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(Circulation Research. 1997;81:493-503.)
© 1997 American Heart Association, Inc.

Articles

Interleukin-1ß Inhibits Phospholamban Gene Expression in Cultured Cardiomyocytes

Charles F. McTiernan, Bonnie H. Lemster, Carole Frye, Steven Brooks, Alain Combes, , Arthur M. Feldman

From the Division of Cardiology, University of Pittsburgh (Pa) Medical Center.

Correspondence to Charles F. McTiernan, PhD, Division of Cardiology, University of Pittsburgh, Biomedical Science Tower 1744.1, 200 Lothrop St, Pittsburgh, PA 15213. E-mail mctier{at}card2.cath.upmc.edu

	Abstract

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Abstract Phospholamban is a key regulatory protein that defines diastolic function. Proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor- (TNF-) can depress contractility and intracellular Ca²⁺ currents and transients. An alteration in phospholamban expression is a possible pathway by which these cytokines modulate cardiac function. To test this hypothesis, primary cultures of neonatal rat cardiomyocytes were incubated with IL-1ß, TNF-, or both, and the level of phospholamban transcripts was examined by Northern blot analyses. Phospholamban transcript levels were decreased 50% (P<.0001) in cells exposed to 2 ng/mL IL-1ß (20 hours), whereas TNF- had no effect. Western blot analyses showed that IL-1ß also reduced phospholamban protein levels (60% of control, P<.0001). The effects on transcript levels were gene specific; IL-1ß induced transcripts for inducible NO synthase (iNOS), did not alter GAPDH transcripts, and reduced sarcoplasmic reticulum Ca²⁺-ATPase (65% of control, P<.001) transcripts. Cardiomyocytes treated with IL-1ß showed no alterations in basal contractile parameters (maximum velocity of contraction and relaxation and maximal amplitude of contraction) but were unresponsive to ß-adrenergic stimulation. Studies performed in the presence of second-messenger inhibitors showed that the effect of IL-1ß on phospholamban transcript levels was blocked by dexamethasone, was insensitive to inhibitors of iNOS, cyclooxygenase, or tyrosine kinases, but was enhanced by the addition of the protein kinase inhibitor staurosporine. These data demonstrate that IL-1ß alters the expression of phospholamban, a key regulator of cardiac contractility, at both the transcript and protein levels. The results suggest novel mechanisms by which IL-1ß may modify cardiac function.

Key Words: molecular biology • cardiomyocyte • proinflammatory cytokine • RNA • phospholamban

	Introduction

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Phospholamban, a small pentameric protein in the SR, regulates myocardial Ca²⁺ homeostasis by decreasing the affinity of SERCA for Ca²⁺ and reducing the rate of Ca²⁺ uptake into the SR.¹ When phosphorylated by cAMP-dependent² or Ca²⁺-calmodulin–dependent protein kinases,³ this inhibition of SERCA is reversed. Therefore, alterations in phospholamban and/or SERCA activity can regulate Ca²⁺ uptake and the duration of Ca²⁺ transients and the diastolic interval. A direct observation of this relationship is seen in phospholamban-deficient mice generated by targeted gene ablation. These mice exhibit SERCA with an increased affinity for Ca²⁺, display enhanced myocardial contractile parameters, and lack responsiveness to ß-adrenergic stimulation.⁴ Several other model systems demonstrate that changes in the level of phospholamban expression are also associated with alterations in contractile function.^{5 6 7} Numerous studies have demonstrated a decreased cardiac expression of the mRNA for phospholamban and SERCA in failing human heart^{8 9 10} and experimental animal models,^{11 12 13 14} although corresponding changes in protein levels remains controversial.^{10 15 16 17} However, it appears clear that variations in SERCA and phospholamban expression may alter the ratio of phospholamban to SERCA protein and modify diastolic function. Although hyperthyroidic and hypothyroidic states have been reported to alter phospholamban expression,¹⁸ the molecular signals that downregulate phospholamban expression during cardiac dysfunction have not been identified.

Recent studies suggest that proinflammatory cytokines (such as IL-1ß and TNF-) participate in depressing cardiac function. These cytokines, traditionally characterized by their role in the inflammatory response, have pleiotropic effects on diverse cell types. Many studies suggest the role of these cytokines in the depressed cardiac function of burn shock,¹⁹ sepsis,^{20 21} myocarditis,²² transplant rejection,²³ and heart failure.^{24 25} Experimentally, IL-1ß and TNF- depress contractile function in intact animals (References 26 and 27^{26 27} and references therein), isolated hearts,^{28 29} isolated papillary muscles,^{30 31} and cardiomyocytes.^{29 32}

IL-1ß and TNF- may alter cardiac function through changes in gene expression. In neonatal rat cardiomyocytes, IL-1ß causes cellular hypertrophy,^{33 34} decreases transcript levels for genes involved in Ca²⁺ movement (voltage-gated Ca²⁺ channel and Ca²⁺ release channel³³ and SERCA^{33 35} ), and induces ß-myosin heavy chain and atrial natriuretic factor transcripts.³³ IL-1ß and TNF- also activate iNOS expression.^{36 37 38 39} These cytokines can alter basal^{21 29} as well as adrenergic-sensitive cardiomyocyte contractility^{32 39} and diminish intracellular Ca²⁺ transients.^{29 40 41} The sum profile of these changes resembles several pathophysiological aspects of heart failure.³³

These findings led us to hypothesize that IL-1ß and TNF- may also modify cardiac function through altered expression of phospholamban. To approach this question, we treated neonatal rat cardiomyocytes with IL-1ß and TNF-, determined phospholamban expression at both the transcript and protein level, and assessed contractile properties of the treated cells.

