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(Circulation Research. 1996;78:839-847.)
© 1996 American Heart Association, Inc.

Articles

Phospholamban Gene Dosage Effects in the Mammalian Heart

Wusheng Luo, Beata M. Wolska, Ingrid L. Grupp, Judy M. Harrer, Kobra Haghighi, Donald G. Ferguson, Jay P. Slack, Gunter Grupp, Thomas Doetschman, R. John Solaro, Evangelia G. Kranias

From the Departments of Pharmacology and Cell Biophysics (W.L., I.L.G., J.M.H., K.H., J.P.S., E.G.K.), Physiology and Biophysics (D.G.F., G.G.), and Molecular Genetics, Biochemistry, and Microbiology (T.D.), University of Cincinnati (Ohio), College of Medicine; and the Department of Physiology and Biophysics (B.M.W., R.J.S.), University of Illinois at Chicago, College of Medicine.

Correspondence to Evangelia G. Kranias, PhD, Department of Pharmacology and 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 ablation has been shown to result in significant increases in cardiac contractile parameters and loss of ß-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40±5%) compared with wild-type mice. The reduced phospholamban levels were associated with significant decreases in the EC₅₀ of the sarcoplasmic reticulum Ca²⁺ pump for Ca²⁺ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC₅₀ of the Ca²⁺-ATPase for Ca²⁺ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and contraction in intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.

Key Words: heart • phospholamban • sarcoplasmic reticulum • cardiomyocytes • working heart preparations

	Introduction

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Evidence from several laboratories indicates that the levels of phospholamban decrease in some cardiac diseases,^{1 2 3 4} but the relative contribution of reduced phospholamban expression in the deterioration of myocardial performance is difficult to evaluate, since several other key cardiac proteins are also downregulated in conjunction with phospholamban. In previous work,⁵ we demonstrated that phospholamban is a key regulator of basal myocardial contractility, and ablation of this protein results in hyperdynamic performance.⁵ However, it is not currently clear whether total ablation of phospholamban is required to obtain significant increases in contractile parameters or whether partial reduction in phospholamban expression levels may also result in significant alterations of the myocardial contractile parameters. In the present study, we have addressed the impact of graded reduction in phospholamban levels by comparing SR and cardiac function of phospholamban-deficient mice to that of mice expressing reduced and native phospholamban levels.

Phospholamban, in the dephosphorylated state, has been shown to be an inhibitor of the SR Ca²⁺-ATPase in vitro, and phosphorylation may relieve this inhibition.⁶ Phospholamban can be phosphorylated at distinct sites by different protein kinases: serine 10 by protein kinase C, serine 16 by cAMP-dependent protein kinase, and threonine 17 by Ca²⁺-calmodulin–dependent protein kinase.⁷ Serine 16 is also believed to be the target of cGMP-dependent protein kinase phosphorylation.⁸ Each phosphorylation is associated with stimulation of SR Ca²⁺ transport.^{9 10 11 12 13 14 15} Studies from several laboratories^{9 10 11 12 13 14 15} have linked the stimulatory effects on SR Ca²⁺ transport to an increase in the affinity of the SR Ca²⁺ pump for Ca²⁺, and in some studies an increase in V_max of the Ca²⁺-ATPase was also reported.^{10 14 15 16}

Phospholamban has also been shown to be phosphorylated in situ, in perfused beating hearts, with isoproterenol stimulation.^{17 18 19 20 21 22} The contribution of phospholamban phosphorylation to the inotropic responses of the heart to ß-adrenergic agonists has been the subject of several studies, and it has been suggested that phosphorylation of phospholamban and the accompanying increases in SR Ca²⁺-pump activity are important in mediating the relaxant effects of ß-adrenergic agonists.^{17 18 19 20 21 22} However, the exact role of phospholamban in the regulation of basal myocardial contractility and the relative significance of phospholamban phosphorylation as a determinant of alterations in cardiac dynamics during ß-adrenergic stimulation is not presently well understood.

