Acta Physiol 2015, 213, 492–504
IL-18 neutralization during alveolar hypoxia improves left ventricular diastolic function in mice V. Hillestad,1,2,3 E. K. S. Espe,1,2,3 F. Cero,1,2,3,4 K. O. Larsen,1,2,3,4 I. Sjaastad,1,2,3 S. Nyg ard,1,2,3,5 O. H. Skjønsberg4 and G. Christensen1,2,3 1 2 3 4 5
Institute for Experimental Medical Research, Oslo University Hospital Ullev al and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center, University of Oslo, Oslo, Norway Center for Heart Failure Research, University of Oslo, Oslo, Norway Departement of Pulmonary Medicine, Oslo University Hospital Ullev al and University of Oslo, Oslo, Norway Bioinformatics Core Facility, Institute for Medical Informatics, Oslo University Hospital and University of Oslo, Oslo, Norway
Received 9 June 2014, revision requested 30 June 2014, revision received 25 August 2014, accepted 27 August 2014 Correspondence: V. Hillestad, Institute for Experimental Medical Research, Oslo University Hospital Ullev al, Kirkeveien 166, 0407 Oslo, Norway. E-mail: vigdis.hillestad@medisin. uio.no
See Editorial Commentary: Toldo, S. et al. 2014. Diastolic dysfunction in chronic hypoxia: IL-18 provides the elusive link. Acta Physiol 213, 298–300.
Abstract Aim: In patients, an association exists between pulmonary diseases and diastolic dysfunction of the left ventricle (LV). We have previously shown that alveolar hypoxia in mice induces LV diastolic dysfunction and that mice exposed to hypoxia have increased levels of circulating interleukin-18 (IL-18), suggesting involvement of IL-18 in development of diastolic dysfunction. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18. In this study, we hypothesized that neutralization of IL-18 during alveolar hypoxia would improve LV diastolic function. Methods: Mice were exposed to 10% oxygen for 2 weeks while treated with IL-18BP or vehicle. Cardiac function and morphology were measured using echocardiography, intraventricular pressure measurements and magnetic resonance imaging (MRI). For characterization of molecular changes in the heart, both real-time PCR and Western blotting were performed. ELISA technique was used to measure levels of circulating cytokines. Results: As expected, exposure to hypoxia-induced LV diastolic dysfunction, as shown by prolonged time constant of isovolumic relaxation (s). Improved relaxation with IL-18BP treatment was demonstrated by a significant reduction towards control s values. Decreased levels of phosphorylated phospholamban (P-PLB) in hypoxia, but normalization by IL-18BP treatment suggest a role for IL-18 in regulation of calcium-handling proteins in hypoxia-induced diastolic dysfunction. In addition, MRI showed less increase in right ventricular (RV) wall thickness in IL-18BP-treated animals exposed to hypoxia, indicating an effect on RV hypertrophy. Conclusion: Neutralization of IL-18 during alveolar hypoxia improves LV diastolic function and partly prevents RV hypertrophy. Keywords diastolic dysfunction, hypoxia, interleukin-18, pulmonary hypertension, right ventricular hypertrophy.
More than 50% of heart failure patients have diastolic heart failure (Lam et al. 2011). Hence, identification of factors causing left ventricular diastolic dysfunction, which might eventually result in diastolic heart 492
failure, is of great importance for development of new therapeutic strategies. Diastolic dysfunction can be the result of several underlying causes, and notably, a relationship exists between diastolic dysfunction and
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diseases affecting the lungs (Baum et al. 1971). It has been documented that exposure to hypoxia leads to right ventricular (RV) hypertrophy (Moore-Gillon & Cameron 1985). Diastolic dysfunction of the right ventricle after chronic hypoxia exposure has also been shown (Larsen et al. 2006). Moreover, in a previous study, we showed that mice exposed to chronic alveolar hypoxia also develop diastolic dysfunction of the LV (Larsen et al. 2006). In previous work, we have demonstrated a role for the pro-inflammatory cytokine interleukin (IL)-18 in the development of diastolic dysfunction after chronic exposure to hypoxia (Larsen et al. 2008), suggesting inflammation as a link between alveolar hypoxia and diastolic dysfunction. In an experimental model, we showed that mice exposed to hypoxia developed diastolic dysfunction (Larsen et al. 2006) and that these animals had markedly increased circulating levels of IL-18 (Larsen et al. 2008). IL-18 is one of the effectors of the innate immune system (Fantuzzi & Dinarello 1999). Other studies support the notion that IL18 might be involved in diastolic dysfunction (Woldbaek et al. 2005, Yu et al. 2009). A naturally occurring soluble inhibitor of IL-18, the IL-18-binding protein (IL-18BP), has been identified, and its ability to inhibit IL-18 has been documented (Kim et al. 2000). In this study, we hypothesized that inhibition of IL-18 in vivo using IL-18BP may attenuate the development of left ventricular diastolic dysfunction induced by exposure to hypoxia.
Materials and methods Animals Male C57Bl/6 mice were obtained from Taconic (Skensved, Denmark) at 6 weeks of age and were acclimated for 7 days before start of any experiments. Animals were housed on standard bedding under day/ night cycles of 12/12 h at 21 °C. Food and water were given ad libidum. All experiments were approved by the Norwegian National Animal Welfare Committee (project ID 1385 and 4432) and conform to the Guide for the Use and the Care of Laboratory Animals published by the US National Institutes of Health (NIH publication No.85-23, revised 1996).
Materials Recombinant human IL-18BP isoform a (rhIL-18BPa) was purchased from R&D systems (Minneapolis, MN, USA) and reconstituted in PBS containing 0.1% serum albumin according to the manufacturer’s recommendations. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 was obtained from LIST
· IL-18 neutralization improves diastolic function
Biological Laboratories (Campbell, CA, USA). Reconstitution in PBS 0.1% serum albumin was achieved by heating at 37 °C with intermittent vortexing according to manufacturer’s recommendation.
Experimental procedures For all invasive procedures anaesthesia was induced and maintained by inhalation of isofluran (Zuurbier et al. 2002). Blood samples for ELISA analysis were drawn from the inferior vena cava under general anaesthesia and collected on ice into lithium–heparin coated tubes (Puls-Norge, Oslo, Norway) before centrifugation at 4000 g at 4 °C for 20 min. Plasma was stored at 70 °C. Heart and lungs were excised after dislocation of the neck in anaesthetized mice. The atria were removed and the right ventricle, LV and septum were separated, weighed and immediately snap frozen in liquid nitrogen.