	Materials and Methods

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Cultured Neonatal Cardiomyocytes
Neonatal cardiomyocytes were prepared from ventricles of 1-day-old Sprague-Dawley rats using a commercially available isolation procedure⁴² (Worthington Biochemical). Recovered cells were preplated on untreated plastic flasks for 1 hour to reduce nonmyocyte cell numbers. Nonadherent cells enriched for cardiomyocytes were cultured in DF-5% medium, which is DMEM/F12 (Mediatech) containing 5% horse serum (Life Technologies Inc), 1 mmol/L glutamine (Mediatech), 10 mmol/L HEPES (Mediatech), 0.1 mmol/L bromodeoxyuridine (Sigma Chemical Co), 5 µg/mL insulin, 5 ng/mL selenium, and 5 µg/mL transferrin (ITS, Sigma), and 10 µg/mL gentamicin (Sigma). To remove the possible effects of thyroid hormone, horse serum was treated with AG1X-10 (Pharmacia) resin as described previously⁴³ to reduce serum triiodothyronine to undetectable levels as determined by radioimmunoassay (Diagnostic Products). All cells were cultured in DF-5% for 24 hours before the initiation of experiments. Studies were also performed in which the cells were switched from DF-5% to DF-ITS (the same medium lacking horse serum) for an additional 24 hours before the initiation of experiments, at which time the cells were maintained in DF-ITS. For experiments performed in the presence of the iNOS inhibitor L-NMMA (see below), cells were placed into arginine-free medium (Select-Amine, Life Technologies Inc) containing 5% horse serum and ITS, HEPES, bromodeoxyuridine, and gentamicin (described above) for 4 hours before the addition of either 1 mmol/L L-arginine or 1 mmol/L L-NMMA. Cells were plated at a density of 1x10⁵ cells/cm² on Pronectin (Promega)–coated tissue culture plates and grown at 37°C in 5% CO₂. Experiments were initiated 24 to 36 hours after cells were plated.

To determine the percentage of nonmyocyte cells in the cardiomyocyte preparations, cells were grown on coated glass coverslips and stained with antibody to myosin heavy chain. Cells were washed twice in ice-cold PBS and fixed with a 1:1 mixture of methanol and acetone. After fixation, cells were incubated in 1:10 diluted goat serum (Sigma) to limit background staining and subsequently stained using a monoclonal anti–sarcomeric myosin antibody (MF20,⁴⁴ Developmental Studies Hybridoma Bank) in a 1:2 dilution. Slides were then washed with PBS and incubated with secondary TRITC-labeled goat anti-mouse antibody (Sigma) in 1:100 dilution. Slides were viewed with an inverted phase immunofluorescence microscope (Nikon). Assessed by this method, the cell preparations routinely contained >95% cardiomyocytes.

Treatment with Cytokines, IL-1ß–Neutralizing Antibody, and Second-Messenger Inhibitors
Experiments were initiated by the addition of fresh medium containing recombinant mouse IL-1ß and/or recombinant rat TNF- (both from Biosource International) or their vehicle (PBS). When necessary, fresh medium containing the same cytokines was added after 24 hours. Experiments were terminated by removal of the medium and freezing of culture plates on liquid nitrogen. Plates were stored at -80°C before isolation of RNA.

To confirm that the effects of IL-1ß treatment were not due to contaminating endotoxin, two separate approaches were taken. Experiments were performed to determine whether the effects induced by IL-1ß could be prevented through pretreatment of the IL-1ß with a rabbit polyclonal neutralizing anti–IL-1ß antibody (BioSource Intl). By following the guidelines of the manufacturer, IL-1ß (4.5 ng) was incubated with 560 µg anti–IL-1ß antibody in 1 mL DF medium without additives at 37°C for 20 minutes and then diluted with complete culture medium to yield a final concentration of 0.5 ng/mL IL-1ß. This medium was then added to neonatal cardiomyocytes for 20 hours; parallel cultures contained 0.5 ng/mL IL-1ß incubated in medium lacking the neutralizing antibody. In the second approach, IL-1ß (5 ng in 0.5 mL of DF medium) was placed in boiling water for 20 minutes, cooled to 37°C, diluted in complete medium to 0.5 ng/mL, and added to neonatal cardiomyocytes for 20 hours. Control cells received only heat-treated vehicle (DF medium). At the end of 20 hours, cells were frozen as described above.

We also performed experiments with specific inhibitors of signal transduction pathways to identify potential mechanisms by which IL-1ß may alter phospholamban expression. These inhibitors were used at the following concentrations: 3.3 µmol/L dexamethasone (Sigma), 1 mmol/L L-NMMA (Chem Biochem Research), 50 µmol/L indomethacin (Sigma), 20 µmol/L genistein (Sigma), and 5 nmol/L staurosporine (Sigma). The solvents and their final concentrations were either <0.02% dimethyl sulfoxide (genistein) or <0.2% ethanol (all other inhibitors). Control cultures were treated with inhibitor alone. Inhibitors were added to cell cultures 30 minutes before the addition of cytokines. After 20 hours of culture, cells were frozen as described.

cGMP Determinations
To demonstrate that the L-NMMA treatment inhibited NO production, cGMP levels were measured as a test for NO synthesis. cGMP levels were determined with a standard radioimmunoassay kit for cGMP (Biomedical Technologies Inc). Cells were cultured in DF-ITS for 24 hours and then placed into arginine-free medium (Select-Amine, Life Technologies Inc) containing the supplements described above for 4 to 6 hours. L-Arginine or L-NMMA (1 mmol/L) was then added to the culture medium 30 minutes before the addition of IL-1ß (2 ng/mL). After an additional 20 hours of culture, 3-isobutyl-1-methylxanthine (Sigma) was added to a final concentration of 0.1 mmol/L for 30 minutes. Medium was then removed, cells were rinsed in PBS, and 0.1 mol/L HCl in PBS was added. Cells were scraped, heated in a boiling water bath for 10 minutes, placed on ice for 10 minutes, frozen in liquid nitrogen, and stored at -80°C. Samples were then thawed, lyophilized to dryness, resuspended in 1 mL acetate buffer (50 mmol/L sodium acetate [pH 6.2]), and centrifuged at 12 000g for 10 minutes, and the supernatant was used in the radioimmunoassay. Pellets were solubilized, and protein values were determined by using a modified Bradford reaction (Bio-Rad) with IgG as a protein standard. Results are reported as picomoles cGMP per milligram protein.

Incorporation of [³H]Leucine
Neonatal cardiomyocytes were cultured in DF-5% medium for 24 hours in 12-well plates. Cells were then rinsed with HBSS (Mediatech), and fresh medium (either DF-5% or DF-ITS) was added. After 24 hours, fresh medium that contained 2 µCi/mL [³H]leucine (DuPont NEN) was added, and the experiment was initiated by the addition of solvent or IL-1ß (final concentration, 2 ng/mL). Cells received fresh medium every 24 hours. At the time of harvest (after 24, 48, or 72 hours of incubation in medium containing [³H]leucine), cells were washed with ice-cold PBS, and 1 mL ice-cold 5% TCA was added to each well. Plates were rocked for 30 minutes at 4°C, the TCA solution was removed, and precipitated proteins were washed with ice-cold 5% TCA. Precipitated proteins were then dissolved in 0.2 mL 1% SDS, and one half of each sample was taken for determination of [³H]leucine incorporation. Results are reported as incorporated ³H dpm/culture well.

