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Am J Physiol Heart Circ Physiol 289: H708-H714, 2005. First published April 8, 2005; doi:10.1152/ajpheart.01179.2004
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Daily administration of interleukin-18 causes myocardial dysfunction in healthy mice

Per Reidar Woldbæk,1,2 Jørn Bodvar Sande,1 Tævje Andreas Strømme,1 Per Kristian Lunde,1 Srdjan Djurovic,4 Torstein Lyberg,3 Geir Christensen,1 and Theis Tønnessen1,2

1Institute for Experimental Medical Research, 2Department of Cardiothoracic Surgery, 3Center for Clinical Research, and 4Institute for Medical Genetics, Ullevål University Hospital, University of Oslo, Oslo, Norway

Submitted 23 November 2004 ; accepted in final form 6 April 2005


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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Although increased levels of circulating interleukin (IL)-18 have been demonstrated in patients with cardiovascular diseases, the functional consequences of chronically increased circulating IL-18 with respect to myocardial function have not been defined. Thus we aimed to examine the effects of chronic IL-18 exposure on left ventricular (LV) function in healthy mice. Moreover, to clarify whether IL-18 has direct effects on the cardiomyocyte, we examined effects of IL-18 on cardiomyocytes in vitro. After 7 days of daily intraperitoneal injections of 0.5 µg IL-18 in healthy mice, a 40% (P < 0.05) reduction in the LV maximal positive derivative, a 25% (P < 0.05) reduction in the LV maximal rate of pressure decay, and a 2.8-fold (P < 0.001) increase in the LV end-diastolic pressure were measured, consistent with myocardial dysfunction. Furthermore, we measured a 75% (P < 0.05) reduction in {beta}-adrenergic responsiveness to isoproterenol. IL-18 induced myocardial hypertrophy, and there was a 2.9-fold increase (P < 0.05) in atrial natriuretic peptide mRNA expression in the LV myocardium. In vitro examinations of isolated adult rat cardiomyocytes being stimulated with IL-18 (0.1 µg/ml) exhibited an increase in peak Ca2+ transients (P < 0.05) and in diastolic Ca2+ concentrations (P < 0.05). In conclusion, this study shows that daily administration of IL-18 in healthy mice causes LV myocardial dysfunction and blunted {beta}-adrenergic responsiveness to isoproterenol. A direct effect of IL-18 on the cardiomyocyte in vitro was demonstrated, suggesting that IL-18 reduces the responsiveness of the myofilaments to Ca2+. Finally, induction of myocardial hypertrophy by IL-18 indicates a role for this cytokine in myocardial remodeling.

cytokines; heart failure; contractile function; calcium

INCREASED LEVELS OF CIRCULATING interleukin (IL)-18 have proven to be a strong and independent predictor of cardiovascular death in patients with coronary artery disease (5). Clinical and experimental studies have demonstrated increased levels of circulating IL-18 following acute myocardial infarction (27) and in the congestive heart failure syndrome (30, 39). Moreover, increased levels of IL-18 and IL-18 receptor-{alpha} have been reported in myocardial tissues from patients with end-stage heart failure (28). Although, from these studies, it is tempting to speculate that IL-18 exerts deleterious effects on myocardial function, thus far no studies have examined whether prolonged IL-18 exposure induces myocardial dysfunction per se.

IL-18 is a proinflammatory cytokine and experimentally has been shown to induce synthesis of tumor necrosis factor (TNF)-{alpha} (35), IL-1{beta} (35), IL-6 (34), and inducible nitric oxide synthase (34), mediators that have all been associated with myocardial dysfunction (6, 13, 24). Also, activation of cytotoxic T lymphocytes (12, 33) and induction of neutrophilic cell infiltration (26), as well as stimulation of intercellular adhesion molecule (ICAM)-1 (23), have been demonstrated. Immunoinflammatory mechanisms induced by IL-18 therefore may play a role during development and progression of myocardial dysfunction.

Although IL-18 might induce myocardial dysfunction indirectly via induction of other cytokines, as is mentioned above, it might also exert direct effects on the cardiomyocyte level. Both alterations in Ca2+ handling as well as reduced myofilament responsiveness to Ca2+ have been reported as mechanisms of myocardial dysfunction (1).

Mediators of myocardial dysfunction might also induce cardiac remodeling, directly or indirectly. One hallmark of cardiac remodeling is myocardial hypertrophy. Recently, Chandrasekar et al. (10) have shown that IL-18 induces hypertrophy of cardiomyocytes in vitro following activation of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase-1 (PDK1), Akt, and GATA-4 signaling. Also, increased atrial natriuretic peptide (ANP) expression in isolated cardiomyocytes has been demonstrated after IL-18 exposure (10, 39), suggesting that IL-18 in vivo also may activate signaling pathways leading to myocardial hypertrophy.

