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(Circulation Research. 1997;81:485-492.)
© 1997 American Heart Association, Inc.

Articles

Monomeric Phospholamban Overexpression in Transgenic Mouse Hearts

Guoxiang Chu, Gerald W. Dorn, II, Wusheng Luo, Judy M. Harrer, Vivek J. Kadambi, Richard A. Walsh, , Evangelia G. Kranias

From the Department of Pharmacology & Cell Biophysics (G.C., W.L., J.M.H., V.J.K., E.G.K.) and the Division of Cardiology of the Department of Internal Medicine (G.W.D., R.A.W.), University of Cincinnati (Ohio), College of Medicine.

Correspondence to Evangelia G. Kranias, PhD, Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave, PO Box 670575, Cincinnati, OH 45267-0575.

	Abstract

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Abstract Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of phospholamban, which is associated with mutating Cys⁴¹ to Phe⁴¹, was shown to be as effective as pentameric phospholamban in inhibiting Ca²⁺ transport in expression systems. To determine whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys⁴¹Phe⁴¹) phospholamban. Quantitative immunoblotting indicated a 2-fold increase in the cardiac phospholamban protein levels compared with wild-type controls, with 50% of phospholamban migrating as monomers and 50% as pentamers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were analyzed in parallel with transgenic hearts overexpressing (2-fold) wild-type phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca²⁺-uptake assays revealed that the EC₅₀ values for Ca²⁺ were as follows: 0.32±0.01 µmol/L in hearts overexpressing monomeric phospholamban, 0.49±0.05 µmol/L in hearts overexpressing wild-type phospholamban, and 0.26±0.01 µmol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca²⁺ kinetics indicated that the inhibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) as assessed by depression of the following: (1) shortening fraction (25% mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), (3) rates of relengthening (25% mt versus 50% ov), (4) amplitude of the Ca²⁺ signal (21% mt versus 40% ov), and (5) time for decay of the Ca²⁺ signal (25% mt versus 106% ov) compared with control (100%) myocytes. The differences in basal cardiac myocyte mechanics and Ca²⁺ transients among the animal groups overexpressing monomeric or wild-type phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.

Key Words: phospholamban • transgenic mouse • Ca²⁺-ATPase • cardiomyocyte • Ca²⁺ transient

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phospholamban, a phosphoprotein in cardiac SR, is a prominent regulator of the SR Ca²⁺-ATPase activity and myocardial contractility. Dephosphorylated phospholamban diminishes the affinity of the SR Ca²⁺-ATPase for Ca²⁺, and phosphorylation restores the high Ca²⁺ affinity state.^{1 2 3} In vitro studies have shown that phospholamban can be phosphorylated at three distinct sites by various protein kinases: serine¹⁰ by protein kinase C, serine¹⁶ by cAMP-dependent protein kinase, and threonine¹⁷ by Ca²⁺-calmodulin–dependent protein kinase.^{3 4 5} Phospholamban has also been shown to be phosphorylated on serine¹⁶ and threonine¹⁷ in vivo during ß-adrenergic stimulation of the heart.^{6 7} Recently, the role of phospholamban in the regulation of basal myocardial contractility has been elucidated using gene targeting and transgenic mouse methodology. Hierarchical studies in animal models with reduced or ablated phospholamban expression using broken cell preparations,⁸ isolated myocytes,⁹ intact hearts,⁸ and intact mice¹⁰ have revealed that phospholamban is a major regulator of (1) the affinity of the cardiac SR Ca²⁺ pump for Ca²⁺, (2) basal myocardial contractility, and (3) cardiac responses to ß-adrenergic agonists. Furthermore, studies in mice with cardiac-specific overexpression of phospholamban indicated that a fraction of the SR Ca²⁺-ATPases is not subject to regulation by phospholamban in vivo. Increased phospholamban expression diminished the affinity of the Ca²⁺-ATPase for Ca²⁺ and decreased the contractile parameters.¹¹ When the relative phospholamban levels were plotted against the affinity of the Ca²⁺-ATPase for Ca²⁺ in hearts overexpressing phospholamban and in wild-type, phospholamban-heterozygous, and phospholamban-knockout hearts, a close linear correlation was observed,^{3 12} indicating that the overexpressed phospholamban was coupled functionally to the SR Ca²⁺-ATPase in the transgenic hearts.