RNA Isolation, Northern Hybridizations, and Analysis
Total RNA was isolated from neonatal cardiomyocytes by an acid-phenol extraction method⁴⁵ and quantified by spectrophotometry. RNA (2 to 5 µg) was electrophoresed in formaldehyde-agarose gels and transferred to nitrocellulose. Hybridization probes were prepared from the following sources: (1) rat phospholamban cDNA,⁴⁶ (2) rabbit slow/cardiac-type SERCA cDNA,⁴⁷ (3) rat GAPDH cDNA,⁴⁸ and (4) a 24-mer oligonucleotide encoding rat 18S ribosomal RNA.⁴⁹ cDNA probes were radiolabeled to at least 1x10⁸ cpm/µg using a random hexamer priming kit (Boehringer-Mannheim). Radiolabeled cDNA probes were hybridized overnight at 42°C in 50% formamide, 10% dextran sulfate, 5x SSC, 25 mmol/L sodium phosphate, 5x Denhardt's solution, and 0.2% SDS. Filters received a final wash in 0.2x SSC/0.1% SDS at 60°C. Radioactive images from hybridizations were obtained with a PhosphorImager (Molecular Dynamics) and quantified with ImageQuant software (Molecular Dynamics). Filters were then stripped and rehybridized with the 18S oligonucleotide. The 18S rRNA oligomer probe (labeled using T4 polynucleotide kinase) was hybridized overnight at 50°C in 6x SSC, 0.1% SDS, 0.05% sodium pyrophosphate, and 1x Denhardt's solution; filters were washed at room temperature in 3x SSC/0.1% SDS. Filters were then reexposed and quantified, and results of the cDNA hybridization were normalized to results of the 18S probe to correct for differences in RNA mass and efficiency of transfer. Data were in turn normalized to the mean of the control samples, arbitrarily set at 100%.

RT-PCR Analysis
A 1 µg aliquot of RNA was reverse-transcribed with Superscript II RT (Life Technologies Inc) in a 20-µL reaction volume using the manufacturer's suggested conditions. A 1 µL aliquot of the RT reaction was used in a 100-µL PCR reaction using 2.5 U Taq polymerase (Life Technologies Inc), 200 ng of each amplification primer, and the manufacturer's suggested reaction buffer. The sense oligonucleotide primer (5'-GAT CAATAACCTGAAGCCCG-3') corresponds to base pairs 2852 to 2871, and the antisense oligonucleotide primer (5'-GCCCTTTTTTGCTCCATAGG-3') is complementary to base pairs 3409 to 3428 of the cDNA sequence (GenBank Accession No. D14051) of the rat vascular smooth muscle iNOS isoform.⁵⁰ The antisense amplification primer was end-labeled with T4 polynucleotide kinase before use in PCR amplification. PCR conditions were 95°C for 45 seconds, 42°C for 40 seconds, and 72°C for 30 seconds and were performed for 24 cycles. PCR reaction aliquots were run on 7% polyacrylamide gels, dried, and exposed as described for Northern blots.

Quantitative Phospholamban Western Blot Analysis
Neonatal cardiac myocytes were treated with IL-1ß (2 ng/mL) or solvent (PBS) for 20 hours before isolation of total cellular protein. Cells were washed with PBS, scraped, and then centrifuged. Cell pellets from 3x10⁶ cells were homogenized in 0.4 mL of 10 mmol/L NaHCO₃. Proteins were quantified as described above. Serial dilutions of the lysates were subjected to SDS–polyacrylamide (12%) gel electrophoresis, and proteins were electrophoretically transferred to nitrocellulose membranes. Filters were blocked for 30 minutes in 3% nonfat dry milk/PBS at room temperature, followed by incubating overnight at 4°C with 1 µg/mL anti-phospholamban monoclonal antibody (Upstate Biotechnology). The membranes were washed and incubated for 1 hour with goat anti-mouse IgG conjugated to horseradish peroxidase. Visualization was achieved using a chemiluminescence Western blotting detection system (Dupont NEN) and x-ray film. Under our sample preparation and gel conditions, phospholamban was detected predominantly as a pentamer. Developed chemiluminescent images were scanned using a Hewlett-Packard Scan Jet 4C and quantified by ImageQuant software (Molecular Dynamics). Data were calculated as pixel value per microgram of protein for each of four protein masses per sample (control and IL-1ß–treated) and normalized to the mean of the control samples, arbitrarily set at 100%. Results are the average of five experimental sets of data.

Analysis of Neonatal Cardiomyocyte Contractile Parameters
Cardiomyocytes were prepared, plated onto coated glass coverslips, and cultured in the presence or absence of IL-1ß (2 ng/mL) in DF-5% for 20 hours as described above. Coverslips were transferred to a temperature-regulated chamber (held at 33°C) mounted on a Nikon Diaphot 300 inverted microscope stage. Cells were perfused with prewarmed modified Tyrode's solution (mmol/L: NaCl 137, KCl 5, glucose 15, MgSO₄ 1.3, NaH₂PO₄ 1.2, HEPES 20, and CaCl₂ 1); perfusion (2 mL/min) was begun 5 minutes before recording contractile parameters. Glass beads (2.1±0.5 µm, Duke Scientific Corp) were added to the neonatal cells to provide high-contrast spots for tracking contractile activity. Although the cultures contained a spontaneously contracting monolayer of cells, they were paced by electrical field stimulation at 1 Hz (15 V/4-millisecond pulse duration, Grass S11 stimulator, Grass Instruments) using platinum electrodes embedded in the wall of the perfusion chamber. A commercially available video edge-detection system (VED 104, Crescent Electronics) was used to follow and record the motion of glass beads attached to the surface of the contracting cells. Data were recorded for 10 consecutive beats from 9 to 12 cells per coverslip, with at least two coverslips prepared per condition (control or IL-1ß, 2 ng/mL), and a total of three separate cell preparations. A data analysis program (IonWizard 4.3, Ionoptix Corp) was used to calculate the maximum speed of contraction, maximum speed of relaxation, and peak amplitude of contraction. Calibration of contractile distance was determined by using Cell-VU grid coverslips (Erie Scientific Corp). To assess the responsiveness of cells to catecholamine challenge, the following protocol was used: The basal contractile motion of a cell was recorded. The perfusion pump was then turned off, and a bolus dose of isoproterenol was added to the perfusion chamber to yield a final dose of 0.1 mmol/L. After 1 minute, contractile parameters were recorded as described above. One cell was recorded per coverslip. Adrenergic responsiveness was determined as the ratio of the peak amplitude of contraction after and before isoproterenol challenge.