The availability of the phospholamban-deficient mouse and its parent, the phospholamban-heterozygous mouse, enabled us to further examine the sensitivity of myocardial contractility to alterations in phospholamban levels. An integrative approach was used and studies were performed at the subcellular, cellular, and intact organ levels. Measurements included assessment of the affinity of the SR Ca²⁺-ATPase for Ca²⁺, the rates of contraction and relaxation in isolated myocytes, and the rates of contraction and relaxation in intact beating hearts.

	Materials and Methods

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Targeted Disruption of the Phospholamban Gene in Mouse Embryonic Stem Cells
The murine phospholamban gene was targeted in murine embryonic stem cells as recently described.⁵ One germ-line chimeric mouse was identified and crossed with CF-1 females. The modified phospholamban allele was thereby transmitted to offspring, which were 129xCF-1 hybrids. All experimental animals were generated by intercrossing the hybrids and therefore had a 129xCF-1 background. Wild-type, heterozygous (phospholamban-heterozygous), and homozygous (phospholamban-deficient) mice for the targeted phospholamban allele were generated. Genotypes of all animals were determined by polymerase chain reaction analysis of tail DNA biopsies. The genotypes of the animals used for the characterization studies were also confirmed by Southern blot analysis of cardiac DNA. Age-matched phospholamban-heterozygous and -homozygous mice were phenotypically indistinguishable from wild-type mice at the gross level.

RNA Preparation and Northern Blot Analysis
Total cellular RNA was isolated from the whole hearts of 8- to 10-week-old mice by the acid guanidinium thiocyanate-phenol-chloroform extraction method.²³ For Northern blot analysis, 20 µg of total RNA from heart was fractionated on 1% agarose gels containing formaldehyde and MOPS buffer. The agarose gel was transferred to the Gene Screen Plus (Du Pont) membrane and hybridized with the ³²P-labeled probes in 1 mol/L NaCl, 0.1% SDS, 10% dextran sulfate, 50% formamide, and 100 µg/mL denatured salmon sperm DNA at 42°C for 18 hours. The filters were washed in 2x SSC and 0.1% SDS at room temperature for 10 minutes and at 60°C for 20 minutes. The blots were autoradiographed for 24 to 72 hours. The probes were a 1-kb cDNA fragment corresponding to the coding region and the 3' untranslated region of the phospholamban gene,²⁴ a 60-mer oligonucleotide (P-1: +17 to +77) representing part of the coding region, and a 60-mer oligonucleotide (P-2: +899 to +959) corresponding to a specific sequence of the 3' untranslated region of the gene. The membranes were stripped between probes in boiling water and exposed for 72 hours to test for the complete removal of the probe before hybridizing to the next probe.

Western Blot Analysis
Mouse hearts from 8- to 10-week-old mice were homogenized in a solution containing (in mmol/L) imidazole (pH 7.0) 10, sucrose 300, DTT 1, sodium metabisulfite 1, and PMSF 0.3. Protein concentration was determined by the Bio-Rad method, using BSA for the standard curve. The relative protein levels of phospholamban in cardiac homogenates from wild-type and phospholamban-heterozygous mice were estimated using quantitative immunoblotting. Cardiac homogenate proteins (6.25, 12.5, 25, and 37.5 µg) were separated by SDS polyacrylamide gel electrophoresis (10% to 20% gradient slab gels) and transferred to nitrocellulose membranes. Transblots were reacted with 10 µg monoclonal antibody that recognizes and is specific for bovine, dog, porcine, and rat phospholamban (PHL-Ab, UBI, Inc). The mouse phospholamban is identical to that in rat, and the PHL-Ab specifically recognizes both the oligomeric and monomeric forms of phospholamban in mouse cardiac homogenates, similar to the phospholamban polyclonal antibody.^{6 13} The secondary antibody was ³⁵S-labeled anti-mouse IgG (Amersham). For SR Ca²⁺-ATPase detection, transblots were reacted with a SR Ca²⁺-ATPase polyclonal antibody (SERCA-ab) at 1:500 dilution and visualized using ³⁵S-labeled anti-rabbit secondary antibody (Amersham), as previously described.⁶ The degree of labeling was determined by PhosphorImager and ImageQuant software (Molecular Dynamics) and expressed in relative PhosphorImager units.