Determination of administration dose and administration frequency of IL-18BP To study pharmacokinetics of the IL-18BP and to establish an administration protocol, two separate sets of experiments were carried out. In the first set of experiments, mice (n = 24) received IL-18BP (12.5 lg per mouse) or vehicle solution only. Plasma levels were determined by ELISA after 6, 10 or 24 h. In the second set of experiments, mice (n = 16) received an initial injection of IL-18BP 1 mg kg1 i.p. followed by i.p. injections of IL-18BP 0.5 mg kg1 every 48 h. Control animals received PBS only. Plasma concentrations of IL-18BP were determined at 2, 4 or 8 days after the initial injections.
Biological effect of IL-18BP In a separate set of experiments, the ability of IL18BP to inhibit IL-18 in vivo was investigated using LPS as previously described (Faggioni et al. 2001). Briefly, mice (n = 12) were injected with LPS (15 mg kg1 i.p.). Twenty-four hours prior to LPS administration, the animals received either IL-18BP (0.5 mg kg1) or vehicle solution only. Control animals received two injections of vehicle solution only. Plasma was collected 8 h after LPS injection and levels of IFN-c and IL-18 were determined by ELISA.
Chronic hypoxia model In the chronic hypoxia model, mice were either exposed to hypoxia (10% O2 in normobaric conditions) or to ambient air (21% O2) (Larsen et al.
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12376
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2006). Duration of hypoxia exposure was 2 weeks. We have previously described the development of elevated pulmonary artery pressure (PAP), RV hypertrophy and left ventricular diastolic dysfunction in this model (Larsen et al. 2006). Half of the animals, both in the hypoxia and the normoxia group, received IL18BP according to the protocol described in the preliminary experiments. The other half received equal volumes of vehicle solution only. After 2 weeks of hypoxia exposure, echocardiography, magnetic resonance imaging (MRI) or cardiac catheterization was performed. For all in vivo measurements of cardiac function, experiments were initiated with n = 8 in each group. The size of the groups was determined to achieve sufficient power and to minimize the number of animals used for the experiments. Some animals died, mainly due to bleeding during the procedure. All group sizes are given in figure legends of the figure corresponding to each experiment. The animals in the various groups were randomly killed, and we did not select one group first. Blood and organs were collected as described in Experimental procedures.
Doppler echocardiography Echocardiography was performed with VEVO 2100 (VisualSonics, Toronto, ON, Canada), which is designed to investigate rodents. Animals exposed to 2 weeks of hypoxia, with or without IL-18BP treatment, as well as their respective controls, were examined as previously described (Larsen et al. 2006). Briefly, animals were anaesthetized and placed in a supine position. An indirect measurement of the PAP was provided by pulmonary artery blood flow and pulmonary artery acceleration time (PAAT). To assess left ventricular diastolic function, mitral flow and tissue velocities were recorded using pulsed-wave Doppler mode. It is recognized that high heart rates in mice might result in fusion of the E and A waves (Collins et al. 2003, Stypmann et al. 2009). Decreased heart rates can be obtained by increasing the amount of anaesthesia. However, we wished to keep the animals as lightly sedated as possible to avoid cardiodepressive effects, as has been recommended by others (Lairez et al. 2013). Hence, for the analysis of maximal mitral inflow and mitral deceleration velocity, 10 of 32 animals had to be excluded due to fused E and A waves.
Magnetic resonance imaging Magnetic resonance imaging experiments were performed on a 9.4T pre-clinical MR system (Agilent Technologies, Palo Alto, CA, USA) with high-performance gradient and RF coils dedicated to mouse 494
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imaging (Espe et al. 2013). Anaesthesia was induced in an anaesthesia chamber with a mixture of O2 and 4% isoflurane and further maintained by administration of a mixture of O2 and 1.5–2.0% isoflurane in freely breathing animals. Heart rate, body temperature and respiration rate were constantly monitored during experiments, and the level of anaesthesia was regulated to keep the heart and respiration rate as stable as practically possible. Animal temperature was maintained by heated air. Magnetic resonance imaging cine data sets were acquired using a motion compensated gradient echo sequence. Left ventricular short-axis slices (7–10) were acquired, covering the heart from base to apex. Central imaging parameters: echo/repetition time = 2.50/3.67 ms; field of view = 25.6 9 25.6 mm; acquisition matrix 128 9 128; slice thickness = 1.00 mm; flip angle 15°; and averages = 3. Acquisition was prospectively gated from ECG and respiration. The total experiment duration was 75 min per animal. MRI data were analysed using MATLAB (The MathWorks, Natick, MA, USA) with the operator blinded to animal groups. While hypoxiainduced LV diastolic dysfunction was primarily described by intraventricular pressure measurements and echocardiography, MRI provided valuable information about RV morphology.
Cardiac catheterization Animals exposed to 2 weeks of hypoxia, with or without IL-18BP treatment, as well as their respective controls, were anaesthetized and placed in a supine position. The investigator was blinded to the experimental groups. A catheter with an optical transducer tip (Samba Prelim 420; Samba Sensors, V€ astra Fr€ olunda, Sweden) was introduced into the right or LV via the surgically exposed right jugular vein or right carotid artery, respectively, and data from 10 consecutive beats were recorded and analysed using DIADEM software (National Instruments, Austin, TX, USA). Right ventricular systolic pressure (RVSP), right ventricular end-diastolic pressure (RVEDP), left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP) and positive/minimum derivative of the pressure curve were registered. The time constant of isovolumetric relaxation (s) was calculated using a custom made script fitting the pressure curve during the isovolumetric relaxation phase to a monoexponential function described by the following formula: P = P0expt/s, where P is left ventricular diastolic pressure, t is time and P0 is pressure at the time point for dP/dtmin. The isovolumetric relaxation phase started at the time point for dP/dtmin and ended when pressure was declined by 1/e.
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Real-time PCR
Enzyme-linked immunosorbent assay
RNA from right and LV was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Extracted RNA was quantified by spectrophotometry (ND-1000 Saveen Werner; Nanodrop, Malm€ o, Sweden), and RNA integrity was evaluated by agarose gel electrophoresis (Bioanalyser 2100; Agilent Technologies). All samples had a RNA integrity number (RIN) higher than 7.5. Quantitative real-time PCR was performed using TaqMan technology (7900 HT real-time PCR; Applied Biosystems, Foster City, CA, USA). All samples were reverse transcribed using iScript Reverse Transcription kit (Bio-Rad laboratories, Hercules, CA, USA). Gene-specific TaqMan probes (Applied Biosystems) for collagen I (Mm00483888_m1) and collagen III (Mm00802331_m1), brain natriuretic peptide (BNP, Mm00435304_g1), Acta1 (a skeletal actin, Mm00808218_g1) and b myosin heavy chain (b-MCH, Mm00600555_m1). All samples were run in duplicates and quantification was done by comparing the average cycle of threshold (Ct) values to a standard curve.