Statistical Analyses
Results are presented as the mean±SE. Statistical analyses were performed using Student's t test (for two groups) or one-way ANOVA (more than two groups). Where appropriate, post hoc multiple comparison testing was performed (Student-Newman-Keuls test) to test for differences between groups. A value of P<.05 was accepted as significant.

	Results

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IL-1ß Decreases the Level of Phospholamban RNA in Neonatal Rat Cardiomyocytes
Neonatal cardiomyocytes were treated with IL-1ß at several different concentrations for different periods of time, and the level of phospholamban transcripts was assessed by Northern blot analysis. Cells treated with IL-1ß (2 ng/mL) for 20 hours (Fig 1A and 1B) showed a marked decrease in the level of phospholamban transcripts relative to untreated (control) neonatal cardiomyocytes (44.2±2.6% of control, P<.0001). Since both IL-1ß⁴⁰ and TNF-^{29 41} can alter Ca²⁺ transients in adult cardiomyocytes, we also examined the ability of TNF- to alter phospholamban transcript levels in neonatal cardiomyocytes (Fig 1C). ANOVA showed highly significant differences (P<.0001) between treatment groups; multiple comparison testing showed significant and similar reductions of phospholamban transcripts in cardiomyocytes exposed simultaneously to either IL-1ß (2 ng/mL) and TNF- (100 U/mL) (55.3±2.7% of control) or IL-1ß alone (48.9±5% of control). However, cells treated with TNF- alone showed no significant changes from untreated control cells (87±7.9%, P=NS). Thus, TNF- by itself did not alter the level of phospholamban transcripts nor did it modify the effect of IL-1ß. Although this concentration of TNF- was reported to decrease contractile activity of adult feline cardiomyocytes,²⁹ no effect on phospholamban transcript levels was observed when TNF- was used at concentrations up to 400 U/mL (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of IL-1ß on phospholamban (PLB) transcript levels in neonatal cardiomyocytes. A, Representative Northern blot hybridization demonstrating PLB transcript levels. B, Quantitative analysis of PLB transcript levels with data obtained from 12 experiments (total of 32 control, 35 IL-1ß–treated [2 ng/mL, 20 hours] samples) (similar to analysis in panel A). *P<.0001 vs control. C, Effect of TNF-. After the indicated treatments (36 hours), PLB transcript levels were quantified; data were from three experiments containing a total of 15 (IL-1ß+TNF-) or 9 (all other) samples per treatment. *P<.05 (post hoc comparison) vs control. NS indicates not significant.

Further confirmation that IL-1ß was the causative agent was obtained by either boiling the IL-1ß or interacting it with an IL-1ß–neutralizing antibody before treatment of cultured neonatal cardiomyocytes (Fig 2). Such treatments should destroy biological activity of IL-1ß but not that of contaminating endotoxins. ANOVA showed highly significant differences (P<.0001) between treatment groups; multiple comparison testing showed that the significant reduction in phospholamban transcripts induced by IL-1ß (40.7±2.3% of control) was blocked when the cytokine was first interacted with neutralizing antibody (IL-1ß+IL-1ß antibody, 88.9±5.2% of control; P=NS) or when boiled (77.3±7.2% of boiled diluent control, P=NS), further indicating that the observed effects on phospholamban transcript levels arose from IL-1ß and not from unidentified contaminants.

View larger version (21K): [in this window] [in a new window]   Figure 2. Effects of IL-1ß are blocked by IL-1ß–neutralizing antibody (IL1Ab) or boiling. Neonatal cardiomyocytes were treated with IL-1ß (20 hours), and phospholamban (PLB) transcript levels were determined. Where indicated (IL-1ß+IL1Ab), IL-1ß was first interacted with IL1Ab; parallel cultures received boiled IL-1ß or diluent. Data were obtained from three experiments, yielding a total of 6 (boiled diluent), 11 (IL-1ß, 0.5 ng/mL), or 9 (all other conditions) samples. *P<.05 (post hoc comparison) vs control (Ctl) or boiled diluent. NS indicates not significant.

The response of phospholamban transcript levels to IL-1ß showed a concentration dependence, with a significant decrease observed at 0.005 ng/mL and a maximal effect (40.4% of control) observed above 0.5 ng/mL (Fig 3A). The decrease in phospholamban transcript levels could be detected as early as 5 hours after the addition of IL-1ß to the culture medium; these effects were maintained through at least 40 hours of exposure to IL-1ß (Fig 3B).

View larger version (14K): [in this window] [in a new window]   Figure 3. IL-1ß decreases phospholamban (PLB) transcript levels in a dose- and time-dependent fashion. A, Neonatal cardiomyocytes were exposed to different IL-1ß concentrations (40 hours), and PLB transcript levels were determined. Data were from two to five experiments (total of 6 to 15 repeats per concentration). PLB transcript levels were significantly reduced at all IL-1ß concentrations (P<.05 relative to control cells). B, Neonatal cardiomyocytes were exposed to IL-1ß (, 2 ng/mL IL-1ß; , control) for 0.5 to 40 hours, and PLB transcript levels were determined. Data were obtained from four experiments, for a total of 6 to 17 repeats per condition except for the 5-hour group (three repeats each, control and IL-1ß–treated cells). *P<.05 vs untreated control cells. C, IL-1ß treatment in the presence or absence of serum is shown. Cells were placed into serum-containing (DF-5%) or serum-free (DF-ITS) medium and cultured for 20 hours in the presence (IL-1ß) or absence (control [Ctl]) of IL-1ß (2 ng/mL), and PLB transcript levels were determined. Data were obtained from three or four experiments containing a total of 8 to 16 samples in each group. *P<.0001 for IL-1ß–treated vs untreated Ctl cells.

Since the nature of culture conditions can markedly affect the results observed with cultured neonatal cardiomyocytes,^{51 52} we examined the effects of IL-1ß on phospholamban transcript levels in cells maintained in either serum-containing (DF-5%) or serum-free medium (DF-ITS) (Fig 3C). A similar reduction in phospholamban transcript levels was observed in response to IL-1ß regardless of the culture medium used (DF-5%, 48.3±4.1% of control [P<.0001]; DF-ITS, 41.4±4.8% of control [P<.0001]).