The functional unit of phospholamban and the mechanism by which it mediates its regulatory effects in vivo are not presently known. Phospholamban consists of 52 amino acids. It migrates as a pentamer with an apparent M_r of 25 000 to 28 000 upon SDS-PAGE, but upon boiling in SDS before electrophoresis, it is dissociated to monomers with an apparent M_r of 6000. The phospholamban monomer has been proposed to consist of two major domains: a hydrophilic domain containing amino acids 1 to 30 and a hydrophobic or transmembrane domain containing amino acids 31 to 52.³ Studies from several laboratories have indicated the importance of the hydrophilic domain in mediating the regulatory effects of phospholamban on the cardiac SR Ca²⁺-ATPase.^{13 14 15 16} Site-directed mutagenesis has identified specific amino acids (amino acids 2 to 4, 7, 9, 12 to 14, and 16 to 18) in the hydrophilic phospholamban domain, which may interact with specific regions (amino acids 336 to 412 and 467 to 712) in the cardiac SR Ca²⁺-ATPase molecule for functional modification.^{14 17} Proteolytic studies and site-directed mutagenesis experiments have shown that the hydrophobic domain of phospholamban is not only important in mediating the regulatory effects of the protein^{3 15 18 19} but that it is also responsible for pentamer formation.^{20 21 22} Early studies suggested that the three cysteine residues at positions 36, 41, and 46 provide for noncovalent interaction between monomeric forms of phospholamban and contribute to stabilization of pentamers,²⁰ with the mutant Cys⁴¹ to Phe⁴¹ resulting in the greatest destabilization of the phospholamban pentamer. Later, analysis of phospholamban pentamers led to the prediction that pentamers form a left-handed coiled-coil helical bundle, with a cylindrical ion pore.^{3 22} Mutations of amino acids Leu³⁷, Ile⁴⁰, Leu⁴⁴, Ile⁴⁷, and Leu⁵¹ led to pentamer instability, and these residues were proposed to form a leucine zipper, which stabilizes the pentameric association of phospholamban monomers and may form a Ca²⁺-selective ion pore.^{21 22 23} Furthermore, interaction between the three leucines of one helix and the two isoleucines of an adjacent helix were suggested to be involved in the interaction of phospholamban monomers for pentameric assembly.²²

It is not presently clear whether pentamer assembly is essential for functional regulation of the cardiac SR Ca²⁺-ATPase. Coexpression studies of phospholamban with the cardiac SR Ca²⁺-ATPase in HEK-293 cells indicated that monomeric phospholamban generated by mutation of Cys⁴¹ to Ser⁴¹ or Phe⁴¹ was capable of regulating SR Ca²⁺ transport in a manner similar to that of pentameric phospholamban.¹⁴ Mutation of Cys⁴¹ to Ser⁴¹ prevented pentameric assembly at temperatures higher than room temperature, whereas mutation of Cys⁴¹ to Phe⁴¹ prevented pentameric formation even at ambient temperatures. To determine whether monomeric phospholamban is also capable of regulating SR function and myocardial contractility in vivo, we generated transgenic mice overexpressing mutant (Cys⁴¹Phe⁴¹) phospholamban in a cardiac-specific manner. Cardiomyocyte mechanics and Ca²⁺ transients were then assessed in parallel in mice overexpressing equal levels of mutant or wild-type phospholamban, in an attempt to gain insight into the functional unit of phospholamban in vivo.

	Materials and Methods

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Site-Directed Mutagenesis
The site-specific mutation of Cys⁴¹Phe⁴¹ (TGTTTT) was introduced into phospholamban cDNA by PCR methodology according to Bowman et al.²⁴ Briefly, a 0.9-kb Sal I fragment containing phospholamban cDNA and the SV40 polyadenylation signal sequence (phospholamban cDNA-SV40-PolyA) was released from the MHCp-phospholamban-SV40 fusion gene, which has been successfully used to generate wild-type phospholamban–overexpressing transgenic mice in our laboratory.¹¹ This Sal I phospholamban cDNA-SV40-PolyA fragment was then subcloned into a pBluescript SK II(-) vector (Stratagene), which has T3 and T7 primer sites flanking the insert. PCR mutagenesis was performed by two consecutive PCR amplifications using two different sets of primers. For the first PCR amplification, 100 pg of the subclone plasmid DNA containing the 0.9-kb Sal I fragment was used as template, along with a 5'-end mutant primer (5'C CTC ATC TTG ATA TTT CTG CTG CTG ATC TG 3'), corresponding to nucleotides 108 to 137 of the phospholamban coding sequence, and a 3'-end T7 primer, to generate a desired mutant-phospholamban cDNA minor product. Subsequently, an aliquot of the first PCR product and the T3 and T7 primers were used for the second PCR, which was designed to amplify the full-length insert with the desired mutation in the phospholamban cDNA. The final amplified product was excised, gel-purified, and resubcloned into the Sal I site of a second pBluescript SK II(-) vector, which was then transformed into XL1-Blue competent cells. Colonies from the transformed cells hosting the desired mutant phospholamban cDNA were identified by DNA sequencing. The mutated phospholamban cDNA-SV40-PolyA sequence was excised by Sal I from the pBluescript SK II(-) vector for sequence analysis and subsequent recloning into the Sal I site of the parent phospholamban overexpression vector pIBI 31.¹¹

Generation and Identification of Mutant Mice
The entire expression construct was contained in vector pIBI 31 as a Kpn I–HindIII fragment, which was composed of the cardiac-specific -MHC promoter (5.5 kb), the phospholamban coding region with the Cys⁴¹Phe⁴¹ mutation (0.6 kb), and the SV40 polyA signal sequence (0.25 kb). The mutation and the expression construct sequence were confirmed by restriction digestion and DNA sequencing. The Kpn I–HindIII fragment was released from the plasmid vector, gel-purified, and used for pronuclear microinjection of fertilized eggs from FVB/N mice to generate transgenic mice according to standard procedures.¹¹ Transgenic mice harboring the mutated phospholamban transgene were identified using PCR methodology and Southern analysis of genomic DNA isolated from tail biopsies, as described previously.¹¹ The transgene expression, driven by the cardiac-specific -MHC promoter, was determined by Northern analysis of total RNA from the transgenic mouse hearts.