To clarify the effects of prolonged IL-18 exposure on cardiac function in vivo, we assessed the effects of 7 days of daily injections of IL-18 on left ventricular (LV) contractile parameters in healthy mice. Moreover, the {beta}-adrenergic responsiveness to isoproterenol was examined. Histological, immunohistochemical, and Western blot analyses of tissues from the LV myocardium of IL-18-treated mice were performed, evaluating the degree of lymphocyte and neutrophilic cell activation as well as the amounts of ICAM-1 and phosphorylation state of Ca2+ handling proteins. To define whether IL-18 might exert a direct effect on the cardiomyocyte, we examined the effect of IL-18 on Ca2+ homeostasis in vitro. Finally, we examined the effects of IL-18 on myocardial hypertrophy.


   METHODS
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 METHODS
RESULTS
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Animal preparation. Thirty male BALB/c mice (Møllegaard and Bomholtgård Breeding and Research Center A/S, Ry, Denmark), 6–8 wk old, were examined in this study. A volume of 1 ml 0.9% NaCl (n = 16 mice) or 1 ml NaCl containing 0.5 µg of mouse IL-18 (B 002–5, R&D Systems; n = 14 mice) was injected daily intraperitoneally during a time period of 7 days. In the mouse, doses of IL-18 administered intraperitoneally have ranged from 0.5 µg/day (9) to 10 µg/day (21). In view of these studies, we decided to use the lowest dose of IL-18 (0.5 µg/day) shown to induce biological effects in vivo. The half-life of IL-18 has been reported to be 16 h (19).

After the 7 days of injections, anesthesia was induced by injections of 0.2 ml (10 mg/ml) propofol (Diprivan; AstraZeneca, Macclesfield, Cheshire, UK) into a tail vein. A cervical midline incision was performed, and a 20-gauge intravenous cannula was inserted into the trachea. The mice were then connected to a rodent ventilator (model 874 092; B. Braun, Melsungen, Germany) and ventilated with a mixture of 2% isoflurane and 98% oxygen. Evaluation of the LV function was performed in a closed-chest model as previously described (41, 42). Briefly, after retrograde cannulation of the left ventricle using a 1.4-Fr Millar microtipped transducer catheter (model SPR-671; Millar Instruments, Houston, TX), values of the LV maximal positive derivative (LV +dP/dt), LV maximal rate of pressure decay (LV –dP/dt), LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), and heart rate (HR) were obtained. To standardize hemodynamic measurements, a depth of anesthesia was chosen to maintain a HR of ~250 beats/min. All animals included in this study received approximately the same amount of anesthetic drugs; thus the influence of anesthesia on the results presented, if any, should be randomly distributed in both experimental groups examined. After the hemodynamic measurements, heart and lungs were removed, blotted dry, and immediately weighed. The remaining heart tissue was divided into right and left ventricles and rapidly snap frozen in liquid nitrogen. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996), is in accordance with the Norwegian Animal Welfare Act, and is approved by the Norwegian Animal Research Authority.

{beta}-Adrenergic responsiveness. For assessment of the {beta}-adrenergic responsiveness, LV pressure recordings were obtained at baseline and then 2 min after intraperitoneal injections of low (0.02 µg) and high (0.5 µg) doses of isoproterenol (Iso) (14). The LV pressure recordings were performed as described in the animal preparation section.

Histological and immunohistochemical examinations. Hearts from mice receiving IL-18 (n = 7 mice) and controls receiving NaCl only (n = 8 mice) were fixed in 4% buffered formaldehyde and embedded in paraffin. The hearts were then sectioned (5 µm) transversely from apex to the left atrium and then conventionally stained with hematoxylin and eosin. For immunohistochemical analyses of T lymphocyte phenotypes, sections from frozen hearts from mice receiving IL-18 (n = 2 mice) and NaCl (n = 2 mice) were fixed in acetone and blocked with 1% bovine serum albumin and the blocking kit (catalog no. SP-2001) from Vector Laboratories (Burlingame, CA). The sections were then immunostained using the Vectastain Elite ABC reagent (Vector) and the peroxidase substrate diaminobenzidine after incubation with rat anti-mouse CD4 or CD8 mAbs (both diluted 1:10; catalog nos. 550278 and 550281, respectively, BD Pharmingen, San Diego, CA) as primary antibodies. Slides were counterstained in hematoxylin and coverslipped.

Western blot analysis. Homogenates of the LV were prepared as previously described by Semb et al. (38), with minor modifications. The following antibodies were used: ICAM-1 (7% SDS gel, catalog no. SC-1511; Santa Cruz Biotechnology, Santa Cruz, CA), sarcoplasmic reticulum Ca2+-ATPase (SERCA2) (7% SDS gel, catalog no. MA-919; Affinity Bioreagents), serine-16-phosphorylated phospholamban (15% SDS gel, catalog no. A010-12; Badrilla, Leeds, UK), threonine-17-phosphorylated phospholamban (15% SDS gel, catalog no. A010-13; Badrilla), and phospholamban (15% SDS gel, catalog no. A010-14, Badrilla).