Immunofluorescence Labeling
Hearts from 8- to 10-week-old mice were excised and rapidly frozen in liquid nitrogen–cooled isopentane, sectioned using a cryostat microtome, and mounted on cooled glass slides. The sections were incubated for 1 hour with goat anti-mouse Fab fragments (Jackson Immunoresearch) in PBS containing 1% BSA to block nonspecific sites and then incubated for 1 hour in PHL-Ab or an IgG fractionated polyclonal antibody raised to a peptide of the SR Ca²⁺-ATPase.⁶ The excess primary antibody was removed by washing three times (10 minutes each) in PBS containing 1% BSA. The sections were then incubated for 1 hour with goat anti-mouse FITC or donkey anti-rabbit TRITC conjugate for the phospholamban and SR Ca²⁺-ATPase reactions, respectively. The excess secondary label was removed by one 10-minute wash in PBS containing 1% BSA and two 10-minute washes in PBS. The sections were mounted with 1,4-diazabicyclo[2.2.2]octane (Sigma Chemical Co). Fluorescent micrographs were obtained using confocal microscopy.

Ca²⁺ Transport Assays
Frozen hearts from 8- to 10-week-old mice were homogenized in a solution containing (in mmol/L) KH₂PO₄ (pH 7.0) 50, NaF 10, EDTA 1, sucrose 300, PMSF 0.3, and DTT 0.5. Oxalate-supported Ca²⁺-uptake rates into SR vesicles in the cardiac homogenates (0.1 mg/mL) were determined as previously described.⁵

Mouse Cardiomyocyte Mechanical Measurements
Cells were isolated essentially as previously described by us,²⁵ with some important modifications to obtain stable, Ca²⁺-tolerant myocytes. Briefly, these modifications included: (1) introduction of 10 mmol/L butanedione monoxime into the solution during cannulation of the heart, (2) addition of 1 mg/mL BSA to the solutions used for isolation, and (3) usage of selected Worthington collagenases type I (75 U/mL) and type II (75 U/mL), based on mechanical stability of the myocytes.

After isolation, the cells were placed in a small perfusion chamber mounted on the stage of an inverted microscope. The cells were allowed to settle on the surface of the chamber, and after 2 to 3 minutes they were perfused with control solution of the following composition (in mmol/L): NaCl 133.5, KCl 4.0, NaH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.0, HEPES 10, and glucose 11 (pH 7.4). To measure cell shortening, the myocyte image was collected by a 40x Nikon objective and transmitted to a multi-image module. The output from the camera was connected into a VCR, and the image of the cell was projected onto a TV monitor. A video-edge detector²⁶ was used to monitor cell length. The cell length signal was recorded on a Gould WindowGraf chart recorder and also on the computer.

Fig 1 depicts a record of cell-length changes and shows schematically how parameters of contraction and relaxation were determined. We compared the time course of the contraction by dividing %L by TTP. We also compared the time course of relaxation by dividing %L_0.5 by RT_0.5 (Fig 1).

View larger version (10K): [in this window] [in a new window]   Figure 1. Schematic representation of the contraction-relaxation cycle in isolated cardiac myocytes.

Mouse Heart Perfusion
Hearts from 8- to 10-week-old mice of either sex were perfused in a work-performing mode under identical load conditions.^{5 27 28 29} The minimum venous return and the afterload required to maintain continuous function of the hearts without any changes in end-diastolic and left atrial pressure were 5 mL/min and 50 mm Hg, respectively. The perfusion fluid, oxygenation, and temperature were constant.

Isoproterenol infusion was achieved by subjecting the work-performing mouse heart preparations to continuous infusion of increasing concentrations of isoproterenol, from 0.8 to 800 nmol/L.⁵

Statistical Analysis
Data are presented as mean±SEM. The number (n) of mice used is indicated. Statistical analysis was performed using the t test of paired comparisons or using an analysis of variance (ANOVA) and the Student-Newman-Keuls test for multiple comparisons. Correlations were tested by least-squares linear regression analyses, using MicroCal Origin software. Values of P<.05 were considered statistically significant.