Commercially available ELISA kits (R&D systems) were used following manufacturer’s instructions to determine the plasma concentrations of human IL18BP, mouse IL-18 and mouse IFN-c (catalogue number 7625 and DY485). ELISA kit used for the detection of mouse IL-18BP was obtained from USCN Life Sciences Inc (Wuhan, China).
Western blot analysis Homogenates were made from left ventricular tissue of mice exposed to 2 weeks of hypoxia and were analysed by SDS page and Western blotting as previously described (Larsen et al. 2006). The following antibodies were used for detection: phospholamban total anti-mouse (#MA3-922), sarcoplasmatic reticulum calcium ATPase anti-mouse (#MA3-910) (AH diagnostic, Oslo, Norway), phospholamban ser16 anti-rabbit (#A010-12) (Badrilla, Leeds, UK) and NCX anti-rabbit [a kind gift from Thomas MJ (Thomas et al. 2003)]. For analysis of the amount of collagen III, left ventricular tissue was homogenized in extraction buffer containing 10 mmol L1 cacodylic acid pH 5.0, 0.15 mol L1 NaCl, 1 lmol L1 ZnCl2, 20 mmol L1, CaCl2, 1.5 mmol L1, NaN3, 0.01% (v/v) Triton X-100 and protease inhibitor. Samples were incubated for 12 h at 4 °C. After centrifugation at 1200 g, supernatants were collected and separated into aliquots. Collagen III anti-rabbit (#Ab 82354) (Abcam, Cambridge, MA, USA) was used for detection. Immunoreactive bands were detected using an enhanced chemiluminescence kit (Amersham, Oakville, ON, Canada) according to manufacturer’s protocol. Blots were detected using a high-resolution video camera system LAS-3000 mini CCD (Fuji Photo Film Europe, D€ usseldorf, Germany) and the staining on the blots was quantified with the IMAGE GAUGE software (Version 2.54; Fuji Photo Film, Japan).
Statistics All statistical analyses were performed using SIGMASversion 12.0 (Systat Software Inc., San Jose, CA, USA). To evaluate the effect of IL-18 neutralization during chronic hypoxia exposure, two-way ANOVA was performed, correcting for multiple comparison using Student–Newman–Keuls’ all pairwise procedures. The two-way ANOVA implementation in SIGMASTAT automatically handles unequal group sizes using a general linear model approach which constructs hypothesis tests using the marginal sums of squares. Student’s t-test was used when comparing two groups only. A P value lower than 0.05 was considered statistically significant. TAT
Results Determination of administration dose and administration frequency of IL-18BP To determine the administration dose and frequency, two sets of experiments were carried out. The first set of experiments showed elevated plasma levels of IL18BP 6 h after administration of 0.5 mg kg1 (Fig. 1a). Very low levels of IL-18BP were measured in plasma of control animals and in animals treated with PBS (Fig. 1a). Previous studies have shown significant biological effects of 0.5 mg kg1 rhIL-18 Pa in vivo (Plater-Zyberk et al. 2001, Wang et al. 2006), and we therefore decided to use this dose in the following experiments. ELISA analysis 6, 10 and 24 h after injection of IL-18BP (0.5 mg kg1) revealed a time-dependent decrease in plasma levels, but the levels were still high 24 h after administration. These findings were used to calculate plasma half-life of IL18BP in vivo in mice, and we found a t1/2 value of 72 h. This relatively high value of t1/2 is seen with peptides linked with antibody Fc domain, as is the case for IL-18BP (Lee et al. 2008). Based on these findings we designed an administration protocol consisting of a bolus dose of 1 mg kg1 followed by injections of 0.5 mg kg1 every 48 h. ELISA performed 2, 4 and 8 days after the primary injection
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Biological effect of IL-18BP Figure 2 shows circulating levels of IFN-c and IL-18 measured 8 h after LPS injection in mice that had received i.p. injections with either 0.5 mg kg1 of IL18BP or vehicle. Decreased levels of IFN-c in the IL18BP group compared with controls receiving vehicle showed that IL-18BP is a potent inhibitor of IL-18 produced in vivo (259 102 vs. 1081 284 pg mL1, P < 0.05, Fig. 2a). The IL-18 concentration in plasma was markedly upregulated by LPS as expected (6396 530 vs. 291 105 pg mL1, P < 0.001, Fig. 2b). A reduction was observed with IL-18BP pretreatment (4067 624 vs. 6396 530 pg mL1, P < 0.01).
IL-18 neutralization attenuates the development of left ventricular diastolic dysfunction We have previously shown left ventricular diastolic dysfunction in animals exposed to hypoxia (Larsen et al.
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2006). Intraventricular pressure measurements confirmed diastolic dysfunction in the present experiments, as shown by a prolongation of the time constant of isovolumetric relaxation s (6.0 0.4 ms vs. 4.8 0.3 ms, P < 0.05, Fig. 3a) and longer isovolumetric relaxation time (IVRT, 7.9 0.4 vs. 5.6 0.3 ms, Fig. 3b) in animals exposed to 2 weeks of hypoxia compared with normoxic controls. Interestingly, in mice treated with IL-18BP during hypoxia exposure, s and IVRT values were significantly shortened compared to the hypoxia vehicle group (4.6 0.3 vs. 6.0 0.4 ms, Fig. 3a, and 5.5 0.2 vs. 7.9 0.4, Fig. 3b, both P < 0.05). A trend towards increased LVEDP in the hypoxia PBS group was also found (6.4 0.8 vs. 4.3 0.7 mmHg, P = 0.10, Fig. 3c). Following treatment with IL-18BP, LVEDP values were similar to control values (4.2 0.6 vs. 4.3 0.7 mmHg, Fig. 3c). No significant differences were found in dP/dtmax relative to peak systolic pressure or heart rate between the four groups (Fig. 3d,e). Representative haemodynamic traces and echocardiographic images are shown in Figure 4. Diastolic parameters analysed by echocardiography are shown in Table 1, and body and heart weights are shown in Table 2.
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Figure 1 Plasma concentrations of IL-18BP. (a) Plasma concentration of recombinant human IL-18BP measured 6, 10 and 24 h (h) after i.p. injections (n = 4 in each group). (b) Plasma concentration of IL-18BP after i.p. injections of IL-18BP (filled symbols) or PBS only (open symbols) every 2. Day (d) (n = 4 in each group).
