+dL/dt); C, maximal rates of re-lengthening (dL/dt). KO+WT, wild-type PLB reintroduced in the PLB knockout hearts; KO+S16A, S16A mutant PLB reintroduced in the PLB knockout hearts; KO+T17A, T17A mutant PLB reintroduced in the PLB knockout hearts. *, p < 0.05 versus KO+WT or KO+T17A in the absence of isoproterenol; #, p < 0.05 versus KO+WT or KO+T17A in the presence of isoproterenol.

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Ca2+ transients in isolated ventricular myocytes loaded with fura-2 and paced at 0.5 Hz in the absence or presence of isoproterenol (0.1 µM). A, Ca2+ amplitude (ratios of 340/380 nm); B, T80 (time to 80% decline of Ca2+ transients). KO+WT, wild-type PLB reintroduced in the PLB knockout hearts; KO+S16A, S16A mutant PLB reintroduced in the PLB knockout hearts; KO+T17A, T17A mutant PLB reintroduced in the PLB knockout hearts. *, p < 0.05 versus KO+WT or KO+T17A in the absence of isoproterenol.

Effects of Isoproterenol on Myocyte Contraction and Ca2+ Transients-- PLB has been implicated to be a major player in the -adrenergic signaling pathway and a critical mediator of cardiac responses to -agonist stimulation (1, 2). To examine the effects of T17A mutant PLB on the myocyte responses to -agonists, cells from these mutant hearts, along with cells from wild-type or S16A mutant PLB hearts, were stimulated at a frequency of 0.5 Hz and sequentially perfused with 1, 10, 30, and 100 nM isoproterenol. Maximal stimulation by isoproterenol (100 nM) was associated with significant increases in the shortening fraction and the rates of shortening (+dL/dt) and re-lengthening (dL/dt) in all three groups (Fig. 5). The maximally stimulated parameters were similar between T17A and wild-type PLB myocytes, whereas these parameters were significantly lower in S16A mutant PLB cells (Fig. 5). In parallel, the increases in the Ca²⁺ transient peaks and the decreases in the rate of Ca²⁺ signal decay (T₈₀, time to 80% decline of Ca²⁺ transients) upon maximal isoproterenol (100 nM) stimulation were similar in myocytes expressing wild-type or T17A mutant PLB (Fig. 6). However, isoproterenol had a small effect on the peak of the Ca²⁺ amplitude and no effect on the decline of the Ca²⁺ transient (T₈₀) in S16A mutant PLB cells (Fig. 6).