	Results

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Phospholamban Levels in Mutant Mice
Phospholamban-deficient mice expressed no phospholamban message or protein, as previously reported.⁵ Phospholamban-heterozygous hearts expressed the same multiple phospholamban transcripts that were present in wild-type hearts,²⁴ but in reduced levels. To determine whether ablation of one of the phospholamban alleles was associated with alterations in the relative abundance of a specific phospholamban transcript, total RNA was isolated in parallel from wild-type and phospholamban-heterozygous hearts and then hybridized with P-1 (Fig 2A). Two major transcripts (0.7- and 2.8-kb) and one minor transcript (1.5-kb) were detected, and the relative abundance of these transcripts was similar in wild-type and heterozygous hearts: 45% for the 2.8-kb, 8% for the 1.5-kb, and 47% for the 0.7-kb species. Similar results were obtained when hybridizations were performed using a phospholamban cDNA probe. Hybridization with P-2 revealed the presence of the 1.5-kb and 2.8-kb transcripts in both wild-type and phospholamban-heterozygous hearts (Fig 2B). The relative abundance of these transcripts was also similar between wild types and heterozygotes. These findings indicate that disruption of one of the phospholamban alleles did not alter the transcriptional regulation of the wild-type allele in phospholamban-heterozygous hearts.

View larger version (45K): [in this window] [in a new window]   Figure 2. Northern blot analysis of RNA isolated from wild-type (+/+) and phospholamban-heterozygous (+/-) hearts. Total RNA was fractionated, transferred onto Gene Screen Plus membranes, and hybridized with P-1 (A) and P-2 (B) as described under "Materials and Methods."

To assess the degree of phospholamban protein reduction in the heterozygous hearts, the phospholamban protein levels were determined using quantitative immunoblotting. The labeling of phospholamban by the PHL-Ab was proportional to the amount of cardiac homogenate protein electrophoresed in the range of 6.25 to 37.5 µg protein (Fig 3). The relative levels of phospholamban in heterozygous hearts were 40±5% of the phospholamban protein levels present in wild-type hearts (Fig 3B). Furthermore, indirect immunofluorescence indicated that the pattern of phospholamban distribution (Fig 4A) was similar to that of the SR Ca²⁺-ATPase (Fig 4B) in phospholamban-heterozygous hearts, suggesting that the reduction in phospholamban expression was uniform among cardiac myocytes.

View larger version (32K): [in this window] [in a new window]   Figure 3. Quantitation of phospholamban protein levels in hearts isolated from wild-type (+/+) and phospholamban-heterozygous (+/-) mice. A, Autoradiogram of the linear range of phospholamban detection in Western blots. Three hearts were pooled from each group, and increasing amounts of cardiac homogenate (6.25, 12.5, 25, and 37.5 µg) were electrophoresed. Only the pentameric form of phospholamban is shown here, since the signal for the monomer was below detection. B, Quantitation of phospholamban protein levels in the phospholamban-heterozygous hearts relative to wild-type hearts. The signal obtained on the blots is expressed in relative PhosphorImager units (pixel values). The symbols represent the mean±SEM of five experiments, each performed in triplicate.

View larger version (107K): [in this window] [in a new window]   Figure 4. Indirect immunofluorescence localization of phospholamban and the SR Ca2+-ATPase in phospholamban-heterozygous mouse hearts. A, Immunolocalization of phospholamban in atrium. All of the cells were stained, indicating that phospholamban is expressed in every cell. Furthermore, the level of staining in each cell appears equivalent, indicating that all of the cells are expressing similar amounts of phospholamban. B, Immunolocalization of the SR Ca2+-ATPase in atrium. All of the cells in this serial longitudinal section are also stained, and the level of staining is the same in each cell. Bar=10 nm.