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Figure 2 Plasma concentration of IFN-c and IL-18. Plasma concentrations of interferon (IFN)-c (a) and endogenous produced mouse interleukin (IL)-18 (b) in mice receiving lipopolysaccharide (LPS). Twenty-four hour prior to LPS injections, the animals received injections with either PBS only (LPS + vehicle, n = 6) or recombinant human IL-18BP (LPS + IL-18BP, n = 6). For mice receiving vehicle only n = 4. *P < 0.05 vs. vehicle only, #P < 0.05 vs. LPS + vehicle.
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12376
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Figure 3 In vivo diastolic function. Mice exposed to hypoxia had increased s (a) and isovolumetric relaxation time (IVRT) (b) compared with control animals, indicating diastolic dysfunction. In the hypoxia IL-18BP group, s (a) and IVRT (b) were normalized to control values. End-diastolic pressure, maximum negative decay of pressure curve relative to peak systolic pressure and heart rate are shown in panel c, d and e respectively. For all panels, n = 7 in control vehicle group, n = 2 in control IL18BP group, n = 5 in the hypoxia vehicle group and n = 8 in the hypoxia IL-18BP group. *P < 0.05 vs. control vehicle, # P < 0.05 vs. hypoxia vehicle
Levels of Ca2+-handling proteins To investigate whether improved diastolic function after IL-18 neutralization could be explained by enhanced relaxation due to an effect on phosphory lation levels of phospholamban in the LV, Western blotting was performed, investigating levels of phosphorylated phospholamban (P-PLB) at serine16 residue relative to total phospholamban levels. Our group has previously identified this as a possible mechanism for diastolic dysfunction during hypoxia (Larsen et al. 2006). We found that levels of serine16 P-PLB were decreased to 55% in the hypoxia group compared with the normoxia group (55 9 vs. 100 8%, P = 0.01, Fig. 5a). A complete normalization was found in the hypoxia group treated with IL-18BP, as the level of P-PLB in the hypoxia IL-18BP group was not significantly different from the control vehicle group (94 9 vs. 100 8%, Fig. 5a). Hypoxia exposure did not induce changes in the levels of SERCA2 protein (110 5 vs. 100 4%, Fig. 5c) or in NCX protein (117 2 vs. 100 6%, Fig. 5e). In animals exposed to hypoxia, there were no significant changes in the level of SERCA2 or NCX between animals receiving IL-18BP and animals receiving vehicle only (111 7 vs. 110 5%, and 119 10 vs. 117 2% respectively, Fig. 5c,e).
Effect of IL-18 neutralization on expression of genes encoding extracellular matrix proteins in the left ventricle Expression of collagen I mRNA was not significantly altered, neither by hypoxia nor by treatment with IL18BP (100 11% in control vehicle, 118 12% in hypoxia vehicle and 108 5% in hypoxia IL-18BP, Fig. 6a). However, the expression of collagen III
mRNA was slightly, but significantly upregulated in the hypoxia vehicle group compared with the control vehicle group (132 4% in hypoxia vehicle vs. 100 9% in control vehicle, P < 0.05, Fig. 6b). Within the time frame examined, we did not find any significant increase in the amount of collagen III protein, neither following hypoxia exposure, nor after IL-18BP treatment (100 14% in control vehicle, 94 16% in hypoxia vehicle, and 113 11% in hypoxia IL-18BP, Fig. 6c).
IL-18 neutralization partly prevents development of right ventricular hypertrophy Two weeks of hypoxia exposure increased RV weight (RVW) normalized to tibia length (RVW/TL) by 35% compared with control animals (1.5 0.1 vs. 1.1 0.1 mg mm1, P < 0.001, Fig. 7b), indicating RV hypertrophy. This was supported by an increased thickness of right ventricle free wall measured by MRI (0.59 0.06 vs. 0.45 0.02 mm, P < 0.001, Fig. 7a). The reduction in RVW/TL did not reach statistical significance in the IL-18BP-treated mice exposed to hypoxia compared with mice exposed to hypoxia receiving vehicle solution (1.4 0.1 vs. 1.5 0.1 mg mm1). However, the IL-18BP treated group had a significantly reduced wall thickness on MRI compared with the hypoxia PBS group (0.51 0.02 vs. 0.59 0.06 mm, Fig. 7a).
IL-18 neutralization and hypoxia-induced pulmonary hypertension Several parameters were measured to investigate the effect of IL-18 neutralization on pulmonary hypertension. In mice exposed to 2 weeks of hypoxia, RVSP was increased by 46% compared with control animals
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Figure 4 Hemodynamic traces and echocardiographic images. Hemodynamic traces, images from flow Doppler and tissue Doppler are shown for one representative animal in each group. Red line indicates the phase of the curve used for s analysis. LVSP, left ventricular peak systolic pressure; dP/dtmin, time point for maximum decay of pressure curve. White arrows indicate maximal tissue velocity.
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(45.0 2.0 vs. 29.3 0.7 mmHg, P < 0.001, Fig. 7c) and PAAT was decreased to 70% (15.2 0.7 vs. 21.9 0.9 ms, P < 0.001, Fig. 7d). These results show that hypoxia exposure induces pulmonary hypertension. The trend towards normalization of RVSP and PAAT in hypoxic animals receiving IL18BP compared with hypoxic animals receiving vehicle only did not reach statistical significance (41.4 1.3 mmHg in hypoxia IL-18BP vs. 45.0 2.0 mmHg in hypoxia vehicle and 16.8 0.6 ms in hypoxia IL-18BP vs. 15.2 0.7 ms in hypoxia vehicle, P = 0.07 and P = 0.16 respectively, Fig. 7c,d). 498
Molecular changes in the right ventricle We also investigated expression of genes known to be regulated in myocardial hypertrophy. Results are shown in Table 3. Although several genes were found to be regulated in animals exposed to hypoxia, only a-skeletal actin was differentially regulated in animals treated with IL-18BP.