IL-1ß Decreases the Level of Phospholamban Protein in Neonatal Rat Cardiomyocytes
Previous studies on failing human myocardium have suggested that a dichotomy may exist between phospholamban transcript and protein level alterations.^{8 9 10 15 16 17} In order to investigate whether a similar result occurs in neonatal cardiomyocytes exposed to IL-1ß, we measured the relative level of phospholamban proteins in neonatal cardiomyocytes exposed to 2 ng/mL IL-1ß for 20 hours (Fig 4A). A 40% reduction in phospholamban protein was observed (P<.0001, Fig 4B), indicating that a parallel decrease in both phospholamban transcripts and proteins occurs in neonatal cardiomyocytes exposed to IL-1ß.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effect of IL-1ß on the level of phospholamban (PLB) protein in neonatal cardiomyocytes. A, Representative Western blot image (see "Materials and Methods") of serial dilutions from control and IL-1ß (2 ng/mL, 20 hours)–treated cardiomyocyte proteins (10 to 80 µg). B, Quantitative analysis of PLB protein levels. Data were from five experiments, each containing four protein dilutions per group (control or IL-1ß–treated cells). *P<.0001 vs control. C, Effect of IL-1ß on [3H]leucine incorporation in cells cultured in medium containing or lacking serum. After 24 hours of culture in DF-5%, cells were washed and placed into DF-5% (triangles) or DF-ITS (circles) containing [3H]leucine. Control (Ctl) cells (open symbols) contained no treatment; IL-1ß–treated cells (closed symbols) received 2 ng/mL IL-1ß; samples were collected after 24, 48, or 72 hours of culture. Data represent the mean of two separate experiments, each containing two to six wells per condition and time point. *P<.0002 for DF-ITS Ctl vs DF-ITS+IL-1ß–treated cells. In DF-5%, there were no significant differences in Ctl vs IL-1ß–treated cells.

Two prior studies (Thaik et al³³ and Palmer et al³⁴ ), but not the study of Harding et al,⁵³ have reported that IL-1ß induces the synthesis of proteins in neonatal cardiomyocytes. Since a relative reduction in phospholamban proteins could reflect a dilution effect from the increased level of other proteins, we assessed the ability of IL-1ß to enhance protein synthesis as determined by the incorporation of radiolabeled amino acids into neonatal cardiomyocyte protein (Fig 4C). When neonatal cardiomyocytes were cultured in DF-5% medium, IL-1ß (2 ng/mL) did not induce a significant increase in [³H]leucine incorporation relative to control medium. However, when cells were cultured in a growth factor–poor medium (DF-ITS), IL-1ß was observed to markedly (P<.0002 for IL-1ß–treated versus control cells) increase [³H]leucine incorporation at all time points investigated. These observations are consistent with the studies of Thaik et al³³ and Palmer et al,³⁴ who reported that IL-1ß induces protein synthesis in a serum-free medium.

IL-1ß Does Not Alter Basal Contractile Properties of Neonatal Cardiomyocytes
Previous studies disagree on the effects that proinflammatory cytokines have on the basal contractile activity of cardiomyocytes. Two groups report that the basal contractility of neonatal³² and adult³⁹ rat cardiomyocytes are not altered after exposure to IL-1ß, TNF-, or products of activated alveolar or peritoneal macrophages, although these agents depress the contractile responses to catecholamine stimulation after 18³⁹ to 72³² hours of continuous exposure. Other studies report that the basal contractile properties of cardiomyocytes are decreased within minutes of exposure to septic serum (neonatal rat cardiomyocytes²¹ ) or TNF- (adult feline cardiomyocytes²⁹ ) or after 18 hours of exposure to IL-1ß or TNF- (adult guinea pig cardiomyocytes³⁶ ). To assess the potential functional consequence of the IL-1ß-induced reduction in phospholamban expression, we determined basal contractile parameters of neonatal cardiomyocytes exposed to 2 ng/mL IL-1ß for 20 hours. A representative trace of basal contractile activity is presented in Fig 5A. Cardiomyocytes exposed to IL-1ß showed no significant changes in the peak contractile amplitude, maximal rate of contraction, or maximal rate of relaxation compared with control cells (Fig 5B). However, the IL-1ß–treated cells were no longer responsive to isoproterenol (P<.002). These observations are consistent with previous reports that IL-1ß (2 ng/mL, 20 hours) does not alter basal contractile parameters in neonatal cardiomyocytes but does limit the ability of cardiomyocytes to respond to catecholamines.^{32 39}

View larger version (32K): [in this window] [in a new window]   Figure 5. Effect of IL-1ß on cardiomyocyte contractile parameters. A, Representative contractile trace from displacement of high-contrast glass bead attached to electrically paced neonatal cardiomyocytes (control). B, Quantitative measurement of contractile parameters derived from neonatal cardiomyocytes exposed for 20 hours to no additive (control) or IL-1ß (2 ng/mL). Isop. indicates isoproterenol. Number of cells examined are in parentheses. Data presented are mean±SEM. NS indicates not significant.

Selective Effects of IL-1ß on Neonatal Cardiomyocyte Gene Expression
To assess the specificity of IL-1ß effects on gene expression, we also determined the level of transcripts for SERCA and GAPDH in neonatal cardiomyocytes treated with IL-1ß. Fig 6A shows Northern blot results indicating a reduction in phospholamban transcripts, a somewhat lesser effect on SERCA, and a lack of effect on GAPDH transcripts. In addition, as reported by other investigators,^{36 37 38 39} we observed that IL-1ß markedly induced the level of iNOS transcripts, which are not typically detected in untreated neonatal cardiomyocytes (Fig 6B). Quantitative analysis (Fig 6C) showed that IL-1ß (2 ng/mL, 20 hours) significantly reduced transcript levels for phospholamban (46.4±3.2% of control, P<.0001) and SERCA (65.1±7.0% of control, P<.0001) and did not alter GAPDH transcript levels (110±5.4% of control, P=NS). Results for iNOS, SERCA, and GAPDH transcript changes are consistent with those previously reported by other investigators.^{33 34 35 36 37 38 39}

View larger version (42K): [in this window] [in a new window]   Figure 6. Effect of IL-1ß on gene transcript levels. A, Representative Northern blot image displays the effects of IL-1ß (2 ng/mL, 20 hours) on phospholamban (PLB), SERCA, and GAPDH transcript levels. B, IL-1ß induces expression of iNOS transcripts. RNA isolated from cells treated with IL-1ß (2 ng/mL), IL-1ß (2 ng/mL)+TNF- (100 U/mL), or diluent (control) was used in RT-PCR with radiolabeled amplification primers, and PCR products were separated on 7% polyacrylamide gels. C, Quantification of Northern blots from experiments similar to those in panel A is shown. Data represent the mean of seven separate experiments, each containing two to nine samples per condition. *P<.0001 for IL-1ß–treated vs control cells; P=NS for IL-1ß–treated vs control cells.