Sarcoplasmic Reticulum Ca²⁺ Uptake
Phospholamban has been shown to be the regulator of the Ca²⁺-ATPase activity in cardiac SR, and ablation of phospholamban resulted in significant increases in the affinity of the SR Ca²⁺ pump for Ca²⁺.⁵ To determine whether the reduction in phospholamban protein levels in heterozygous mice was also associated with alterations in SR Ca²⁺ uptake, the initial rates of Ca²⁺ uptake were assayed in unfractionated SR vesicles in homogenates of heterozygous hearts (Fig 5). The incubation conditions under which Ca²⁺ uptake is restricted to SR vesicles in the homogenate have been defined, and the validity and advantages of this approach have been previously reported.^{5 17 30} Ca²⁺-uptake rates by phospholamban-heterozygous hearts were higher than those by wild-type hearts, especially at low [Ca²⁺]. Analysis of these data indicates that the EC₅₀ of the SR Ca²⁺ pump for Ca²⁺ was 0.18±0.02 µmol/L (n=6) for phospholamban-heterozygous compared with 0.24±0.02 µmol/L (n=7) for wild-type hearts. However, there was no effect on V_max of the SR Ca²⁺-ATPase (89±4 versus 92±4 nmol Ca²⁺·mg^-1·min^-1, n=6, for heterozygous versus wild-type hearts), consistent with previous observations in phospholamban-deficient hearts.⁵ These findings, together with studies on the SR Ca²⁺-ATPase protein levels using quantitative immunoblotting (data not shown), indicated that reduction or ablation of phospholamban protein levels had no effect on SR Ca²⁺-ATPase expression levels. Thus, the relative ratio of phospholamban to SR Ca²⁺-ATPase was set as 1.0 in wild-type hearts, 0.4±0.05 in phospholamban-heterozygous hearts, and 0 in phospholamban-deficient hearts. When the EC₅₀ values of the SR Ca²⁺ pump for Ca²⁺ in wild-type, phospholamban-heterozygous, and phospholamban-deficient hearts (EC₅₀=0.10±0.02, n=8) were plotted against the relative ratios of phospholamban to the SR Ca²⁺-ATPase in these hearts, a close correlation was observed (Fig 5, inset).

View larger version (18K): [in this window] [in a new window]   Figure 5. Calcium uptake in cardiac homogenates from phospholamban-heterozygous () and wild-type () hearts. The initial rates of Ca2+ uptake by sarcoplasmic reticulum vesicles in homogenates were assayed over a wide range of Ca2+ concentrations. Values are mean±SEM of six different hearts in each group. Inset shows relation between the ratio of phospholamban (PLB) to SR Ca2+-ATPase levels in wild-type (), phospholamban-heterozygous (), and phospholamban-deficient () hearts and the EC50 of the SR Ca2+-ATPase for Ca2+. The linear relation between the PLB/SR Ca2+-ATPase and EC50 is given by the regression line y=0.108+0.147x (r=.99).

Cardiac Myocyte Contractile Parameters
To assess the physiological significance of the SR biochemical changes in phospholamban-heterozygous and phospholamban-deficient hearts, cardiac myocytes were isolated from these animal models in parallel with wild-type hearts, and their contractile parameters were compared (Fig 6). The phospholamban-deficient cardiac myocytes demonstrated a significant increase in cell shortening (21.5±1.6, n=8) compared with phospholamban-heterozygous (11.9±1.4, n=9) and wild-type (9.3±1.6, n=7) cardiac myocytes; there was no significant difference between phospholamban-heterozygous and wild-type cardiac myocytes. Both the phospholamban-heterozygous and phospholamban-deficient cardiac myocytes exhibited significant decreases in TTP and RT_0.5 compared with wild types (Table 1). To further characterize contractile parameters, we compared the rates of contraction (%L/TTP) and relaxation (%L_0.5/RT_0.5) in cardiac myocytes from the three groups. The rate of relaxation increased by 2.2-fold and the rate of shortening increased by 2.0-fold in the phospholamban-heterozygous hearts compared with wild-type hearts. The increases in these parameters were even higher in the phospholamban-deficient myocytes, in which the rates of relaxation and shortening increased by 4.5-fold and 3.6-fold, respectively, compared with wild-type myocytes. The mechanical parameters corresponding to the rates of myocyte shortening and relaxation were plotted against the relative levels of phospholamban to SR Ca²⁺-ATPase in wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts (Fig 7). Although the levels of phospholamban were assessed in whole hearts composed of atrial and ventricular muscle, which differentially express phospholamban,³¹ they correlated well with mechanical function in ventricular myocytes. This is probably because the ventricular contribution in protein determinations using whole hearts is estimated to be >95% of the total.