Endogenous mouse plasma IL-18BP To investigate whether the injected human IL-18BP interfered with the in vivo production of IL-18BP, we
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Table 1 Cardiac function and morphology Control vehicle n=8 Cardiac dimensions measured by MRI RV-free wall ED (mm) IVS ES (mm) LV-free wall ED (mm) RV-free wall ES (mm) IVS ES (mm) LV-free wall ES (mm) Ejection fraction (%) Echocardiography: (tissue) Doppler Heart rate Maximum mitral velocity (mm s1) Diastolic tissue velocity (m s1) E/E0 Mitral flow deceleration (m s2) Cardiac output (mL min1)
Control IL-18BP n=8
Hypoxia vehicle n=8
Hypoxia IL-18BP n=8
0.46 1.10 1.06 0.59 1.33 1.50 73.56
0.02 0.06 0.05 0.02 0.10 0.08 2.49
0.40 1.13 0.95 0.53 1.39 1.52 67.66
0.02 0.04 0.04 0.03 0.05 0.08 2.33
0.59 1.23 1.05 0.70 1.40 1.38 65.77
0.02 *,*** 0.05 0.02 0.03*,*** 0.07 0.06 1.49
0.51 1.13 1.05 0.66 1.26 1.64 64.17
0.02 *,***,** 0.03 0.03 0.04*,*** 0.02 0.07 2.57
454.5 586.8 16.1 39.6 24.7 27.2
13.3 29.6 1.4 4.4 2.8 2.8
453.5 632.0 14.9 43.2 23.6 25.7
8.0 22.9 0.7 2.4 2.4 1.8
475.8 559.1 13.5 42.6 26.0 18.1
20.0 42.3 0.9 3.7 2.4 1.4
521.8 531.5 16.1 34.6 22.5 25.2
20.5 11.0 1.2 2.8 1.4 2.2
RV, right ventricle; LV, left ventricle; IVS, interventricular septum; ES, end systole; ED, end diastole; MRI, magnetic resonance imaging. *P < 0.05 vs. control vehicle, **P < 0.05 vs. hypoxia vehicle, ***P < 0.05 vs. control IL-18BP.
Table 2 Organ weights Control vehicle n = 16 Body weight (g) RVW (mg) RVW/TL (mg mm1) LVW/TL (mg mm1) LW/TL (mg mm1)
27.05 19.57 1.15 4.97 8.09
0.52 0.69 0.04 0.12 0.25
Control IL-18BP n = 16 27.03 20.26 1.19 5.21 8.65
0.56 0.76 0.04 0.14 0.24
Hypoxia vehicle n = 16 25.08 27.36 1.62 4.76 11.10
0.51*,** 1.02*,** 0.06*,** 0.13 0.25*,**
Hypoxia IL-18BP n = 16 25.22 25.44 1.50 4.76 10.94
0.27*,** 1.1*,** 0.06*,** 0.12 0.33*,**
RVW, right ventricular weight; TL, tibia length; LVW, left ventricular weight; LW, lung weight. *P < 0.05 vs. control vehicle, **P < 0.05 vs. control IL-18BP.
measured endogenous mouse IL-18BP in animals exposed to hypoxia for 2 weeks. No significant changes were induced, neither by hypoxia, nor by IL18BP injections (488 57 pg mL1 in control vehicle, 404 57 in pg mL1 control IL-18BP, 488 47 pg mL1 in hypoxia vehicle and 506 66 pg mL1 in hypoxia IL-18BP, Fig. 8). These results show that the concentration of the injected human IL-18BP was about 20-fold higher than the levels of endogenous mouse IL-18BP during hypoxia.
Discussion The present study shows that in vivo injection of IL18BP is an efficient method for IL-18 neutralization. Moreover, inhibition of IL-18 during hypoxia exposure attenuates development of left ventricular
diastolic dysfunction. We demonstrate that the dephosphorylation of phospholamban previously observed after hypoxia exposure (Larsen et al. 2006) can be prevented by IL-18 neutralization, which may explain the observed improvement in diastolic function. Moreover, IL-18 neutralization inhibits the development of RV hypertrophy. Diastolic dysfunction secondary to lung disease has received relatively little attention, and currently, no available treatment exists to prevent the development of diastolic dysfunction secondary to pulmonary disease. It has been shown that exposure to hypoxia can lead to diastolic dysfunction of both ventricles in healthy human beings (Huez et al. 2005). Furthermore, although hypoxia leads to RV hypertrophy, systolic function is generally preserved (Huez et al. 2005, Kolb & Hassoun 2012). Development of diastolic dysfunction of both right and LV in the experimental
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(a)
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(b)
(c)
(d)
(e)
(f)
Figure 5 Calcium-handling proteins. Two weeks of hypoxia exposure induced a decrease in serine16 phosphorylated phospholamban (PLN) which was normalized to control levels in the hypoxia IL-18BP group (panel a and d, n = 4 in each group). Protein levels of sarcoplasmatic reticulum calcium ATPase 2 (SERCA2, panel b and e, n = 4 in each group) and sodium–calcium exchanger (NCX, panel c and f, n = 4 in each group) and were not significantly changed, neither by hypoxia nor by IL-18BP treatment. Results are shown as per cent of control vehicle. Vinculin was used as loading control. *P < 0.05 vs. control vehicle.
mouse model of hypoxia has previously been described by us and these animals also have increased circulating levels of IL-18 (Larsen et al. 2008). We here show that specific neutralization of IL-18 during hypoxia exposure can partly prevent development of diastolic dysfunction, as shown by normalization of the time constant of isovolumic relaxation (s). s is considered to be one of the most reliable parameters 500
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(a)
(b)
(c)
(d)
Figure 6 Collagen content in the left ventricle (LV). Hypoxia exposure did not induce any significant changes in left ventricular gene expression of collagen I (a, n = 8 in each group) or collagen III (b, n = 8 in each group), neither did IL-18BP treatment. The amount of collagen III protein in the LV was not altered (panel c, n = 4 in each group), Vinculin was used as loading control (panel d). *P < 0.05 vs. control vehicle.
of isovolumic relaxation as it is considered independent of load and heart rate. This is not the case for the maximal rate of pressure decay (dP/dtmax) which is considered dependent on the prevailing load of the intact circulation. To our knowledge, this is the first study showing that inhibition of a specific cytokine can improve diastolic function due to alveolar hypoxia, and our findings are in agreement with other studies which have shown that administration of IL18 can cause and aggravate diastolic dysfunction (Woldbaek et al. 2005, Yu et al. 2009, Xing et al. 2010). Improved cardiac function following IL-18 neutralization has been shown in a model of endotoxaemia by a reduction in LVEDP, which does not reflect diastolic function per se (Raeburn et al. 2002). We sought to determine the mechanisms behind the positive effect of IL-18 neutralization on diastolic function. Several theories have been raised to explain the mechanisms causing diastolic dysfunction in chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (Schena et al. 1996, Tutar et al. 1999, Ozer et al. 2001). In a previous study
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$#
0.5 0.4 0.3 0.2 0.1
(c)
Control
50
RV weight/TL (mg mm–1)
RV free wall (mm)
0.6
(d)
0.8 0.6 0.4 0.2 Hypoxia
25
30
20
500
400
300
200
100
PAAT (ms)
RVSP (mmHg)
1.0
20
*
$
Hypoxia
0
15
10
5
10
Control
600
$
40
0
IL-18BP
$
1.2
Control
Vehicle
700
1.4
0.0
Hypoxia
*
*
1.6
*
0.0
Vehicle IL-18BP
(b)
0.7
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Mouse plasma IL-18BP (pg mL–1)
(a)
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0
Control
Hypoxia
Figure 7 Right ventricular (RV) hypertrophy and pulmonary hypertension. Hypoxia exposure induced RV hypertrophy and pulmonary hypertension, as shown by increased RV wall thickness (panel a, n = 8 in each group), increased RV weight (panel b, n = 16 in each group), increased right ventricular systolic pressure (RVSP, panel c, n = 8 in each group) and shortened pulmonary acceleration time (PAAT, panel d, n = 8 in each group). *P < 0.05 vs. control vehicle, $ P < 0.05 vs. control IL-18BP, #P < 0.05 vs. hypoxia vehicle.