Real-time quantitative PCR. Real-time (RT) quantitative Taqman PCR analysis was performed to determine the relative mRNA expression levels of ANP and TNF-{alpha}. Primers and Taqman probes were designed by using the Primer Express software version 1.5 (Applied Biosystems, Foster City, CA). All probes were labeled 5' 6-FAM and 3' Dark Quencher. The actual sequences of primers and probes used for RT-PCR were as follows: ANP (forward primer 5'-GTGGCTTGTGGGAAAATAGTTGA-3'; reverse primer 5'-CTGGCTTGATGATCTGCCTTTAC-3'; probe 5'- CGCTGTTCAAGTGGAACAATTTAGCC-3'), TNF-{alpha} (forward primer 5'-CATCTTCTCAAAATTCGAGTGACAA-3'; reverse primer 5'-TGGGAGTAGACAAGGTACAACCC-3'; probe 5'- CACGTCGTAGCAAACCACCAAGTGGA-3') P0 (forward primer 5'-CATTGAAATTCTGAGTGATGT-3'; reverse primer 5'-AGATGTTCAGCATGTTCACAGTGT-3'; probe 5'-CTGGCTCCCACCTTGTCTCCAGTCTTTATC-3'). RT-PCR (Ambion, Austin, TX) and Taqman PCR (Applied) were performed according to the manufacturers' instructions, using 2x Taqman Universal Master Mix (Applied) and 20 µM sense and antisense primers. Quantification of mRNA was done using the Sequence Detector version 1.6.3 program (Applied). Gene expression of the 60S acidic ribosomal protein P0 (Applied) was used for normalization.

Myocyte isolation and intracellular Ca2+ measurements. Both isolation of adult rat myocytes and Ca2+ measurements were performed as described in previous articles from our laboratory (18, 37). Briefly, myocytes were plated on coverslips and loaded with fluo-3 acetoxymethyl ester (10 µmol/l; Molecular Probes, Eugene, OR) at room temperature for 40 min. After being washed for 15 min in standard Tyrode solution (in mmol/l: 5 HEPES, 140 NaCl, 1.8 CaCl2, 5.4 KCl, 0.5 MgCl2, 5.5 glucose, 0.4 NaH2PO4; pH was adjusted to 7.4 with NaOH), the coverslips were placed in an open perfusion chamber located on an inverted microscope (Diaphot-TMD; Nikon, Tokyo, Japan), which was attached to a PTI Deltaram fluorescence system (Photon Technology International, Monmouth Junction, NJ). The myocytes were stimulated at 0.5 Hz, and the temperature was 32 ± 0.5°C. The protocol used for measuring Ca2+ transients (peak and diastolic) and the rate of decline in Ca2+ transients was performed as follows: Myocytes were superfused with HEPES-Tyrode solution for 4 min (control phase). The perfusion was then switched to a HEPES-Tyrode solution containing IL-18 (0.1 µg/ml) for 4 min (IL-18 phase), followed by an additional 4 min of superfusion with HEPES-Tyrode solution (recovery phase). The dose of IL-18 used in this experiment was based on previous studies in which IL-18 was shown to exert effects in vitro (26, 42, 44). The Ca2+ transients were calculated as a mean of four consecutive transients, and the intracellular calcium concentration values measured in the IL-18 and recovery phases were all normalized to the control phase. The mean intracellular calcium concentration values of the control phase were set to 100%. Eight myocytes from four separate isolations were included in the study.

Statistical analysis. The data are expressed as means ± SE. Statistical analysis was performed using Student's t-test and one-way analysis of variance when appropriate (SigmaStat version 2.0; Jandel Scientific, Erkrath, Germany). Multiple comparisons were corrected for by using the Student-Newman-Keuls test. A P < 0.05 was considered statistically significant.


   RESULTS
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Characterization of the animals. Mean body weights, heart weights, lung weights, and heart and lung-to-body weight ratios are shown in Table 1. After 7 days of IL-18 administration, body weights remained unchanged compared with controls. However, a significant increase in lung weights and in the heart and lung-to-body weight ratios was found in mice receiving IL-18, consistent with pulmonary edema and cardiac hypertrophy.


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  		Table 1. Body, heart, and lung weights

 
Effects of IL-18 on LV function. Figure 1, A–E, shows the in vivo effects of chronic IL-18 administration on LV function (LVSP, LVEDP, LV +dP/dt, LV –dP/dt, and HR). Mice receiving IL-18 demonstrated significantly reduced values of both LV contractility (LV +dP/dt; 2,590.3 ± 202.1 vs. 4,326.2 ± 297.9, P = 0.001) and LV relaxation (LV –dP/dt; 2,101.0 ± 237.7 vs. 2,794.8 ± 101.1, P < 0.05) compared with controls. In addition, mice receiving IL-18 demonstrated a 2.8-fold (P < 0.05) increase in LVEDP, which is consistent with LV dysfunction. We found, however, no significant changes in LVSPs or HRs when comparing the two experimental groups.



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  		Fig. 1. In vivo effects of daily peritoneal administration of 0.5 µg/ml interleukin (IL)-18 (n = 5 mice) or 1 ml NaCl (n = 6 mice) during a time period of 7 days on left ventricular (LV) systolic pressure (A), LV end-diastolic pressure (B), maximal positive derivative of LV pressure (LV +dP/dt, C), maximal rate of LV pressure decay (LV –dP/dt, D), and heart rate (HR, E). Data represent means ± SE. *P < 0.05 vs. NaCl; **P < 0.001 vs. NaCl.