Quantitative Immunoblotting of Phospholamban and SR Ca²⁺-ATPase
Quantitative immunoblotting of cardiac homogenates was performed to determine the protein levels of phospholamban and SR Ca²⁺-ATPase as described previously.²⁵ Briefly, hearts were homogenized in buffer (pH 7.0) containing (mmol/L) imidazole 10, sucrose 300, dithiothreitol 1, sodium metabisulfite 1, and phenylmethylsulfonyl fluoride 0.3. Protein concentrations were determined by the Bio-Rad method using bovine serum albumin as a standard. The cardiac homogenates (6 to 15 µg protein) were incubated with equal volumes of loading buffer (20% glycerol, 2% ß-mercaptoethanol, 4% SDS, 0.001% bromophenol blue, and 130 mmol/L Tris-Cl, pH 6.8), subjected to 13% SDS-PAGE,²⁰ 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) at a dilution of 1:1000. After washing out the unbound antibody with Tris-buffered saline (10 mmol/L Tris-HCl and 150 mmol/L 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 nitro blue 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 the ImageQuant software. Quantification of pentameric and monomeric forms of phospholamban were also carried out to calculate the percentage of monomer in sample preparations.

SR Ca²⁺ Uptake Assay
Mouse hearts were excised, frozen in liquid nitrogen, and stored at -80°C. The frozen hearts were powdered and homogenized in (mmol/L) KH₂PO₄ 50 (pH 7.0), NaF 10, EDTA 1, sucrose 0.3, phenylmethylsulfonyl fluoride 0.3, and dithiothreitol 0.5. The initial rates of Ca²⁺ uptake in whole-heart homogenates were obtained and calculated as previously described.⁸

Cardiac Myocyte Isolation
Ten- to 12-week-old wild-type FVB/N mice and transgenic mice overexpressing either mutant (Cys⁴¹Phe⁴¹) phospholamban or wild-type phospholamban in the heart were used in parallel for isolation of left ventricular myocytes, which were subsequently used for measurements of myocyte mechanics and Ca²⁺ transients. The procedure for isolation of ventricular myocytes has been previously described.^{11 26} Briefly, animals were anesthetized with methoxyflurane (Pitman-Moore Inc), and hearts were rapidly excised and perfused on a Langendorff apparatus with a Ca²⁺-free Joklik buffer (S-MEM, Joklik-modified, pH 7.2, GIBCO-BRL) at 2.2 mL/min for 5 minutes. Perfusion was then switched to the Joklik buffer (pH 7.2) supplemented with 25 µmol/L Ca²⁺, 75 U/mL collagenase I (Worthington), 75 U/mL collagenase II (Worthington), 1% albumin (Sigma Chemical Co), and 2% DCS. All buffers were kept at 37°C and continuously oxygenated with mixed gas of 95% O₂ and 5% CO₂. After perfusion for an additional 15 minutes or so, the heart became flaccid and was transferred into low-Ca²⁺ Joklik buffer (25 µmol/L Ca²⁺ and 2% DCS), and the left ventricle was excised and minced gently. The resultant cell suspension was rinsed several times with the above buffer and resuspended in physiological buffer composed of (mmol/L) NaCl 132, KCl 4.8, MgCl₂ 1.2, glucose 5, HEPES 10, and Ca²⁺ 1.8.

Measurements of Mechanical Parameters and Ca²⁺ Transients
The resuspended isolated myocytes were placed in a specially designed superfusion chamber on the stage of an inverted epifluorescence microscope (Olympus IMT-2) and continuously superfused with oxygenated physiological buffer. Cells were equilibrated for 15 minutes before experimental use, and all experimental protocols were carried out at room temperature (27°C). The myocytes were field-stimulated (0.25 Hz, square waves with a pulse duration of 2 milliseconds) through a pair of platinum electrodes positioned in the chamber and coupled to a Grass S9 stimulator. The imaging of the myocytes was acquired through a charge-coupled device (model GP-CD60, Panasonic). Data were collected and recorded on videotape and subsequently digitized on a computer. Myocyte dimensions (width and length) were measured from the videotaped images calibrated with a micrometer. A video motion edge detector (Crescent Electronics) and ImagePro computer software (developed by the Division of Cardiology of the University of Cincinnati) were used to analyze and calculate myocyte shortening fraction, the rate of shortening (+dL/dt), and the rate of relengthening (-dL/dt).