carried out by our group, decreased level of P-PLB was suggested to be responsible for the observed impairment in relaxation (Larsen et al. 2006). Phospholamban is tightly coupled to SERCA at the sarcoplasmatic reticulum membrane and plays a crucial role in regulating calcium reuptake during diastole (MacLennan & Kranias 2003). When phospholamban is phosphorylated at the serine16 residue, conformational changes in the protein lead to a relief of the
Control
Hypoxia
Figure 8 Endogenous mouse plasma IL-18BP. Circulating levels of endogenous mouse IL-18BP in mice exposed to 2 weeks of normoxia (control) or hypoxia and receiving injections with either human IL-18BP or vehicle, n = 4 in each group.
inhibitory effect of phospholamban on SERCA activity. A relationship between circulating IL-18 and phosphorylation levels of phospholamban has already been suggested (Woldbaek et al. 2003, Larsen et al. 2008). Here, we show that phosphorylation levels of phospholamban were completely normalized when circulating IL-18 was inhibited by IL-18BP treatment. Hence, our finding indicates that IL-18 is directly involved in regulation of phosphorylation levels of phospholamban and that this response can be inhibited by neutralizing the cytokine. To our knowledge, this physiological effect of specific anti-inflammatory treatment has not previously been reported. As our group has already demonstrated that the hypoxiainduced decline in P-PLB is related to an IL-18 induced increase in protein phosphatase 2A activity, IL-18BP treatment may reverse that increase. Phospholamban phosphorylation may also be regulated by beta-adrenergic stimulation, but we have previously
Table 3 Markers of hypertrophy
BNP/rpl32 Acta1/rpl32 b-MHC/rpl32
Control vehicle n = 16
Control IL-18BP n = 16
Hypoxia vehicle n = 16
Hypoxia IL-18BP n = 16
1.00 0.16 1.00 0.19 1.00 0.13
1.93 0.38 1.69 0.65 1.17 0.17
5.25 1.32*,** 3.32 0.57*,** 1.33 0.16
7.09 1.4*,** 6.19 1.16*,**,*** 1.06 0.18
BNP, brain natriuretic peptide; RPL32, ribosomal protein L32; Acta1, alpha sceletal actin; b-MCH, b-myosin heavy chain. All data are normalized to the expression of ribosomal protein L32 (RPL32) and are shown as percent of control vehicle. *P < 0.05 vs. control vehicle, **P < 0.05 vs. control IL-18BP, ***P < 0.05 vs. hypoxia vehicle.
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shown that alveolar hypoxia exposure does not alter levels of beta-adrenoceptors and their activity in the LV (Larsen et al. 2008). In a previous study we examined the effect of IL-18 on isolated cardiomyocytes and found that IL-18 induced decreased levels of P-PLB (Larsen et al. 2008), supporting the notion that IL-18 inhibition might have beneficial effects on cardiac cells. A study published during progress of our study shows that IL18 can cause cardiac dysfunction by direct effects on the heart and that blocking of IL-18 with IL-18BP prevents the negative effects on cardiac function (Toldo et al. 2014). IL-18BP is considered to exert its effect through IL-18 inhibition, and no off-target effects of IL-18BP have been described. However, we acknowledge that general off-target effects cannot be excluded, including other direct effects of IL-18BP and non-cardiac effects of IL-18 inhibition. Prolongation of the time constant of isovolumetric relaxation reflects slowed relaxation of the cardiomyocytes (Paulus et al. 2007). However, increased stiffness of the ventricle due to extracellular matrix remodelling could also lead to impaired relaxation. Increased fibrosis and remodelling is an important cause of ventricular stiffness, and evidence exists for a link between left ventricular fibrosis and inflammation (Paulus et al. 2007, Wei 2011). Hence, an additional explanation for diastolic dysfunction secondary to hypoxia could be alterations in extracellular matrix, leading to increased myocardial stiffness, affecting the filling phase of diastole. To assess ventricular stiffness measurements of pressure–volume relationships could have been performed. However, we examined extracellular matrix composition by gene expression analysis and determination of protein levels of collagen and did not find alterations indicating altered myocardial stiffness. Apart from a slight increase in collagen III mRNA expression, we could not show any increased amount of collagen at the protein level in animals exposed to hypoxia within the time frame examined. In addition, Larsen et al. (2008) could not observe any sign of fibrosis in this model on histological preparations. Although there is evidence that IL-18 can mediate cardiac fibrosis (Platis et al. 2008, Yu et al. 2009), fibrosis does not seem to play an important role in the development of diastolic dysfunction induced by alveolar hypoxia within the time frame examined in this study. We also found reduced RV hypertrophy after IL18BP treatment. Exposure to hypoxia leads to increased pressure in the pulmonary vasculature which constitutes increased afterload on the right ventricle and stimulates a hypertrophic response of the latter. Diastolic dysfunction has also been shown to be associated with pulmonary hypertension (Tutar et al. 502
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1999). Thus, an effect of IL-18BP on PAP could be a plausible explanation for the observed effects on the heart. Although we did not find a significant reduction of RV systolic pressure in the IL-18BP-treated group compared with the non-treated group, we cannot exclude this mechanism. In addition, IL-18 has been shown to directly affect myocardial hypertrophy (Chandrasekar et al. 2005, Vanderheyden et al. 2005). Findings in those previous studies support the notion that IL-18 may directly affect the hypertrophy of the right ventricle. Our gene expression data did not show a reduction in hypertrophy markers. However, such divergence between hypertrophic growth and foetal gene expression has also been shown by others. Notably, in one study, suppression of hypertrophy in transverse aortic banding led to a further increase in BNP expression (Patrizio et al. 2007). A tendency towards increased expression of hypertrophy markers was present in the control IL-18BP group compared to the control vehicle group. However, we did not observe any increase in RV wall thickness measured by MRI in the control IL-18BP group compared with the control vehicle group. Hence, we do not believe that the control IL-18BP animals have hypertrophy of the right ventricle. Inhibition of IL-18 with IL-18BP could constitute a new therapeutic approach. In our experiments using LPS, we demonstrate the potency of IL-18BP as an inhibitor of inflammation. Indeed, in animals pre-treated with IL-18BP, IFN-c levels were reduced to 20% of levels observed in animals pre-treated with vehicle solution only. Strong effects of IL-18BP on LPSinduced mortality have also been shown by others (Faggioni et al. 2001). Plasma concentrations of IL18BP used in the LPS experiments were similar to the steady-state concentrations we obtained when treating the animals exposed to hypoxia. Hence, we know that the actual plasma concentration is efficient for inhibiting IL-18 in vivo. This is of particular importance, as too large concentrations of IL-18BP may lead to a pooling effect and increased activity of the targeted cytokine, as has been shown to be the case with TNFa and etanercept (Mann et al. 2008). Such effects might explain the lack of effect in heart failure patients treated with etanercept. This should also be considered when designing treatment with IL-18BP. IL-18BP already exists for clinical use and has been tested in patients with rheumatic disease (Tak et al. 2006). IL-18 inhibition might thus offer new possibilities for treatment of diastolic heart failure in hypoxic COPD patients, but beforehand, careful study of dosage of the inhibitor is needed. In conclusion, in this study, we show that neutralization of IL-18 during alveolar hypoxia improves LV diastolic function and partly prevents RV hypertrophy.