 
{beta}-Adrenergic responsiveness. Figure 2 shows the effects of Iso on LV +dP/dt in controls and in IL-18-treated mice. When compared with baseline conditions, control mice demonstrated a 3.6-fold (P < 0.05) and a 4.1-fold (P < 0.05) higher increase in LV +dP/dt following low and high doses of Iso compared with the response in IL-18-treated mice, indicating a substantial blunted LV {beta}-adrenergic response to Iso in mice receiving IL-18.



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  		Fig. 2. In vivo effects of low (0.02 µg) and high (0.5 µg) doses of isoproterenol (Iso) on LV +dP/dt in mice treated with IL-18 (n = 6 mice) or NaCl (n = 6 mice) only. Increase in LV +dP/dt values represents the relative increase in LV +dP/dt assessed before and 2 min after injection of low and high doses of Iso, respectively. Data represent means ± SE. *P < 0.05 vs. NaCl.

 
Histological examination. Histological examination of the excised hearts from mice receiving IL-18 showed no increase in the number of infiltrating lymphocytes, granulocytes, or macrophages in the LV myocardium compared with controls. Neither were there any signs of increased adhesion of leukocytes to the endothelial surface of the myocardial microvessels. The few lymphocytes demonstrated in each histological section were found almost exclusively intravascularly, and the CD4+-to-CD8+ ratio was not different in IL-18-treated mice and controls and was well within the normal range.

ICAM-1 protein. Western blot examinations revealed no differences in the amounts of ICAM-1 protein between tissues from LV myocardium in mice treated with IL-18 (112 ± 5.5 arbitrary units; n = 6 mice) and controls treated with NaCl only (100 ± 3.3 arbitrary units; n = 6 mice).

Phospholamban and SERCA2 protein. Figure 3, A–E, shows the relative levels of the monomeric and pentameric forms of serine-16-phosphorylated phospholamban (PLB), threonine-17-phosphorylated PLB, total PLB, and SERCA2 proteins from the LV myocardium in controls and IL-18-treated mice. Mice receiving IL-18 showed a significant increase in both the monomeric (1.8-fold; P = 0.001) and pentameric (1.9-fold; P < 0.05) forms of the serine-16-phosphorylated PLB when compared with controls. In contrast, there were no significant differences between the two experimental groups regarding the amount of threonine-17-phosphorylated PLB, total PLB, or SERCA2 protein.



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  		Fig. 3. Representative Western blot analyses and bar graphs of the relative abundance of phosphorylated phospholamban (PLB) at serine-16 and threonine-17 and total PLB as well as sarcoplasmic reticulum Ca2+-ATPase (SERCA2) proteins in tissues from the LV myocardium in mice treated with IL-18 (n = 5 mice) and in controls (Ctr, n = 6 mice) treated with NaCl only. A: monomeric form of serine-16-phosphorylated PLB (PSer16-PLB monomeric form); B: pentameric form of serine-16-phosphorylated PLB (Pser16-PLB pentameric form); C: pentameric form of threonine-17-phosphorylated PLB (PThr17-PLB pentameric form); D: total PLB; E: SERCA2. Bar graph values represent data from IL-18 mice normalized to control mice (set to 100%). Data represent means ± SE. *P < 0.05 vs. NaCl; **P = 0.001.

 
ANP and TNF-{alpha} gene expression. RT-PCR analysis showed a significant increase in the ANP mRNA expression in the LV myocardium in mice receiving IL-18 compared with controls (0.56 ± 0.16 vs. 0.19 ± 0.06 arbitrary units; P < 0.05). In contrast, the analysis revealed no significant differences in the levels of TNF-{alpha} mRNA when comparing IL-18-treated mice (0.023 ± 0.012 arbitrary units) and controls (0.017 ± 0.008 arbitrary units).

Effects of IL-18 on Ca2+ transients in rat cardiomyocytes. Figure 4, A–C, shows the effects of IL-18 on Ca2+ transients (peak and diastolic) and on the rate of decline in Ca2+ transients. After IL-18 exposure, the peak Ca2+ transient increased by 12.8 ± 8.3% (P < 0.05) compared with the peak Ca2+ transients obtained during the control phase. During the recovery phase, however, there was a further increase of the peak Ca2+ transient to 29.1 ± 4.8%. To exclude the possibility that this result represented an artifact, we also paced cardiomyocytes at 0.5 Hz for 10 min in HEPES Tyrode solution only. However, the peak Ca2+ transients obtained during this time period remained unchanged (data not shown). Consistent with an increase in peak Ca2+ transients, an increase of 35% (P < 0.05) in the rate of decline in Ca2+ transients was measured in the recovery phase. After IL-18 exposure, increased diastolic Ca2+ transients were also measured in both the IL-18 phase (10%, P < 0.05) and recovery phase (14%, P < 0.05) compared with the control values obtained during superfusion with HT solution.