For measurements of cytosolic free Ca²⁺, the isolated cardiac myocytes were incubated with 7 µmol/L fura 2-AM for 30 minutes at 37°C in low-Ca²⁺ Joklik buffer (25 µmol/L Ca²⁺ and 2% DCS, pH 7.2). After loading, the myocytes were resuspended in physiological buffer, placed in a superfusion chamber on the epifluorescence microscope stage (Olympus IMT-2), superfused constantly with oxygenated physiological buffer, and stimulated electronically to contract at room temperature, as described above. The intracellular free Ca²⁺ was then monitored as the 340/380-nm fluorescence ratio, which was acquired by alternating excitation wavelengths of 340 nm and 380 nm, using a PTI Delta Scan-1 dual-beam spectrophotofluorometer (Photon Technology International Inc). The background fluorescence, measured initially, was subtracted from subsequent fluorescence measurements from each cell. The baseline, amplitude, and T80 were calculated using Calcium Imaging Tool computer software (developed by the Division of Cardiology of the University of Cincinnati).

The effects of isoproterenol (Sanofi Winthrop Pharmaceuticals) on the myocyte mechanics and Ca²⁺ transients were also examined. Intact ventricular myocytes were stimulated by field electrodes at a default rate of 0.25 Hz. After the steady state values of myocyte mechanics or Ca²⁺ transients were obtained, the myocytes were superfused sequentially by 1, 10, 30, or 100 nmol/L isoproterenol in oxygenated physiological buffer under identical conditions. After each dose of isoproterenol, the myocytes were allowed to stabilize for 2 minutes, and then data were collected continuously for 50 seconds. Measurements from myocytes with spontaneous contraction or hypercontracture upon isoproterenol were discarded.

Statistical Analysis
Data are expressed as mean±SEM, and n indicates the number of mice. Statistical analysis was performed using one-way ANOVA followed by Dunnett's test for multiple comparisons. Values of P<.05 were considered statistically significant. For the myocyte experiments, 3 to 5 cells from individual hearts were used for replicate measurements, and each animal was treated as a single n.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation and Identification of Mutant Mice
Site-directed mutagenesis of Cys⁴¹ to Phe⁴¹ in phospholamban has been shown previously to result in monomer formation even at ambient temperature,²⁰ and the mutant phospholamban was equally effective as wild-type phospholamban in regulating the affinity of the SR Ca²⁺ transport for Ca²⁺ in expression systems.¹⁴ To determine whether this mutant phospholamban can also regulate cardiac function in vivo, Cys⁴¹ was mutated to Phe⁴¹ (TGTTTT) in the mouse phospholamban cDNA, and cardiac-specific expression of mutant phospholamban was driven using the -MHC gene promoter (Fig 1A). Six founder mice harboring the mutated phospholamban transgene were identified by PCR analysis, using primers corresponding to the -MHC promoter (primer 1, 5'CAC ATA GAA GCC TAG CCC ACA C3') and phospholamban-encoding sequence (primer 2, 5'GAT TCT GAC GTG CTT GCT GAG G3'). The resulting PCR product was 150 bp in size. These six transgenic mice were further analyzed using Southern blot hybridization. Genomic DNA isolated from tail biopsies was digested with BamHI and EcoRI and probed with a ³²P-labeled phospholamban cDNA. Autoradiography indicated that the endogenous wild-type phospholamban DNA migrated at 7 kb and that the phospholamban transgene migrated at 3 kb. The six transgenic lines were bred for further analysis. Northern blot analysis (Fig 1B) of total RNA from their hearts revealed the presence of the two endogenous phospholamban transcripts at 2.8 and 0.7 kb, as previously described,¹¹ and three of the six transgenic lines also demonstrated strong signals of the transgenic transcript migrating at 1.0 kb. The transgenic lines were then bred and propagated for further characterization of the phospholamban protein expression levels.

View larger version (49K): [in this window] [in a new window]   Figure 1. A, Schematic representation of the construct harboring the Cys41-Phe41 mutation in the phospholamban (PLB) cDNA. The -MHC promoter was used to drive cardiac-specific overexpression of the mutant PLB. The construct also includes the SV40 polyA site at the 3' end. TGT (Cys41) in the PLB cDNA was converted to TTT (Phe41) (underlined) by site-specific mutagenesis. B, Cardiac expression of the -MHC–PLB transgene. Representative Northern blots of transgenic (TG) progeny from lines TG41, TG30, and TG28 as well as wild-type (WT) littermates are shown. Total RNA was prepared from individual hearts, and the denatured RNA was separated by electrophoresis on a 1.2% agarose gel, blotted onto a GeneScreen membrane, and hybridized with a 32P-labeled PLB cDNA probe. Washing conditions were as follows: 0.5x SSC and 0.5% SDS for 30 minutes at 60°C. Each line expresses the expected 1.0-kb TG transcript in addition to the endogenous WT mouse PLB transcripts migrating at 2.8 and 0.7 kb.