View larger version (8K): [in this window] [in a new window]   Figure 6. Typical examples of shortening of cardiac myocytes isolated from wild-type (A), phospholamban-heterozygous (B), and phospholamban-deficient (C) mouse hearts.

View this table: [in this window] [in a new window]   Table 1. Effect of Phospholamban Levels on Contractile Parameters in Cardiac Myocytes

View larger version (17K): [in this window] [in a new window]   Figure 7. Graph showing the relationships between the rates of myocyte shortening and relengthening and the relative ratio of phospholamban (PLB) to SR Ca2+-ATPase levels in the heart. The myocyte values of contraction (%L/TTP, ) and relaxation (%L0.5/RT0.5, ) in the three groups of mice, assessed in parallel (Table 1), were plotted against the phospholamban levels in age-matched phospholamban-deficient, phospholamban-heterozygous, and wild-type hearts. The linear relations between PLB/SR Ca2+-ATPase and myocyte contractile parameters are given by the regression lines y=0.234-0.175x (r=.97) for %L/TTP and y=0.224-0.182x (r=.96) for %L0.5/RT0.5.

Cardiac Contractile Parameters in Work-Performing Hearts
Hearts from phospholamban-deficient mice have previously been shown to exhibit significant increases in their contractile parameters compared with wild types.⁵ To determine whether decreased phospholamban expression would also result in enhanced myocardial performance, phospholamban-heterozygous hearts were studied in parallel with wild-type hearts under identical afterload (50 mm Hg mean aortic pressure) and venous return (an approximation of preload: 5 mL/min) conditions. Although the body weight (26 to 28 g), heart weight (200 mg), heart rate (380 bpm), cardiac power (250 mm Hg·mL^-1·min^-1), and stroke volume (13 µL) were similar between phospholamban-heterozygous and wild-type hearts, the left intraventricular pressure was significantly different (Table 2). The maximal rates of pressure development (dP/dt) were also significantly higher in the phospholamban-heterozygous hearts compared with wild-type hearts (Table 2). In addition, contraction and relaxation times normalized per millimeter Hg of peak pressure (TPP per millimeter Hg and RT_0.5 per millimeter Hg, both expressed in milliseconds per millimeter Hg) were calculated.²⁷ These contractile parameters were significantly shorter than the values exhibited by wild-type hearts (Table 2). However, both the contraction and relaxation times were significantly longer than the corresponding values in phospholamban-deficient hearts (Table 2).

View this table: [in this window] [in a new window]   Table 2. Effect of Phospholamban Levels on Contractile Parameters in Mouse Hearts

When the contraction and relaxation parameters in wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts were plotted against the relative levels of phospholamban to SR Ca²⁺-ATPase in these hearts, a close linear correlation was obtained for both the rates of relaxation (-dP/dt) and contraction (+dP/dt) (Fig 8) and the times for relaxation (RT_0.5) and contraction (TPP) (data not shown). These observations in intact beating hearts were similar to those in isolated myocytes (Fig 7), described above.

View larger version (19K): [in this window] [in a new window]   Figure 8. Graph showing the relationships between the contractile parameters in work-performing heart preparations and the relative ratio of phospholamban (PLB) to SR Ca2+-ATPase levels. The rates of contraction (+dP/dt, ) and relaxation (-dP/dt, ) in age-matched phospholamban-deficient, phospholamban-heterozygous, and wild-type hearts were recorded in parallel at similar heart rate, mean aortic pressure (afterload), cardiac output (venous return), cardiac power (mean aortic pressurexcardiac output), and stroke volume (cardiac output/heart rate). These contractile values, shown in Table 2, were plotted against the PLB/SR Ca2+-ATPase in phospholamban-deficient, phospholamban-heterozygous, and wild-type hearts. The linear relations between PLB/SR Ca2+-ATPase and cardiac contractile parameters are given by the regression lines y=5098-1952x (r=.99) for +dP/dt and y=4907-2315x (r=.99) for -dP/dt.