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  		Fig. 4. In vitro effects of IL-18 on peak Ca2+ transients (A), rate of decline in Ca2+ transients (B) and diastolic Ca2+ (C) in isolated cardiomyocytes (n = 8) after a stabilization period of 4 min (Ctr), then 4 min after stimulation with IL-18 (0.1 µg/100 ml), and finally 4 min after ending the IL-18 stimulation (recovery) are shown. Data represent means ± SE. *P < 0.05 vs. Ctr; {dagger}P < 0.05 vs. IL-18.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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Heart failure of different etiologies is commonly characterized by depressed myocardial contractility and relaxation (7). In our study we are demonstrating that daily administration of IL-18 in healthy mice diminishes LV function. Moreover, IL-18 increases lung weight and induces myocardial hypertrophy. These findings are consistent with the animals being in heart failure. Our observations may explain why Mallat et al. (27) reported in patients with acute coronary artery disease a correlation between increased circulating IL-18 and reduced LV ejection fraction, although in that study a causal relationship was not demonstrated. Furthermore, Raeburn et al. (36) showed in an experimental model of sepsis in mice that neutralization of IL-18 protects against LPS-induced myocardial dysfunction. These findings indicate that IL-18 activates cardiodepressive pathways within the LV myocardium, thus contributing to development of cardiac contractile dysfunction.

IL-18 may directly activate cytotoxic T cells (12, 33) and inflammatory cells (26), as well as ICAM-1 (23), within the LV myocardium. Activation of cytotoxic T cells and ICAM-1 has been shown to be associated with cardiomyocyte injury (40, 43). However, in our study, neither increased accumulation of neutrophils or cytotoxic T cells within the myocardial tissue nor evidence for increased leukocyte-endothelium adhesion within myocardial vessels or increased amounts of ICAM-1 were demonstrated in IL-18-treated mice. Similarly, Okamota et al. (32) demonstrated no changes in inflammatory cells in lung tissue following chronic exposure to a lower dose of IL-18 (0.1 µg). However, that study showed that IL-18 in synergy with IL-2 induced lethal lung injury because of activation of mononuclear lymphocytes such as cytotoxic T cells. Consequently, the lack of cardiac neutrophils or cytotoxic T cells observed in our experimental model does not exclude that IL-18 may act in synergy with other cytokines such as IL-2 in promoting cardiomyocyte injury.

Heart failure of different etiologies has been shown to be associated with reduced inotropic responsiveness to {beta}-adrenergic stimulation (7). Mice receiving IL-18 chronically show a substantially blunted LV {beta}-adrenergic responsiveness to Iso. It is well recognized that other inflammatory mediators such as TNF-{alpha}, IL-1{beta}, and nitric oxide (NO) may contribute to {beta}-adrenergic hyporesponsiveness because of impaired coupling of the {beta}-adrenergic receptor to adenylate cyclase and/or alterations in G proteins (2, 4, 16). Previously, IL-18 has been shown to facilitate the synthesis of TNF-{alpha} (35) and IL-1{beta} (35) and stimulate NO production (34) in noncardiac cells. Our study, however, revealed no significant increase in the levels of TNF-{alpha} mRNA in the LV myocardium after prolonged IL-18 exposure. Moreover, Ogura et al. (31) have shown that daily administration of IL-18 failed to induce IL-1{beta} in sera of mice. On the other hand, that study demonstrated increased circulating levels of IL-6, a cytokine, which previously has been shown to depress myocardial contractility via NO production (13). It is therefore possible that the observed cardiodepressive effect from IL-18 may result from increased production of the myocardial depressant NO.

In the present study we have shown a direct effect of IL-18 on the cardiomyocyte. After short-time IL-18 exposure in vitro, we demonstrated increased Ca2+ transients (diastolic and peak) and reduced contractility of cardiomyocytes (42). The precise mechanisms underlying the IL-18-induced increase in cytosolic Ca2+ are not presently defined. However, IL-1{beta} has been shown to increase the influx of Ca2+ into the cardiomyocytes because of an interaction with L-type Ca2+ channel proteins (4). Because IL-18 structurally belongs to the IL-1 receptor family sharing common intracellular signal pathways with IL-1{beta} (3), we find it conceivable that IL-18 also may increase Ca2+ through the same mechanism. In support of our results it should be noted that Wyman et al. (44) reported a rapid increase in intracellular Ca2+ when stimulating neutrophils with IL-18. The immediate reduction of cardiomyocyte contractility in the presence of increased intracellular Ca2+ is intriguing, and it might be speculated that increased cytosolic Ca2+ initiates reduced responsiveness of the myofilaments to Ca2+ and promotes myofilament proteolysis (15, 22).

Mice treated with IL-18 chronically demonstrated a significant increase in the phosphorylation state of PLB at serine-16 in the LV myocardium compared with mice receiving NaCl only. Regarding the physiological significance of PLB serine-16 phosphorylation, previous studies (17) have clearly demonstrated that serine-16 is phosphorylated by cAMP-dependent protein kinase and that this phosphorylation process reflects a {beta}-adrenoceptor-mediated response. As IL-18-treated mice are in heart failure and a common physiological response to cardiac dysfunction is an increased activation of the sympathetic nervous system (11), we are suggesting that increased phosphorylation of PLB at serine-16, as observed in this study, most likely has to be interpreted as a compensatory regulation of the {beta}-adrenergic signaling pathway in restoring cardiac function.