Dexamethasone Prevents the Effect of IL-1ß on Phospholamban Transcript Levels
In many cells, the effects of IL-1ß can be blocked with the synthetic glucocorticoid dexamethasone.^{31 54 55} Neonatal cardiomyocytes were treated with a combination of IL-1ß (10 ng/mL) and TNF- (100 U/mL) for 36 hours in the presence or absence of 3.3 µmol/L dexamethasone (Fig 7), and the level of phospholamban transcripts was quantified. ANOVA showed highly significant differences (P<.0001) between treatment groups; multiple comparison testing showed that IL-1ß+TNF-–treated cells had a significant decrease in phospholamban transcript levels (58.1±3.5% of control). This effect was prevented when cells were cotreated with dexamethasone (98.7±9.7% of control, P=NS). However, the level of phospholamban transcripts was significantly increased by dexamethasone alone (154±19.8% of control), making it difficult to ascertain the direct effects of dexamethasone on proinflammatory cytokine-induced changes in phospholamban transcript levels.

View larger version (73K): [in this window] [in a new window]   Figure 7. Dexamethasone (DEX) blocks the effect of proinflammatory cytokines on phospholamban (PLB) transcript levels. Neonatal cardiomyocytes were treated with IL-1ß (2 ng/mL)+TNF- (100 U/mL) or the diluent (control) for 24 hours in the presence or absence of 3.3 µmol/L DEX. A, Representative Northern blot image. B, Quantification of Northern blot images. Data are from two or three separate experiments containing three to six replicate samples per experiment. *P<.05 (post hoc comparison) vs control. NS indicates not significant.

One pathway by which dexamethasone may block the effects of cytokines is through inhibition of cytokine-stimulated iNOS expression.^{54 55} Since IL-1ß induces iNOS expression^{36 37 38 39} (Fig 6B) and since dexamethasone can inhibit both iNOS expression^{54 55} and the decrease in phospholamban transcript levels (Fig 7), we examined the possible role of iNOS expression in the IL-1ß effect on phospholamban transcripts.

IL-1ß Does Not Alter Phospholamban Transcripts Through NO-Dependent Mechanisms
IL-1ß can stimulate iNOS expression and the production of NO and cGMP,^{38 54} leading to alterations of cardiac and cardiomyocyte contractile parameters.^{28 29 30 31 32} We examined the potential role of NO in mediating the effects of IL-1ß on phospholamban transcript levels through the use of an inhibitor of iNOS activity (Fig 8A). Neonatal cardiomyocytes were placed in medium containing either 1 mmol/L arginine or 1 mmol/L L-NMMA and then treated with either diluent (control) or IL-1ß (2 ng/mL, 20 hours). In both media, 2 ng/mL IL-1ß caused a similar decrease in the level of phospholamban transcripts (1 mmol/L arginine medium+IL-1ß, 68.7±8.9% of control [P<.005]; 1 mmol/L L-NMMA medium+IL-1ß, 56±4.1% of control [P<.005]), suggesting that the IL-1ß effects on phospholamban transcript levels are not blocked by an inhibitor of iNOS.

View larger version (16K): [in this window] [in a new window]   Figure 8. IL-1ß decreases phospholamban (PLB) transcripts through a NO-independent pathway. A, Neonatal cardiomyocytes were cultured in medium containing 1 mmol/L L-arginine (Arg) or 1 mmol/L L-NMMA (NMMA) and exposed to IL-1ß (2 ng/mL) or diluent for 20 hours. PLB transcript levels were quantified as described. Data are from three experiments containing two to four replicates. *P<.005 for IL-1ß–treated vs control cells. B, Effect of NMMA on IL-1ß–induced cGMP production. Cells were cultured and treated as described above, and cGMP was extracted and measured by radioimmunoassay. Data represent the mean of three experiments, each containing triplicate samples. *P<.004 for IL-1ß–treated (Arg) vs control (Arg) cells. P=NS for IL-1ß–treated (NMMA) vs control (NMMA) cells.

To confirm that the culture condition in 1 mmol/L L-NMMA was sufficient to block NO production, we measured cGMP levels as a marker of IL-1ß–induced NOS activity (Fig 8B). Cells grown in 1 mmol/L arginine showed a 3-fold induction of cGMP levels when cultured in the presence of 2 ng/mL IL-1ß for 20 hours (arginine [control], 24.6±3.3 pmol/mg protein; arginine+IL-1ß, 75.7±14.7 pmol/mg protein; P<.004). This induction of cGMP levels was completely blocked when cells were cultured in 1 mmol/L L-NMMA (L-NMMA [control], 5.23±1.27 pmol/mg protein; L-NMMA+IL-1ß, 6.79±1.56 pmol/mg protein; P=NS), indicating that the selected culture conditions in 1 mmol/L L-NMMA were sufficient to block NOS activity.