Since phospholamban has been suggested to be an important determinant in the isoproterenol responses in vivo, phospholamban-heterozygous hearts were also subjected to perfusion with various concentrations of this ß-agonist, and the stimulatory effects on contractile parameters were determined. Maximal isoproterenol stimulation was associated with a 1.5-fold increase in the rate of relaxation and a 1.3-fold increase in the rate of contraction in phospholamban-heterozygous hearts. In parallel studies, the rates of relaxation and contraction were increased by 2.1-fold and 1.7-fold, respectively, in isoproterenol-stimulated wild-type hearts. The maximally stimulated contractile parameters were similar between phospholamban-heterozygous and wild-type hearts (Table 2). Furthermore, these parameters in isoproterenol-stimulated hearts were similar to the basal parameters in phospholamban-deficient hearts (Table 2), which could not be further stimulated by ß-agonists.⁵ When the maximal increases in the rates of relaxation and contraction on isoproterenol stimulation were plotted against the relative levels of phospholamban to SR Ca²⁺-ATPase in wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts, close linear correlations were observed (Fig 9).

View larger version (23K): [in this window] [in a new window]   Figure 9. Graph showing the relationships between the maximally stimulated contractile parameters by isoproterenol and the relative ratio of phospholamban (PLB) to SR Ca2+-ATPase in the hearts. The increases in the rates of contraction (+dP/dt, ) and relaxation (-dP/dt, ) in phospholamban-deficient, phospholamban-heterozygous, and wild-type hearts were recorded in parallel under maximal isoproterenol stimulation. The linear relations between PLB/SR Ca2+-ATPase and contractile responses are given by the regression lines y=99.5+67.5x (r=.99) for +dP/dt and y=104.5+106.8x (r=.99) for -dP/dt in these hearts.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our experiments are the first to demonstrate a close linear correlation between the relative levels of phospholamban in the mouse heart and (1) the affinity of the SR Ca²⁺ pump for Ca²⁺; (2) the rates of contraction and relaxation in isolated, beating hearts; and (3) the rates of contraction and relaxation in isolated ventricular myocytes. The development of mutant mice, using gene-targeting technology in embryonic stem cells, and the ability to assess murine cardiac function at the cellular and organ levels have provided the opportunity to unambiguously determine the function of phospholamban in myocardial contractility.

The phospholamban-mutant mice were generated by targeting the phospholamban-coding region in exon 2 of the phospholamban gene in embryonic stem cells.⁵ Mice heterozygous for the disrupted allele were generated, and mating of the phospholamban-heterozygous mice yielded offspring homozygous for the mutation, or phospholamban-deficient mice. The phospholamban-heterozygous mice expressed reduced phospholamban message levels, but the relative abundance of each of the phospholamban multiple transcripts²⁴ was similar between heterozygous and wild-type hearts. Thus, disruption of one of the phospholamban alleles did not alter the transcriptional regulation of the wild-type allele in the phospholamban-heterozygous mice. Multiple phospholamban mRNA species generated by different polyadenylation signals have also been previously observed in dog heart,³² rabbit heart and rabbit slow-twitch skeletal muscle,³³ and pig atrium,³⁴ but the significance of these multiple phospholamban mRNA species is not presently known. Reduction in phospholamban mRNA levels was associated with reduced phospholamban protein levels in heterozygous hearts. It is interesting to note that disruption of one of the phospholamban alleles resulted in about half (40±5%) of the phospholamban protein levels present in wild-type hearts. The reduction in phospholamban protein levels reflected a significant decrease in the EC₅₀ of the SR Ca²⁺ pump for Ca²⁺ in phospholamban-heterozygous hearts. There were no alterations in the maximal velocity of the SR Ca²⁺-ATPase, consistent with previous findings in phospholamban-deficient hearts.⁵ A comparison between the relative levels of phospholamban to SR Ca²⁺-ATPase and the EC₅₀ values of the SR Ca²⁺ pump for Ca²⁺ in wild-type, phospholamban-heterozygous, and phospholamban-deficient hearts revealed a close linear correlation, indicating the importance of phospholamban in modulating this property of the SR Ca²⁺-transport system. However, the exact stoichiometric relationship between phospholamban and the SR Ca²⁺-ATPase is not presently known. Values ranging from 2:1 to 1:5 for phospholamban:Ca²⁺-ATPase have been previously reported.^{35 36 37 38} Furthermore, we have recently shown that a twofold increase in phospholamban expression levels in vivo is associated with an increase in the EC₅₀ of the SR Ca²⁺ pump for Ca²⁺.³⁹