Cardiac hypertrophy is an important adaptive response to cardiac dysfunction (7). Recent studies have shown that IL-18 activates intracellular signaling proteins such as p38 mitogen-activated protein kinase (44), extracellular signal-regulated kinase (29), and signal transducer and activator of transcription STAT3 (20), all of which have been shown to be associated with myocardial hypertrophy (8, 25, 45). More recently, one in vitro study (10) has convincingly demonstrated that IL-18 induces cardiomyocyte hypertrophy through activation of the PI3K-PDK1-Akt-GATA4 signaling pathway. Interestingly, mice receiving IL-18 chronically showed both increased heart-to-body weight ratios and increased expression of the myocardial hypertrophy marker ANP. In accordance with this finding, increased expression of ANP mRNA has previously been reported after IL-18 exposure to isolated cardiomyocytes in vitro (10, 39). Consequently, our in vivo data from IL-18-treated mice confirm that IL-18 is a prohypertrophic cytokine.

In conclusion, our study shows that daily administration of IL-18 in healthy mice induces LV contractile dysfunction and blunted {beta}-adrenergic responsiveness to isoproterenol. Moreover, induction of myocardial hypertrophy by IL-18 indicates a role for this cytokine in myocardial remodeling. We have also demonstrated a direct effect of IL-18 on the cardiomyocyte in vitro, suggesting that IL-18 reduces the responsiveness of the myofilaments to Ca2+. However, further in vivo studies are required to elucidate the precise role of IL-18 in the pathogenesis of myocardial dysfunction.


   GRANTS
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 METHODS
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This work was supported by Anders Jahre's Fund for Promotion of Science, Prof. Carl Semb's Medical Research Fund, the Norwegian Foundation for Health and Rehabilitation, Rakel and Otto Bruun's Fund, and the Norwegian Research Council.


   ACKNOWLEDGMENTS
 
We thank Unni Lie Henriksen, Annlaug Ødegaard, Hilde Dishington, Lisbeth Winer, Roy Trondsen, and Morten Eriksen, Institute for Experimental Medical Research; Else Marit Løberg and Tove Norèn, Department of Pathology; and Jamie Cameron, Institute for Medical Genetics, Ullevål University Hospital, 0407 Oslo, Norway, for skillful technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. R. Woldbæk, Institute for Experimental Medical Research, Ullevål University Hospital, 0407 Oslo, Norway (e-mail: pewo{at}uus.no)