IL-1ß Effects on Phospholamban Transcripts Are Not Blocked by Inhibitors of Tyrosine Kinase, Cyclooxygenase, or PKC
Besides enhancement of NO and cGMP production via iNOS, a variety of other second-messenger pathways have been proposed to mediate the effects of IL-1ß on different cells. In neonatal cardiomyocytes, the hypertrophy-inducing effects of IL-1ß have been reported to involve a tyrosine kinase–dependent pathway.³⁴ In addition to NO, IL-1ß–signaling pathways dependent on prostaglandins⁵⁶ and PKC^{57 58} have also been reported. To assess the role of these pathways in the signaling of IL-1ß effects on phospholamban expression, neonatal cardiomyocytes were challenged with IL-1ß in the absence and presence of the tyrosine kinase inhibitor genistein, the cyclooxygenase inhibitor indomethacin, and the PKC inhibitor staurosporine (Fig 9A). Indomethacin (50 µmol/L) failed to block the effects of IL-1ß on phospholamban transcripts; this concentration is at least five times higher than that previously used to block prostaglandin synthesis in other cell types, including cardiomyocytes.³⁴ The tyrosine kinase inhibitor genistein (20 µmol/L) also failed to inhibit the effects of IL-1ß on phospholamban transcripts. This concentration is twice that previously used to reduce the protein synthesis induced in neonatal cardiomyocytes by either IL-1ß or platelet-derived growth factor.³⁴ Finally, the PKC inhibitor staurosporine (5 nmol/L) appeared to augment the extent to which IL-1ß decreased phospholamban transcripts (Fig 9). This concentration of staurosporine was previously shown to limit norepinephrine-induced hypertrophy in neonatal cardiomyocytes.⁵⁹

View larger version (62K): [in this window] [in a new window]   Figure 9. Second-messenger pathways in the IL-1ß–induced decrease of phospholamban (PLB) transcripts. Neonatal cardiomyocytes were treated with IL-1ß (2 ng/mL, 20 hours), diluent (control [Ctl]), the indicated inhibitor, or IL-1ß plus inhibitor. Inhibitors used were staurosporine (Stauro, 5 nmol/L), indomethacin (Indom, 50 µmol/L), or genistein (Genist, 20 µmol/L). A, Relative PLB transcript levels were determined as described; samples treated with inhibitor alone served as controls for inhibitor plus IL-1ß–treated samples. Data represent the mean of two to five experiments, each containing three or four replicate samples. *P<.0001 for Ctl (diluent or inhibitor only) vs treated (IL-1ß alone or with inhibitor) cells. B, Northern blot image of RNA isolated from neonatal cardiomyocytes treated with IL-1ß (2 ng/mL, 20 hours) in the presence or absence of 5 nmol/L Stauro is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies have shown that IL-1ß and TNF- can alter the contractile properties (either adrenergic responsive^{32 39} or basal^{21 36} ) of cultured cardiomyocytes and modify the expression of the genes that regulate contraction.^{33 35} The major finding of the present study extends these observations to identify a decreased expression of phospholamban at both the transcript and protein levels in cardiomyocytes exposed to physiologically relevant levels of IL-1ß. By contrast, we could not observe any alteration in phospholamban expression in response to TNF-, another proinflammatory cytokine that reportedly depresses myocardial contractile activity.^{26 29} In addition, the response to IL-1ß occurred at physiologically relevant concentrations. For example, a significant decrease in phospholamban transcripts was observed at 10 to 100 pg/mL IL-1ß (0.6 to 6.0 pmol/L); these levels are comparable to the serum levels observed in some patients with end-stage heart failure (20 pg/mL, 1.2 pmol/L²⁵ ) and are below those seen in sepsis (170 pg/mL, 10 pmol/L⁶⁰ ). Admittedly, the variety of circulating proteins related to the IL-1ß and IL-1 receptors that modulate the biological activity of IL-1ß²⁷ must be considered when making a comparison between the biologically effective concentrations observed in vitro and in vivo.

The observation that serum levels of IL-1ß and TNF- are elevated in many situations of depressed cardiac function^{19 20 21 22 23 24 25} has suggested their role in mediating some of the changes in myocardial gene expression observed in heart failure. Although much recent attention has focused on the role of TNF- in the alteration of cardiac function, significant evidence points to the importance of IL-1ß as well. Circulating IL-1ß is elevated in many patients with end-stage heart failure.²⁵ Administration of an IL-1 receptor antagonist can limit the cardiovascular effects of septic challenge²⁰ and viral myocarditis.²² In a murine model of viral myocarditis leading to cardiomyopathy, an elevated expression of IL-1ß in the myocardium persists from the acute through the chronic cardiomyopathic phase, whereas an elevated cardiac level of TNF- is limited to the acute phase of viral infection.⁶¹ Finally, there is a consensus that IL-1ß is required for the induction of iNOS expression in cardiomyocytes. However, by contrast, the requirement for TNF- is less clear.^{36 37 38 39}

Are Changes in Phospholamban Expression Antithetical to Changes in Cardiac Function?
Since IL-1ß may contribute to depressed cardiac function in whole animals and isolated cells and tissues,^{20 21 22 28 31 36} it is somewhat unexpected that cardiomyocytes exposed to IL-1ß express lower levels of phospholamban. This decrease could relieve inhibition of the SR Ca²⁺ pump, which should enhance and not diminish contractility. In addition, the decrease in phospholamban transcript levels appears somewhat greater than that observed for SERCA, potentially leading to alterations in the ratio of phospholamban to SERCA protein and diastolic properties. Furthermore, one might expect that the elevation in cGMP induced by IL-1ß could lead to enhanced phosphorylation of phospholamban,⁶² further diminishing the inhibitory effect that phospholamban has on the SR Ca²⁺ pump. Thus, the biochemical changes observed in the present study may appear to counter those that might accompany decreased cardiac contractility.

However, several additional observations concerning the effects of IL-1ß on cardiomyocyte contractility should be considered. First, although it has been reported that septic patient serum as well as proinflammatory cytokines can diminish the basal contractility of neonatal rat cardiomyocytes in culture,²¹ other reports have found that products of activated splenocytes,³² activated alveolar macrophages,³⁹ or recombinant IL-1ß and/or TNF-^{32 39} do not alter the basal contractility of neonatal or adult rat cardiomyocytes. Indeed, the present study concurs with these latter findings; 20 hours of exposure to 2 ng/mL IL-1ß did not cause changes in the basal contractile parameters of neonatal rat cardiomyocytes. Instead, exposure to immune products may alter cardiomyocyte contractility by blocking the ability of cardiomyocytes to respond to adrenergic stimulation, as evidenced by a lack of both enhanced contractility and elevation of cAMP levels.³² It is likely that a reduction in phospholamban expression could also contribute to the inability of adrenergic agents to stimulate contractility in cardiomyocytes exposed to proinflammatory cytokines. Indeed, in a study on the developmental alterations in SERCA and phospholamban expression and adrenergic responsiveness in the developing rabbit heart,⁶³ it was found that fetal hearts have a phospholamban-to-SERCA protein ratio that is only 1/5 that of the adult rabbit, yet the fetal hearts had slower maximal rates of relaxation despite a lower phospholamban-to-SERCA ratio and were nonresponsive to adrenergic stimulation, as determined by cardiac relaxation or Ca²⁺ uptake in cardiac homogenates. Hence, a lower phospholamban-to-SERCA ratio is not always associated with increased basal rates of relaxation, although it may be expected to alter adrenergic responsiveness. Finally, when exposed to IL-1ß, guinea pig papillary muscles³¹ and adult cardiomyocytes⁶⁴ did not show alterations in relaxation properties; instead, they showed a diminished sensitivity of the myofibrillar element to Ca²⁺, mediated through the production of cGMP. Thus, the effects of IL-1ß on cardiomyocyte contractility may arise from a loss of adrenergic responsiveness,^{32 39} mediated partially through an uncoupling of cAMP production³² and a decreased expression of phospholamban, a diminished Ca²⁺ pump capacity arising from reduced expression of SERCA,^{33 35} and a loss of myofiber Ca²⁺ sensitivity.⁶⁴ However, the diverse physiological changes and immunomodulators present in the intact animal, as well as the number of potential biochemical targets that IL-1ß may alter within the cardiomyocyte, make it unclear just how alterations in phospholamban expression contribute to the changes in cardiac function that occur after challenge with IL-1ß and other inflammatory stimuli.