The functional significance of alterations in phospholamban levels and SR Ca²⁺-transport activity was reflected by alterations in basal cardiac contractile parameters and their responses to ß-adrenergic stimulation, assessed in intact beating heart preparations. In the present study, an inverse relationship between the levels of phospholamban in the heart and the rates of both relaxation and contraction was observed. However, on isoproterenol stimulation and full reversal of the phospholamban inhibitory effects, the contractile parameters between wild-type, phospholamban-heterozygous, and phospholamban-deficient hearts were similar. The maximal increases in the rates of left ventricular relaxation and contraction elicited by isoproterenol correlated well with the relative levels of phospholamban in the heart. Although phosphorylation of other regulatory proteins, such as SR Ca²⁺-ATPase,⁴⁰ troponin I,^{17 18} ryanodine receptor,⁴¹ and phospholemman,⁴² may also contribute to the stimulatory effects of ß-agonists, our findings suggest that phospholamban is a key determinant of the ß-adrenergic responses in the heart.

An important question is whether the phospholamban regulatory effects on cardiac basal contractile parameters may also be observed in isolated cardiac myocytes. The myocytes provide a preparation devoid of the effects of left ventricular chamber geometry, fiber orientation, and the extracellular matrix for examining the effects of altered phospholamban gene expression. Reduction or ablation of phospholamban was associated with enhancement of the rates of both contraction and relaxation in isolated myocytes, similar to findings in work-performing heart preparations. Furthermore, comparison of the phospholamban levels and the contractile parameters revealed close linear correlations in myocytes from wild-type, phospholamban-heterozygous, and phospholamban-deficient hearts. Thus, the ability to assess contractile parameters in isolated ventricular myocytes and isolated work-performing heart preparations enabled us to link biochemical alterations at the SR level with physiological alterations at the cellular and organ levels. However, it remains to be determined whether compensatory mechanisms and differences in Ca²⁺ currents or the expression levels of other proteins involved in the excitation-contraction pathway may also contribute to the observed alterations in contractile parameters.

In summary, our findings indicate that there is a direct relationship between the relative phospholamban levels and the contractile parameters in the mammalian heart. Thus, control of the levels or activity of phospholamban should permit fine regulation of SR Ca²⁺-pump function and contractility, which may be beneficial in heart disease. The role of phospholamban in heart disease and specifically heart failure is not presently well understood. In some studies, there were no apparent alterations in phospholamban levels,^{43 44 45} whereas in other studies, a reduction in phospholamban mRNA^{1 2 3} or protein⁴ levels was noted in human failing hearts. Our data suggest that even a small reduction in phospholamban levels would translate into enhancement of cardiac contractile parameters, and it is interesting to propose that downregulation of phospholamban, either through cellular responses or therapeutic devices, would provide an important compensatory mechanism in the failing heart.

	Selected Abbreviations and Acronyms

 

%L = percent shortening %L0.5 = half-amplitude of shortening kb = kilobase P-1, P-2 = probes 1 and 2, respectively RT0.5 = half-time of relaxation SR = sarcoplasmic reticulum TTP = time to peak contraction

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL-26057, HL-22619, and HL-07382 and by a fellowship (Dr Wolska) from the American Heart Association of metropolitan Chicago. We wish to thank Z. Zhou, G. Newman, and M. Tosun for excellent technical assistance.

Received September 29, 1995; accepted February 12, 1996.

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