Phospholamban Protein Expression Levels: Monomers Versus Pentamers
To determine the levels of phospholamban protein in transgenic mice, cardiac homogenates were subjected to Western blot analysis. Quantitative immunoblotting of cardiac homogenates (Fig 2A) revealed a 2-fold increase in phospholamban protein levels in transgenic mice compared with wild-type mice (Fig 2B). However, 50% of the phospholamban protein detected in mutant-phospholamban mouse hearts migrated as monomers and 50% migrated as pentamers, whereas the phospholamban present in wild-type control mice and mice overexpressing wild-type phospholamban¹¹ migrated as pentamers (Fig 2A and 2C). Boiling of the samples before electrophoresis resulted in migration of all phospholamban as monomers (Fig 3A). Quantification of the monomeric phospholamban protein levels revealed a 2-fold increase in both the mutant-phospholamban–as well as the wild-type phospholamban–overexpressing hearts compared with wild-type hearts (Fig 3B). To demonstrate that the overexpressed mutant phospholamban was actually incorporated into the SR membrane, microsomal fractions enriched in SR membranes were isolated by differential centrifugation and subsequently subjected to Western analysis (Fig 4A). The relative levels of phospholamban (210±15% [mutant] versus 100% [wild-type], n=4) and the ratio of monomer to pentamer (52:48) in the microsomal preparations from mutant mouse hearts were similar to those in the corresponding cardiac homogenates shown in Fig 2A, indicating that the overexpressed phospholamban monomers were incorporated into the SR membranes. Two of the mutant-phospholamban germ lines, with 2-fold protein expression levels, were bred in parallel with wild-type control mice as well as transgenic mice overexpressing wild-type phospholamban¹¹ for further functional analysis. The wild-type phospholamban–overexpressing transgenic mice served as controls in these studies, since the total protein levels of phospholamban in their hearts were similar to those in mutant-phospholamban transgenic mice (2-fold increase compared with wild-type mice).

View larger version (43K): [in this window] [in a new window]   Figure 2. A, Western blot analysis of phospholamban (PLB) expression levels in wild-type mice (WT, n=6) and transgenic mice (transgenic mice overexpressing mutant PLB [OV-MU, n=6] and transgenic mice overexpressing wild-type PLB [OV-WT, n=6]). Increasing concentrations of cardiac homogenates (6, 10, and 15 µg) were separated by 15% SDS-PAGE, electroblotted onto a nitrocellulose membrane, and incubated with an anti-mouse PLB monoclonal antibody. The PLB bands were visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates for the alkaline phosphatase reaction. Note that PLB monomers and pentamers migrated at 6 and 28 kD on SDS-PAGE, respectively. B, Quantification of total PLB protein expression levels in hearts from transgenic mice relative to WT. C, Levels of PLB pentamers in transgenic mouse hearts relative to WT, as detected by SDS-PAGE and quantitative immunoblotting.

View larger version (30K): [in this window] [in a new window]   Figure 3. Effect of boiling on phospholamban (PLB) expressed in transgenic mouse hearts. Cardiac homogenates (10 and 15 µg) from wild-type mice (WT, n=6) and transgenic mice (transgenic mice overexpressing mutant PLB [OV-MU, n=6] and transgenic mice overexpressing wild-type PLB [OV-WT, n=6]) were boiled for 5 minutes immediately before electrophoresis, transferred to a nitrocellulose membrane, and incubated with an anti-PLB monoclonal antibody. A, Representative Western blots show that all the PLB migrated as monomers under these conditions. B, Quantification of PLB monomers in hearts from OV-MU or OV-WT relative to hearts from WT littermates.

View larger version (30K): [in this window] [in a new window]   Figure 4. Representative Western blots of phospholamban (A) and the SR Ca2+-ATPase (B) using the cardiac SR-enriched microsomal preparations from mutant mice (transgenic mice overexpressing mutant phospholamban [OV-MU] and transgenic mice overexpressing wild-type phospholamban [OV-WT]) as well as from their wild-type (WT) littermates. Mouse hearts were powdered in liquid nitrogen and homogenized at 4°C in buffer (pH 7.0) composed of (mmol/L) imidazole 10, sucrose 300, dithiothreitol 1, sodium metabisulfite 10, and phenylmethylsulfonyl fluoride 0.3. SR-enriched microsomal fractions were obtained using differential centrifugation of the cardiac homogenates. The SR membrane protein was subjected to 13% SDS-PAGE, blotted onto a nitrocellulose membrane, and then reacted with a mouse monoclonal antibody to phospholamban or a polyclonal antibody to the SR Ca2+-ATPase.

To determine whether overexpression of mutant phospholamban was associated with any alteration in the SR Ca²⁺-ATPase expression levels, in an attempt to compensate for the increased levels of its regulatory protein phospholamban, we performed quantitative immunoblotting of the SR-enriched microsomal preparations (Fig 4B) and cardiac homogenates. The SR Ca²⁺-ATPase protein levels were similar between transgenic mice and wild-type littermates.