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We thank Almira Hasic, Ulla Enger, Hilde Dishington and Lisbeth Winer for skilful technical work and Marita Martinsen and the rest of the staff at the animal facility for expert animal care. We also thank Finn Olav Levy for useful advice concerning pharmacological calculations.
Conflict of interest None to declare.
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Larsen, K.O., Sjaastad, I., Svindland, A., Krobert, K.A., Skjønsberg, O.H. & Christensen, G. 2006. Alveolar hypoxia induces left ventricular diastolic dysfunction and reduces phosphorylation of phospholamban in mice. Am J Physiol Heart Circ Physiol 291, H507–H516. Larsen, K.O., Lygren, B., Sjaastad, I., Krobert, K.A., Arnkværn, K., Florholmen, G., Larsen, A.K., Levy, F.O., Tasken, K., Skjønsberg, O.H. & Christensen, G. 2008. Diastolic dysfunction in alveolar hypoxia: a role for interleukin-18-mediated increase in protein phosphatase 2A. Cardiovasc Res 80, 47–54. Lee, T.Y., Tjin Tham Sjin, R.M., Movahedi, S., Ahmed, B., Pravda, E.A., Lo, K.M., Gillies, S.D., Folkman, J. & Javaherian, K. 2008. Linking antibody Fc domain to endostatin significantly improves endostatin half-life and efficacy. Clin Cancer Res, 14, 1487–1493. MacLennan, D.H. & Kranias, E.G. 2003. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol 4, 566–577. Mann, D.L., Bozkurt, B., Torre-Amione, G., Soran, O.Z. & Sivasubramanian, N. 2008. Effect of the soluble TNFantagonist etanercept on tumor necrosis factor bioactivity and stability. Clin Transl Sci 1, 142–145. Moore-Gillon, J.C. & Cameron, I.R. 1985. Right ventricular hypertrophy and polycythaemia in rats after intermittent exposure to hypoxia. Clin Sci (Lond) 69, 595–599. Ozer, N., Tokgozoglu, L., Coplu, L. & Kes, S. 2001. Echocardiographic evaluation of left and right ventricular diastolic function in patients with chronic obstructive pulmonary disease. J Am Soc Echocardiogr 14, 557–561. Patrizio, M., Musumeci, M., Stati, T., Fasanaro, P., Palazzesi, S., Catalano, L. & Marano, G. 2007. Propranolol causes a paradoxical enhancement of cardiomyocyte foetal gene response to hypertrophic stimuli. Br J Pharmacol 152, 216– 222. Paulus, W.J., Tschope, C., Sanderson, J.E., Rusconi, C., Flachskampf, F.A., Rademakers, F.E., Marino, P., Smiseth, O.A., De Keulenaer, G., Leite-Moreira, A.F. et al. 2007. How to diagnose diastolic heart failure: a consensus statement on the diagnosis of heart failure with normal left ventricular ejection fraction by the Heart Failure and Echocardiography Associations of the European Society of Cardiology. Eur Heart J 28, 2539–2550. Plater-Zyberk, C., Joosten, L.A., Helsen, M.M., SattonnetRoche, P., Siegfried, C., Alouani, S., van De Loo, F.A., Graber, P., Aloni, S., Cirillo, R., Lubberts, E., Dinarello, C.A., van Den Berg, W.B. & Chvatchko, Y. 2001. Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis. J Clin Invest 108, 1825–1832. Platis, A., Yu, Q., Moore, D., Khojeini, E., Tsau, P. & Larson, D. 2008. The effect of daily administration of IL-18 on cardiac structure and function. Perfusion 23, 237–242. Raeburn, C.D., Dinarello, C.A., Zimmerman, M.A., Calkins, C.M., Pomerantz, B.J., McIntyre, R.C. Jr, Harken, A.H. & Meng, X. 2002. Neutralization of IL-18 attenuates lipopolysaccharide-induced myocardial dysfunction. Am J Physiol Heart Circ Physiol 283, H650–H657.