Isoproterenol stimulation was associated with significant phosphorylation of both Ser¹⁶ and Thr¹⁷ in myocytes expressing wild-type PLB, as detected by the phosphorylation site-specific antibodies to PLB (Fig. 7). Note that Ser¹⁶ or Thr¹⁷ phosphorylation was barely detectable in isolated wild-type cardiomyocytes under basal conditions. In myocytes expressing T17A mutant PLB, Ser¹⁶ was capable of being phosphorylated upon isoproterenol stimulation (Fig. 7). Consistent with mutation of Thr¹⁷ to Ala, there was no phosphorylation of Thr¹⁷ detected in these cells. Similar findings were also obtained in SR-enriched membranes isolated from isoproterenol-stimulated Langendorff-perfused hearts expressing T17A mutant PLB in the null background (data not shown). In myocytes expressing S16A mutant PLB, there was no phosphorylation of Thr¹⁷, consistent with previous observations in Langendorff-perfused hearts (7). Moreover, isoproterenol stimulation (1 µM) was not associated with any detectable phosphorylation of Thr¹⁷ in S16A mutant myocytes, even in the presence of high Ca²⁺ (3.6 mM) and the phosphatase inhibitor okadaic acid (1 µM) (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Site-specific phosphorylation of PLB upon isoproterenol (ISO) stimulation. Cardiomyocytes were stimulated with isoproterenol (0.1 µM) for 5 min. Myocyte protein (50 µg) was subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The phosphoserine 16 (PSer16-PLB) and phosphothreonine 17 (PThr17-PLB) in PLB were detected using the phosphorylation site-specific antibodies, which specifically recognize PLB phosphorylated at Ser16 or Thr17, in conjunction with an enhanced chemiluminescence detection system. KO+WT, wild-type PLB reintroduced in the PLB knockout hearts; KO+S16A, S16A mutant PLB reintroduced in the PLB knockout hearts; KO+T17A, T17A mutant PLB reintroduced in the PLB knockout hearts.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The availability of the PLB knockout mouse in combination with transgenesis, allowing reintroduction of wild-type or mutant PLB in the null background, offered a unique opportunity to examine the functional significance of each phosphorylation site in PLB, the interrelationship between the PKA and CaMKII pathways at the level of PLB phosphorylation, and their stimulatory effects on cardiac contractility in vivo. Cardiac-specific expression of a PLB mutant, in which one of the phosphorylation sites (Thr¹⁷) was mutated to Ala, was achieved using the -myosin heavy chain promoter. The contractile properties and Ca²⁺ kinetics of T17A mutant PLB cardiomyocytes were characterized in parallel with cardiomyocytes expressing similar levels of wild-type or S16A mutant PLB (7) in the absence or presence of maximal isoproterenol stimulation. Mutation of Thr¹⁷ to Ala did not alter the ability of the adjacent Ser¹⁶ to be phosphorylated by PKA in vitro. Similarly, mutation of Ser¹⁶ to Ala did not affect phosphorylation of Thr¹⁷ by CaMKII in vitro (7). Taken together, these findings indicate that Ser¹⁶ and Thr¹⁷ can be phosphorylated independently in SR membranes, consistent with previous observations in expression systems and native SR membranes (13-15), although one study has proposed that phosphorylation of Ser¹⁶ by PKA proceeds via a random process, whereas that of Thr¹⁷ by CaMKII proceeds in a cooperative manner (16). The mechanism by which phosphorylation of PLB mediates its regulatory effects has been suggested to involve enhancement of the interaction between individual SERCA2 polypeptide chains due to spatial rearrangement and protein-protein interactions, thus allowing for the removal of inhibition of SERCA2 activity by PLB (2, 31, 32).

The mutant T17A PLB was able to reverse the hyperdynamic cardiac myocyte function to the same extent as wild-type PLB upon its reinsertion in the null background, indicating that mutation of threonine 17 to alanine did not alter the ability of PLB to interact with and inhibit SERCA2. Isoproterenol administration was associated with increases in the rates of shortening and re-lengthening as well as abbreviation of Ca²⁺ kinetics in T17A mutant PLB cardiomyocytes. Furthermore, the maximal stimulatory effects on mechanics and Ca²⁺ kinetics in T17A PLB mutant cardiomyocytes were similar to those in wild types. Thus, mutation of T17A in PLB was not associated with any significant alterations in the stimulatory effects of PLB in response to isoproterenol. However, in parallel studies, mutation of serine 16 to alanine in PLB diminished the stimulatory effects of isoproterenol in cardiomyocytes, consistent with our previous observations in Langendorff-perfused hearts (7). These findings suggest that phosphorylation of Ser¹⁶ in PLB may be sufficient to mediate the maximal cardiac responses to -agonists, and the effects of dual-site (Ser¹⁶ and Thr¹⁷) PLB phosphorylation do not appear to be additive in vivo.

It is interesting to note that cardiomyocytes expressing the S16A mutant PLB exhibited increases in the shortening fraction and the maximal rates of shortening and relaxation compared with wild types upon isoproterenol stimulation, even though the S16A PLB was not phosphorylated. Thus, the functional changes in S16A mutant myocytes were associated with mechanisms independent of PLB phosphorylation, such as phosphorylation and functional modification of cardiac TnI, the ryanodine receptor, or L-type Ca²⁺ channels. However, the relative contribution of these phosphoproteins in the cardiac responses to -agonists remains to be elucidated.