SR Ca²⁺-Uptake Rates
To examine the effects of mutant-phospholamban overexpression on SR function, cardiac homogenates from mice overexpressing mutant or wild-type phospholamban were processed with those from control mice, and the initial rates of SR Ca²⁺ uptake were assessed over a wide range of Ca²⁺ concentration (Fig 5). In hearts overexpressing wild-type phospholamban, the affinity (EC₅₀, 0.49±0.05 µmol/L; n=7) of the SR Ca²⁺ uptake for Ca²⁺ was significantly reduced compared with wild-type hearts (EC₅₀, 0.26±0.01 µmol/L; n=6), in agreement with our previous findings.¹¹ Interestingly, the affinity of the SR Ca²⁺ uptake for Ca²⁺ in hearts overexpressing mutant phospholamban was lower (EC₅₀, 0.32±0.01 µmol/L; n=9) but not significantly different from that in wild-type hearts. The maximal velocities of SR Ca²⁺ uptake were similar among the transgenic and wild-type hearts (Fig 5).

View larger version (16K): [in this window] [in a new window]   Figure 5. Initial rates of SR Ca2+ uptake in transgenic and wild-type mouse hearts. Cardiac homogenates from transgenic mice overexpressing mutant phospholamban (, n=9), transgenic mice overexpressing wild-type phopholamban (, n=7), and wild-type mice (, n=6) were prepared, and the initial rates of the SR Ca2+ uptake were assessed as a function of [Ca2+]. Points represent the mean±SEM of three to five experiments, using either individual or pooled hearts. Each experiment was performed in triplicate. The data were analyzed by nonlinear regression using Microcal Origin (version 6.0) software.

Mechanical Properties of Isolated Cardiomyocytes
Overexpression of wild-type phospholamban was shown to result in significant depression of cardiomyocyte mechanics and basal left ventricular function.¹¹ Thus, it was of special interest to examine whether overexpression of monomeric phospholamban was also associated with similar functional alterations in vivo. Cardiomyocytes from wild-type, mutant-phospholamban–overexpressing, and wild-type phospholamban–overexpressing mice were studied in parallel, and their mechanical properties as well as Ca²⁺ transients were examined. The shortening fraction was decreased by 25% in the cardiomyocytes overexpressing monomeric phospholamban and by 45% in the cardiomyocytes overexpressing pentameric phospholamban compared with wild-type cardiomyocytes (Fig 6A). The maximal velocities of myocyte shortening and relengthening were also decreased (+dL/dt, 27%; -dL/dt, 25%) in the myocytes overexpressing monomeric phospholamban; these decreases (+dL/dt, 48%; -dL/dt, 50%) were more pronounced in the myocytes overexpressing pentameric phospholamban (Fig 6B and 6C). The intermediate contractile properties in cardiomyocytes overexpressing mutant phospholamban were significantly distinct from wild-type cardiomyocytes (Fig 6A and 6B).

View larger version (31K): [in this window] [in a new window]   Figure 6. Shortening fractions (A) and rates of shortening (+dL/dt) (B) and relengthening (-dL/dt) (C) in isolated unloaded cardiomyocytes (paced at 0.25 Hz) from wild-type mice (WT, n=11) and transgenic mice (transgenic mice overexpressing mutant phospholamban [OV-MU, n=13] and transgenic mice overexpressing wild-type phospholamban [OV-WT, n=7]). Values are mean±SEM. *P<.05 vs WT; #P<.05 vs WT.

Ca²⁺ Transients in Isolated Cardiomyocytes
The alterations detected in contractile parameters of the cardiomyocytes overexpressing phospholamban prompted further examination of the Ca²⁺ kinetics in these hearts. The Table presents the Ca²⁺ transients in the cardiomyocytes loaded with fura 2-AM and paced at 0.25 Hz, similar to our previous studies.¹¹ The amplitudes of the Ca²⁺ signals during systole were depressed by 21% in the cardiomyocytes overexpressing mutant phospholamban and 40% in the cardiomyocytes overexpressing wild-type phospholamban compared with wild-type cardiomyocytes. However, the baseline values, which represent diastolic free Ca²⁺ concentration, were similar among the three groups. T80 was determined to evaluate the rate of decline of the free Ca²⁺ concentration, which is an index of the function of the SR Ca²⁺ transport system. In the cardiomyocytes overexpressing monomeric phospholamban, the T80 was prolonged (25%), and prolongation of this parameter was more pronounced (106%) in the cardiomyocytes overexpressing pentameric phospholamban than in wild-type cardiomyocytes (Table).