Mechanism of IL-1ß–Induced Alterations in Phospholamban Expression
We also performed studies with inhibitors of signal transduction to identify the pathways that may mediate the effects of IL-1ß on phospholamban transcript levels. Although some effects of IL-1ß are mediated via prostaglandins,^{27 56} an inhibitor of prostaglandin synthesis (indomethacin) did not alter the IL-1ß effect on phospholamban transcript levels. This observation is congruent with the report that prostaglandins do not mediate the effects of IL-1ß on enhancing protein synthesis in cardiomyocytes.³⁴ Similarly, IL-1ß enhances iNOS expression in cardiomyocytes and cardiac muscle,^{36 37 38 39} an observation also confirmed by the present study. However, an inhibitor of iNOS activity (L-NMMA) did not affect the reduction in phospholamban transcripts caused by IL-1ß. Again, these results are consistent with the reports that in cardiomyocytes, inhibitors of iNOS do not diminish the effect that IL-1ß has on either transcript levels³⁵ or protein synthesis.^{33 34} Tyrosine kinase–dependent pathways have been implicated in the ability of IL-1ß to increase protein synthesis,³⁴ as well as in the induction of iNOS^{38 65} in neonatal cardiomyocytes. Nonetheless, we could not demonstrate that the tyrosine kinase inhibitor genistein would alter the IL-1ß effect on phospholamban expression.

We did observe that the PKC inhibitor staurosporine enhanced the effects of IL-1ß on phospholamban transcript levels. Several studies suggest that PKC is involved in the signal transduction of IL-1ß.^{57 58} However, since staurosporine has potent inhibitory effects on multiple kinases,⁶⁶ we cannot exclude the possibility that other non-PKC staurosporine-sensitive kinases participate in IL-1ß signal transduction. Interestingly, two laboratories have detected novel kinases that can be immunoprecipitated with antibodies directed against the IL-1 receptor.^{67 68} In one case, the IL-1 receptor–associated kinase was inhibited by staurosporine, but not by either of two more specific PKC inhibitors, nor by two tyrosine kinase inhibitors.⁶⁸ Thus, further studies will be required to ascertain whether the effect of staurosporine observed in the present study arises from inhibition of PKC or other novel kinases involved in IL-1ß signal transduction.

At this time, we do not know if the IL-1ß–induced decrease in phospholamban expression occurs at the transcriptional or posttranscriptional level. Previous studies found no evidence that IL-1ß treatment altered the half life of mRNA for either skeletal -actin³⁵ or iNOS,⁶⁵ arguing against changes in transcript half life. Conversely, several studies indicate that IL-1ß acts at a transcriptional level, increasing the promoter activity of an iNOS reporter plasmid⁶⁵ and decreasing the basal transcriptional activity of a SERCA promoter reporter plasmid as well as the ₁-adrenergic stimulation of skeletal -actin promoter reporter plasmid transiently transfected into neonatal cardiomyocytes.³⁵ IL-1ß increases the expression of at least two transcriptional regulatory proteins, YY1³⁵ and NF-B.⁶⁹ Since the functional promoter elements of the rat phospholamban gene remain incompletely defined, the role of YY1 or NF-B in the regulation of phospholamban expression is not known. Analysis of the rat and rabbit phospholamban gene sequences^{46 70} likely to contain promoter elements (from -1200 to +200 with respect to transcription initiation) do not reveal any consensus YY1 or NF-B sites (data not shown). However, YY1, NF-B, and other IL-1ß–responsive elements could be present in the large (6-kb) incompletely sequenced intron that is present after the short (<100-nt) first exon. Hence, although IL-1ß may alter transcriptional activity in neonatal cardiomyocytes, we cannot exclude the possibility that changes in RNA stability or protein turnover mediate the effects of IL-1ß on phospholamban expression.

In summary, we have demonstrated that in vitro, the proinflammatory cytokine IL-1ß alters both the transcript and protein level of a gene that regulates cardiac relaxation. Although the mechanism by which a decrease in phospholamban expression would lead to a decrease in basal cardiac contractility is not clear, it could help to reduce the ability of adrenergic stimulation to enhance SR Ca²⁺ uptake and thus complement other biochemical changes (such as a cGMP-induced reduction in myofiber Ca²⁺ sensitivity) that diminish cardiac function. Additionally, these changes provide an NO-independent pathway by which inflammatory cytokines could alter myocardial function. Thus, the observation that phospholamban and other functionally important myocardial genes are responsive to IL-1ß may lead to new insights as to how IL-1ß alters contractile function.

	Selected Abbreviations and Acronyms

 

IL = interleukin iNOS = inducible NO synthase L-NMMA = NG-monomethyl-L-arginine NF-B = nuclear factor-B PCR = polymerase chain reaction PKC = protein kinase C RT = reverse transcriptase SERCA = SR Ca2+-ATPase SR = sarcoplasmic reticulum TCA = trichloroacetic acid TNF- = tumor necrosis factor-

	Acknowledgments

 
Dr Combes was supported by the AMSTER association, the Fédération Française de Cardiologie, the Laboratoires SERVIER, and the funds from the Bill Hillgrove Cardiology Fellowship.

Received March 4, 1997; accepted June 27, 1997.

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