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


   REFERENCES
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  1. Atar D, Gao WD, and Marban E. Alterations of excitation-contraction coupling in stunned myocardium and in failing myocardium. J Mol Cell Cardiol 27: 783–791, 1995.[CrossRef][ISI][Medline]
  2. Balligand JL. Regulation of cardiac beta-adrenergic response by nitric oxide. Cardiovasc Res 43: 607–620, 1999.[Free Full Text]
  3. Bazan JF, Timans JC, and Kastelein RA. A newly defined interleukin-1? Nature 379: 591, 1996.[CrossRef][Medline]
  4. Bick RJ, Liao JP, King TW, LeMaistre A, McMillin JB, and Buja LM. Temporal effects of cytokines on neonatal cardiac myocyte Ca2+ transients and adenylate cyclase activity. Am J Physiol Heart Circ Physiol 272: H1937–H1944, 1997.[Abstract/Free Full Text]
  5. Blankenberg S, Tiret L, Bickel C, Peetz D, Cambien F, Meyer J, and Rupprecht HJ. Interleukin-18 is a strong predictor of cardiovascular death in stable and unstable angina. Circulation 106: 24–30, 2002.[Abstract/Free Full Text]
  6. Brady AJ, Warren JB, Poole-Wilson PA, Williams TJ, and Harding SE. Nitric oxide attenuates cardiac myocyte contraction. Am J Physiol Heart Circ Physiol 265: H176–H182, 1993.[Abstract/Free Full Text]
  7. Braunwald E and Bristow MR. Congestive heart failure: fifty years of progress. Circulation 102: IV14–IV23, 2000.[Medline]
  8. Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R, Hewett TE, Jones SP, Lefer DJ, Peng CF, Kitsis RN, and Molkentin JD. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 19: 6341–6350, 2000.[CrossRef][ISI][Medline]
  9. Carson WE, Dierksheide JE, Jabbour S, Anghelina M, Bouchard P, Ku G, Yu H, Baumann H, Shah MH, Cooper MA, Durbin J, and Caligiuri MA. Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferon-gamma production and STAT-mediated signal transduction. Blood 96: 1465–1473, 2000.[Abstract/Free Full Text]
  10. Chandrasekar B, Mummidi SM, Claycomb WC, Mestril R, and Nemer MD. Interleukin-18 is a pro-hypertrophic cytokine that acts through a phosphatidylinositol 3-kinase- phosphoinositide-dependent kinase-1-Akt-GATA4 signaling pathway in cardiomyocytes. J Biol Chem 280: 4553–4567, 2005.[Abstract/Free Full Text]
  11. Cohn JN. The sympathetic nervous system in heart failure. J Cardiovasc Pharmacol 14, Suppl 5: S57–S61, 1989.
  12. Dao T, Mehal WZ, and Crispe IN. IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells. J Immunol 161: 2217–2222, 1998.[Abstract/Free Full Text]
  13. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, and Simmons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 257: 387–389, 1992.[Abstract/Free Full Text]
  14. Funakoshi H, Kubota T, Kawamura N, Machida Y, Feldman AM, Tsutsui H, Shimokawa H, and Takeshita A. Disruption of inducible nitric oxide synthase improves beta-adrenergic inotropic responsiveness but not the survival of mice with cytokine-induced cardiomyopathy. Circ Res 90: 959–965, 2002.[Abstract/Free Full Text]
  15. Gao WD, Liu Y, Mellgren R, and Marban E. Intrinsic myofilament alterations underlying the decreased contractility of stunned myocardium. A consequence of Ca2+-dependent proteolysis? Circ Res 78: 455–465, 1996.[Abstract/Free Full Text]
  16. Gulick T, Chung MK, Pieper SJ, Lange LG, and Schreiner GF. Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte beta-adrenergic responsiveness. Proc Natl Acad Sci USA 86: 6753–6757, 1989.[Abstract/Free Full Text]
  17. Hagemann D and Xiao RP. Dual site phospholamban phosphorylation and its physiological relevance in the heart. Trends Cardiovasc Med 12: 51–56, 2002.[CrossRef][ISI][Medline]
  18. Holt E and Christensen G. Transient Ca2+ overload alters Ca2+ handling in rat cardiomyocytes: effects on shortening and relaxation. Am J Physiol Heart Circ Physiol 273: H573–H582, 1997.[Abstract/Free Full Text]
  19. Hosohara K, Ueda H, Kashiwamura S, Yano T, Ogura T, Marukawa S, and Okamura H. Interleukin-18 induces acute biphasic reduction in the levels of circulating leukocytes in mice. Clin Diagn Lab Immunol 9: 777–783, 2002.[Medline]
  20. Kalina U, Kauschat D, Koyama N, Nuernberger H, Ballas K, Koschmieder S, Bug G, Hofmann WK, Hoelzer D, and Ottmann OG. IL-18 activates STAT3 in the natural killer cell line 92, augments cytotoxic activity, and mediates IFN-gamma production by the stress kinase p38 and by the extracellular regulated kinases p44erk-1 and p42erk-21. J Immunol 165: 1307–1313, 2000.[Abstract/Free Full Text]
  21. Kawakami K, Koguchi Y, Qureshi MH, Miyazato A, Yara S, Kinjo Y, Iwakura Y, Takeda K, Akira S, Kurimoto M, and Saito A. IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells. J Immunol 165: 941–947, 2000.[Abstract/Free Full Text]
  22. Kitakaze M, Weisman HF, and Marban E. Contractile dysfunction and ATP depletion after transient calcium overload in perfused ferret hearts. Circulation 77: 685–695, 1988.[Abstract/Free Full Text]
  23. Kohka H, Yoshino T, Iwagaki H, Sakuma I, Tanimoto T, Matsuo Y, Kurimoto M, Orita K, Akagi T, and Tanaka N. Interleukin-18/interferon-gamma-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells. J Leukoc Biol 64: 519–527, 1998.[Abstract]
  24. Kumar A, Thota V, Dee L, Olson J, Uretz E, and Parrillo JE. Tumor necrosis factor alpha and interleukin 1beta are responsible for in vitro myocardial cell depression induced by human septic shock serum. J Exp Med 183: 949–958, 1996.[Abstract/Free Full Text]