View this table: [in this window] [in a new window]   Table 1. Ca2+ Transients in Isolated, Fura 2–Loaded Ventricular Cardiomyocytes From Transgenic and Wild-Type Mice

Effects of Isoproterenol on Cardiomyocyte Mechanics and Ca²⁺ Transients
Phospholamban has been implicated as the major player in the ß-adrenergic signaling pathway and a prominent regulator of the cardiac responses to ß-agonist stimulation.^{3 8} Thus, cardiomyocytes were isolated from mutant-phospholamban– and wild-type phospholamban–overexpressing mice as well as from control mice. The cardiomyocytes were superfused with sequential concentrations of isoproterenol to assess their mechanical and Ca²⁺ transient responses. Isoproterenol stimulation was associated with increases in the shortening fraction of all three groups, reaching similar maximal values at optimal ß-agonist concentrations (Fig 7A). Similar patterns of responses were observed in the rates of shortening (Fig 7B) and relengthening (Fig 7C) upon isoproterenol stimulation. The maximally stimulated values, obtained at 0.1 µmol/L isoproterenol, were similar in all groups. Furthermore, there were no significant differences in the slopes of the various curves between control, wild-type phospholamban–overexpressing, and mutant-phospholamban–overexpressing cardiac myocytes. In parallel studies, the effects of isoproterenol on T80, the parameter assessing the rate of decay of the Ca²⁺ signal, were also examined. Isoproterenol stimulation was associated with dose-dependent decreases in T80 in all three groups, reaching the same values at maximal stimulation (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 7. Dose-response curves of isoproterenol on shortening fractions (A) and the rates of shortening (+dL/dt) (B) and relengthening (-dL/dt) (C) in isolated unloaded cardiomyocytes (paced at 0.25 Hz) from wild-type (, n=11) and transgenic mice overexpressing either mutant phospholamban (, n=13) or wild-type phospholamban (, n=7).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first study to present evidence of the functional significance of monomeric phospholamban overexpression in vivo. Cardiac-specific overexpression of phospholamban harboring the Cys⁴¹ to Phe⁴¹ mutation was achieved using the -MHC promoter, which is both developmentally and hormonally regulated in vivo.¹¹ The mutation of Cys⁴¹ to Phe⁴¹ in phospholamban was chosen, since this substitution has been shown previously to prevent pentamer formation, even at ambient temperature,²⁰ and it does not compromise the inhibitory effects of phospholamban on SR Ca²⁺ transport, assessed in expression systems.¹⁴ Although overexpression of monomeric phospholamban in vivo was associated with depression of contractile parameters and Ca²⁺ transients in isolated cardiomyocytes, these inhibitory effects were more pronounced upon overexpression of wild-type (pentameric) phospholamban. These findings suggest that pentameric assembly of phospholamban may be necessary for mediating its optimal regulatory effects in vivo.

Phospholamban has been proposed to exist as pentamers in native SR membrane, and it is stabilized through interactions between the transmembrane domains. Evidence from several laboratories has indicated that Cys⁴¹ may play an important role in the higher order structure of phospholamban.¹⁹ This site has been suggested to be involved in the packing interface between adjacent helixes, and it appears to be the most intolerant to changes.^{22 27} Cys⁴¹ has been proposed to pack against Leu³⁹ on the neighboring phospholamban helix,²⁷ and it has also been proposed to be located in the cleft within the phospholamban leucine-zipper helical structure.²² However, previous in vitro expression studies indicated that mutation of Cys⁴¹ to Phe⁴¹ in phospholamban did not diminish its inhibitory effects on the affinity of the SR Ca²⁺ transport system for Ca²⁺.¹⁴ This result suggests that substitution of the bulky phenylalanine residue did not alter the phospholamban regulatory effects. Based on these findings, it was hypothesized that the monomeric form of phospholamban is as effective as the pentameric form in regulating SR Ca²⁺ transport. Thus, overexpression of the Cys⁴¹ to Phe⁴¹ mutant phospholamban in the heart was expected to have similar inhibitory effects as overexpression of wild-type phospholamban. However, biochemical assays of cardiac SR Ca²⁺ transport indicated that overexpression of monomeric phospholamban was associated with a decrease in the affinity of the Ca²⁺-ATPase for Ca²⁺, but its effects were not as pronounced as those observed by overexpressing similar levels of wild-type (pentameric) phospholamban. Moreover, overexpression of either monomeric or pentameric phospholamban did not affect the maximal velocity of SR Ca²⁺ transport, in agreement with our previous studies in transgenic mice.^{8 11 12} Consistent with the findings at the subcellular level, the mechanical properties and Ca²⁺ transients in cardiomyocytes from transgenic mice overexpressing monomeric phospholamban were not as depressed as those in cardiomyocytes overexpressing the pentameric form of the protein. Compared with wild-type control phospholamban, overexpression of monomeric phospholamban resulted in an 25% decrease in cardiomyocyte mechanics. By contrast, cardiac-specific overexpression of wild-type phospholamban was associated with dramatic depression (45% to 50%) in the mechanical parameters of the isolated cardiomyocytes, similar to previous observations.¹¹ The alterations in contractile parameters reflected changes in Ca²⁺ kinetics of these cardiomyocytes. The amplitude and the rate of decline of [Ca²⁺]_i were depressed in mutant-phospholamban–overexpressing myocytes, and these inhibitory effects were more pronounced in wild-type phospholamban–overexpressing cardiomyocytes compared with control cardiomyocytes.