The effect of -agonist stimulation on PLB phosphorylation is mainly associated with activation of the cAMP-dependent signaling pathway. Stimulation of adenylase cyclase and the consequent increase of cAMP lead to phosphorylation of Ser¹⁶ in PLB via PKA. Stimulation of -receptors also elevates intracellular Ca²⁺, which is expected to contribute to phosphorylation of Thr¹⁷ in PLB via CaMKII. Thus, it becomes difficult to distinguish between the two protein kinase pathways and determine the significance of single-site PLB phosphorylation in vivo. The significance of Ser¹⁶ phosphorylation in the -agonist stimulatory effects in vivo has been proposed by several studies (7, 22, 33), and a close correlation between the degree of phosphorylation of Ser¹⁶ and contractile parameters has been observed in perfused hearts (12, 24) and isolated myocytes (22). However, in all these studies, Thr¹⁷ was also phosphorylated, and the importance of Ser¹⁶ single-site phosphorylation was not conclusive. The significance of CaMKII phosphorylation of PLB, independent of PKA phosphorylation, has been more difficult to assess in vivo. Phosphorylation of PLB by CaMKII was suggested to increase the V_max of SERCA2 (34), and inhibition of this kinase resulted in prolongation of the Ca²⁺ decline in rat cardiac myocytes (34, 35). CaMKII phosphorylation of PLB has also been implicated in the regulation of the cardiac force-frequency relationship by enhancing SR Ca²⁺ uptake and Ca²⁺ kinetics. However, opposite results have been observed in studies using isolated rat cardiomyocytes. In an earlier study (36), no significant changes in phosphorylation levels of Ser¹⁶ and/or Thr¹⁷ sites of PLB were obtained with increasing stimulation frequency, whereas Thr¹⁷ phosphorylation was recently reported to increase in a frequency-dependent manner, and the increases in Thr¹⁷ phosphorylation were correlated with enhanced rates of myocyte contraction and relaxation in rat ventricular myocytes (37). In perfused rat hearts, phosphorylation of Thr¹⁷ occurred upon inhibition of protein phosphatase-1 in the presence of elevated extracellular Ca²⁺ or under acidic conditions (24, 25, 38), suggesting the possible involvement of Thr¹⁷ phosphorylation under pathophysiological conditions.

In summary, the present study further elucidates the regulatory role of dual-site PLB phosphorylation by specifically addressing: 1) the interdependence of Ser¹⁶ and Thr¹⁷ phosphorylation in PLB, and 2) the contribution of each phosphorylation site to Ca²⁺ handling and contractile parameters in cardiac myocytes in response to -adrenergic stimulation. Our findings indicate that Ser¹⁶ in PLB can be phosphorylated independently of Thr¹⁷ in vitro and in vivo, whereas Thr¹⁷ can only be independently phosphorylated in vitro. Phosphorylation of Ser¹⁶ is sufficient to mediate the maximal cardiac calcium kinetic and contractile responses to -adrenergic stimulation, suggesting that the effects of dual-site (Ser¹⁶ and Thr¹⁷) phosphorylation in PLB are not additive in vivo. Based on these results, we conclude that PKA-dependent phosphorylation of Ser¹⁶ in PLB plays a dominant role in mediating the cardiac contractile responses to -agonists.

	ACKNOWLEDGEMENT

We thank Dr. Alicia Mattiazzi for helpful discussion and critical evaluation of the manuscript.

	FOOTNOTES

^* This study was supported by the National Institutes of Health Grants HL26057, HL52318, HL07382, and P40RR12358.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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0575. Tel.: 513-558-2377; Fax: 513-558-2269; E-mail: kraniaeg@email.uc.edu.

Published, JBC Papers in Press, September 14, 2000, DOI 10.1074/jbc.M004079200

	ABBREVIATIONS

The abbreviations used are: PLB, phospholamban; cAMP, cyclic AMP; SR, sarcoplasmic reticulum; PKA, cAMP-dependent protein kinase; CaMKII, Ca²⁺-calmodulin-dependent protein kinase; PCR, polymerase chain reaction; kb, kilobase pair(s); -MHC, -myosin heavy chain; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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