  25. Kunisada K, Negoro S, Tone E, Funamoto M, Osugi T, Yamada S, Okabe M, Kishimoto T, and Yamauchi-Takihara K. Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy. Proc Natl Acad Sci USA 97: 315–319, 2000.[Abstract/Free Full Text]
  26. Leung BP, Culshaw S, Gracie JA, Hunter D, Canetti CA, Campbell C, Cunha F, Liew FY, and McInnes IB. A role for IL-18 in neutrophil activation. J Immunol 167: 2879–2886, 2001.[Abstract/Free Full Text]
  27. Mallat Z, Henry P, Fressonnet R, Alouani S, Scoazec A, Beaufils P, Chvatchko Y, and Tedgui A. Increased plasma concentrations of interleukin-18 in acute coronary syndromes. Heart 88: 467–469, 2002.[Abstract/Free Full Text]
  28. Mallat Z, Heymes C, Corbaz A, Logeart D, Alouani S, Cohen-Solal A, Seidler T, Hasenfuss G, Chvatchko Y, Shah AM, and Tedgui A. Evidence for altered interleukin 18 (IL)-18 pathway in human heart failure. FASEB J 18: 1752–1754, 2004.[Abstract/Free Full Text]
  29. Morel JC, Park CC, Zhu K, Kumar P, Ruth JH, and Koch AE. Signal transduction pathways involved in rheumatoid arthritis synovial fibroblast interleukin-18-induced vascular cell adhesion molecule-1 expression. J Biol Chem 277: 34679–34691, 2002.[Abstract/Free Full Text]
  30. Naito Y, Tsujino T, Fujioka Y, Ohyanagi M, Okamura H, and Iwasaki T. Increased circulating interleukin-18 in patients with congestive heart failure. Heart 88: 296–297, 2002.[Free Full Text]
  31. Ogura T, Ueda H, Hosohara K, Tsuji R, Nagata Y, Kashiwamura S, and Okamura H. Interleukin-18 stimulates hematopoietic cytokine and growth factor formation and augments circulating granulocytes in mice. Blood 98: 2101–2107, 2001.[Abstract/Free Full Text]
  32. Okamoto M, Kato S, Oizumi K, Kinoshita M, Inoue Y, Hoshino K, Akira S, McKenzie AN, Young HA, and Hoshino T. Interleukin 18 (IL-18) in synergy with IL-2 induces lethal lung injury in mice: a potential role for cytokines, chemokines, and natural killer cells in the pathogenesis of interstitial pneumonia. Blood 99: 1289–1298, 2002.[Abstract/Free Full Text]
  33. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K, Akita K, Namba M, Tanabe F, Konishi K, Fukuda S, and Kurimoto M. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 378: 88–91, 1995.[CrossRef][Medline]
  34. Olee T, Hashimoto S, Quach J, and Lotz M. IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses. J Immunol 162: 1096–1100, 1999.[Abstract/Free Full Text]
  35. Puren AJ, Fantuzzi G, Gu Y, Su MS, and Dinarello CA. Interleukin-18 (IFNgamma-inducing factor) induces IL-8 and IL-1beta via TNFalpha production from non-CD14+ human blood mononuclear cells. J Clin Invest 101: 711–721, 1998.[ISI][Medline]
  36. Raeburn CD, Dinarello CA, Zimmerman MA, Calkins CM, Pomerantz BJ, McIntyre RC Jr, Harken AH, and Meng X. Neutralization of IL-18 attenuates lipopolysaccharide-induced myocardial dysfunction. Am J Physiol Heart Circ Physiol 283: H650–H657, 2002.[Abstract/Free Full Text]
  37. Sande JB, Sjaastad I, Hoen IB, Bokenes J, Tønnessen T, Holt E, Lunde PK, and Christensen G. Reduced level of serine(16) phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity. Cardiovasc Res 53: 382–391, 2002.[Abstract/Free Full Text]
  38. Semb SO, Lunde PK, Holt E, Tønnessen T, Christensen G, and Sejersted OM. Reduced myocardial Na+, K+-pump capacity in congestive heart failure following myocardial infarction in rats. J Mol Cell Cardiol 30: 1311–1328, 1998.[CrossRef][ISI][Medline]
  39. Seta Y, Kanda T, Tanaka T, Arai M, Sekiguchi K, Yokoyama T, Kurimoto M, Tamura J, and Kurabayashi M. Interleukin-18 in patients with congestive heart failure: induction of atrial natriuretic peptide gene expression. Res Commun Mol Pathol Pharmacol 108: 87–95, 2000.[ISI][Medline]
  40. Simms MG and Walley KR. Activated macrophages decrease rat cardiac myocyte contractility: importance of ICAM-1-dependent adhesion. Am J Physiol Heart Circ Physiol 277: H253–H260, 1999.[Abstract/Free Full Text]
  41. Woldbæk PR, Hoen IB, Christensen G, and Tønnessen T. Gene expression of colony-stimulating factors and stem cell factor after myocardial infarction in the mouse. Acta Physiol Scand 175: 173–181, 2002.[CrossRef][ISI][Medline]
  42. Woldbæk PR, Tønnessen T, Henriksen UL, Florholmen G, Lunde PK, Lyberg T, and Christensen G. Increased cardiac IL-18 mRNA, pro-IL-18 and plasma IL-18 after myocardial infarction in the mouse: a potential role in cardiac dysfunction. Cardiovasc Res 59: 122–131, 2003.[CrossRef][ISI][Medline]
  43. Woodley SL, McMillan M, Shelby J, Lynch DH, Roberts LK, Ensley RD, and Barry WH. Myocyte injury and contraction abnormalities produced by cytotoxic T lymphocytes. Circulation 83: 1410–1418, 1991.[Abstract/Free Full Text]
  44. Wyman TH, Dinarello CA, Banerjee A, Gamboni-Robertson F, Hiester AA, England KM, Kelher M, and Silliman CC. Physiological levels of interleukin-18 stimulate multiple neutrophil functions through p38 MAP kinase activation. J Leukoc Biol 72: 401–409, 2002.[Abstract/Free Full Text]
  45. Zechner D, Thuerauf DJ, Hanford DS, McDonough PM, and Glembotski CC. A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression. J Cell Biol 139: 115–127, 1997.[Abstract/Free Full Text]



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