The mechanism by which pentameric assembly of phospholamban facilitates its inhibitory effects in vivo is not presently known. Multiple studies using reconstitution and expression systems as well as computational structure modeling have suggested that oligomerization of phospholamban monomers may serve as a fine-tuning mechanism for its regulatory effects on the Ca²⁺-ATPase in vivo and that the pentameric assembly may facilitate allosteric regulation of phospholamban or/and its geometrical coordination with the Ca²⁺ pump.²⁸ Thus, it is possible that the physical interaction between a single monomeric unit of phospholamban and the "regulatory motif" of the Ca²⁺ pump may be less effective for their functional association and signal transduction compared with pentameric assembly. Furthermore, previous studies have indicated that phospholamban may also form Ca²⁺-selective channels in lipid bilayers.²³ The putative transmembrane domain responsible for the pentamerization and the channel properties of phospholamban is composed of bulky hydrophobic amino acids and the three cysteines (Cys³⁶, Cys⁴¹, and Cys⁴⁶). Extensive mutagenesis studies and consequent modeling by Arkin et al²¹ revealed that phospholamban pentameric formation was a left-handed coiled-coil configuration, with a cylindrical ion pore. A recent report has also suggested that phospholamban pentameric association contained a central pore, defined and stabilized by a leucine zipper, with potential relevance to an ion channel.²² Therefore, it is interesting to propose that the differences in the cardiac regulatory effects between pentameric and monomeric phospholamban observed in the present study may be due to the Ca²⁺-selective ion-transfer properties of the pentameric form. Overexpression of phospholamban pentamers would be expected to result in formation of additional Ca²⁺-selective ion channels in the SR membrane, whereas overexpression of mutant phospholamban (eg, phospholamban monomers) would prevent channel formation and thus Ca²⁺ leakage from the SR, compromising its overall inhibitory effects in vivo. Alternatively, phospholamban monomers may be as effective as pentamers in exerting the inhibitory effects of the protein,¹⁴ but substitution of the bulky phenylalanine for cysteine may not allow for proper interaction between phospholamban and the Ca²⁺ pump, resulting in a compromise of the phospholamban regulatory effects. Future studies using other more conservative amino acid substitutions for Cys⁴¹ in phospholamban may help distinguish between the necessity of pentameric structure versus inefficient interaction of mutant phospholamban with the SR Ca²⁺ pump.

Phospholamban has been also shown to be a prominent mediator of the ß-adrenergic responses in the mammalian heart.^{3 8} Thus, it was important to evaluate the effects of isoproterenol on the altered cardiomyocyte mechanics and Ca²⁺ transients upon overexpression of monomeric phospholamban. Isoproterenol stimulated the contractile parameters and Ca²⁺ kinetics in a dose-dependent manner, and the values under maximal isoproterenol stimulation were similar between cardiomyocytes overexpressing monomeric or pentameric phospholamban and control cardiomyocytes. These studies suggest that the depressed mechanics and Ca²⁺ transients in phospholamban-overexpressing myocytes could be relieved by phosphorylation of this protein. Actually, phosphorylation of mutant phospholamban appeared to be similar to that of wild-type phospholamban in vivo (authors' unpublished data, 1997). The present results are in accordance with previous reports in wild-type phospholamban–overexpressing¹¹ and phospholamban-knockout⁸ mouse hearts, indicating that phospholamban is the most important mediator of the cardiac responses to ß-adrenergic receptor stimulation.

In conclusion, the present findings demonstrate that mutation of Cys⁴¹ to Phe⁴¹ in phospholamban prevents its pentameric assembly in native membranes, leading to formation of monomers, as determined by SDS-PAGE. Compared with overexpression of pentameric phospholamban, cardiac-specific overexpression of monomeric phospholamban is associated with less depression of the affinity of the SR Ca²⁺ pump for Ca²⁺, cardiomyocyte mechanics, and Ca²⁺ kinetics, providing evidence that pentameric assembly of phospholamban may be important for the efficient regulation of SR function and cardiac contractility in vivo. However, it remains to be elucidated whether the diminished inhibitory effects of the mutated phospholamban in vivo are due to its monomeric nature, or/and inefficient interaction of this specific mutant with the SR Ca²⁺ pump, or/and its inability to form Ca²⁺-selective ion channels. Ultimately, elucidating the three-dimensional structure of phospholamban and the SR Ca²⁺ pump in native membranes will provide insights into the nature of the phospholamban regulatory effects.

	Selected Abbreviations and Acronyms

 

DCS = donor calf serum MHC = myosin heavy chain PCR = polymerase chain reaction SR = sarcoplasmic reticulum SV = simian virus T80 = time for 80% decay of the Ca2+ signal

	Acknowledgments

 
This study was supported by the National Institutes of Health grants HL-26057, HL-22619, and HL-52318. We wish to thank Dr J. Robbins for providing the murine -MHC promoter, J.C. Neumann for pronuclear microinjection of the transgenic construct, and D.L. Kirkpatrick for excellent technical assistance. We are also grateful to Dr David MacLennan for helpful discussions and critical evaluation of this manuscript.

Received April 23, 1997; accepted July 5, 1997.

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