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Schena, M., Clini, E., Errera, D. & Quadri, A. 1996. EchoDoppler evaluation of left ventricular impairment in chronic cor pulmonale. Chest 109, 1446–1451. Stypmann, J., Engelen, M.A., Troatz, C., Rothenburger, M., Eckardt, L. & Tiemann, K. 2009. Echocardiographic assessment of global left ventricular function in mice. Lab Anim 43, 127–137. Tak, P.P., Bacchi, M. & Bertolino, M. 2006. Pharmacokinetics of IL-18 binding protein in healthy volunteers and subjects with rheumatoid arthritis or plaque psoriasis. Eur J Drug Metab Pharmacokinet 31, 109–116. Thomas, M.J., Sjaastad, I., Andersen, K., Helm, P.J., Wasserstrøm, J.A., Sejersted, O.M. & Ottersen, O.P. 2003. Localization and function of the Na+/Ca2+-exchanger in normal and detubulated rat cardiomyocytes. J Mol Cell Cardiol 35, 1325–1337. Toldo, S., Mezzaroma, E., O’Brien, L., Marchetti, C., Seropian, I.M., Voelkel, N.F., Van Tassell, B.W., Dinarello, C.A. & Abbate, A. 2014. Interleukin-18 mediates Interleukin-1induced cardiac dysfunction. Am J Physiol Heart Circ Physiol 306, H1025–H1031. Tutar, E., Kaya, A., Gulec, S., Ertas, F., Erol, C., Ozdemir, O. & Oral, D. 1999. Echocardiographic evaluation of left ventricular diastolic function in chronic cor pulmonale. Am J Cardiol 83, 1414–1417, A9. Vanderheyden, M., Paulus, W.J., Voss, M., Knuefermann, P., Sivasubramanian, N., Mann, D. & Baumgarten, G. 2005. Myocardial cytokine gene expression is higher in aortic stenosis than in idiopathic dilated cardiomyopathy. Heart 91, 926–931. Wang, X., Hagberg, H., Mallard, C., Zhu, C., Hedtjarn, M., Tiger, C.F., Eriksson, K., Rosen, A. & Jacobsson, B. 2006.
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Disruption of interleukin-18, but not interleukin-1, increases vulnerability to preterm delivery and fetal mortality after intrauterine inflammation. Am J Pathol 169, 967– 976. Wei, L. 2011. Immunological aspect of cardiac remodeling: T lymphocyte subsets in inflammation-mediated cardiac fibrosis. Exp Mol Pathol 90, 74–78. Woldbaek, P.R., Tønnessen, T., Henriksen, U.L., Florholmen, G., Lunde, P.K., Lyberg, T. & Christensen, G. 2003. Increased cardiac IL-18 mRNA, pro-IL-18 and plasma IL18 after myocardial infarction in the mouse; a potential role in cardiac dysfunction. Cardiovasc Res 59, 122–131. Woldbaek, P.R., Sande, J.B., Strømme, T.A., Lunde, P.K., Djurovic, S., Lyberg, T., Christensen, G. & Tønnessen, T. 2005. Daily administration of interleukin-18 causes myocardial dysfunction in healthy mice. Am J Physiol Heart Circ Physiol 289, H708–H714. Xing, S.S., Bi, X.P., Tan, H.W., Zhang, Y., Xing, Q.C. & Zhang, W. 2010. Overexpression of interleukin-18 aggravates cardiac fibrosis and diastolic dysfunction in fructosefed rats. Mol Med 16, 465–470. Yu, Q., Vazquez, R., Khojeini, E.V., Patel, C., Venkataramani, R. & Larson, D.F. 2009. IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice. Am J Physiol Heart Circ Physiol 297, H76–H85. Zuurbier, C.J., Emons, V.M. & Ince, C. 2002. Hemodynamics of anesthetized ventilated mouse models: aspects of anesthetics, fluid support, and strain. Am J Physiol Heart Circ Physiol 282, H2099–H2105.
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IL-18 neutralization during alveolar hypoxia improves left ventricular diastolic function in mice V. Hillestad,1,2,3 E. K. S. Espe,1,2,3 F. Cero,1,2,3,4 K. O. Larsen,1,2,3,4 I. Sjaastad,1,2,3 S. Nyg ard,1,2,3,5 O. H. Skjønsberg4 and G. Christensen1,2,3 1 2 3 4 5
Institute for Experimental Medical Research, Oslo University Hospital Ullev al and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center, University of Oslo, Oslo, Norway Center for Heart Failure Research, University of Oslo, Oslo, Norway Departement of Pulmonary Medicine, Oslo University Hospital Ullev al and University of Oslo, Oslo, Norway Bioinformatics Core Facility, Institute for Medical Informatics, Oslo University Hospital and University of Oslo, Oslo, Norway
Received 9 June 2014, revision requested 30 June 2014, revision received 25 August 2014, accepted 27 August 2014 Correspondence: V. Hillestad, Institute for Experimental Medical Research, Oslo University Hospital Ullev al, Kirkeveien 166, 0407 Oslo, Norway. E-mail: vigdis.hillestad@medisin. uio.no
See Editorial Commentary: Toldo, S. et al. 2014. Diastolic dysfunction in chronic hypoxia: IL-18 provides the elusive link. Acta Physiol 213, 298–300.
Abstract Aim: In patients, an association exists between pulmonary diseases and diastolic dysfunction of the left ventricle (LV). We have previously shown that alveolar hypoxia in mice induces LV diastolic dysfunction and that mice exposed to hypoxia have increased levels of circulating interleukin-18 (IL-18), suggesting involvement of IL-18 in development of diastolic dysfunction. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18. In this study, we hypothesized that neutralization of IL-18 during alveolar hypoxia would improve LV diastolic function. Methods: Mice were exposed to 10% oxygen for 2 weeks while treated with IL-18BP or vehicle. Cardiac function and morphology were measured using echocardiography, intraventricular pressure measurements and magnetic resonance imaging (MRI). For characterization of molecular changes in the heart, both real-time PCR and Western blotting were performed. ELISA technique was used to measure levels of circulating cytokines. Results: As expected, exposure to hypoxia-induced LV diastolic dysfunction, as shown by prolonged time constant of isovolumic relaxation (s). Improved relaxation with IL-18BP treatment was demonstrated by a significant reduction towards control s values. Decreased levels of phosphorylated phospholamban (P-PLB) in hypoxia, but normalization by IL-18BP treatment suggest a role for IL-18 in regulation of calcium-handling proteins in hypoxia-induced diastolic dysfunction. In addition, MRI showed less increase in right ventricular (RV) wall thickness in IL-18BP-treated animals exposed to hypoxia, indicating an effect on RV hypertrophy. Conclusion: Neutralization of IL-18 during alveolar hypoxia improves LV diastolic function and partly prevents RV hypertrophy. Keywords diastolic dysfunction, hypoxia, interleukin-18, pulmonary hypertension, right ventricular hypertrophy.
More than 50% of heart failure patients have diastolic heart failure (Lam et al. 2011). Hence, identification of factors causing left ventricular diastolic dysfunction, which might eventually result in diastolic heart 492
failure, is of great importance for development of new therapeutic strategies. Diastolic dysfunction can be the result of several underlying causes, and notably, a relationship exists between diastolic dysfunction and
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diseases affecting the lungs (Baum et al. 1971). It has been documented that exposure to hypoxia leads to right ventricular (RV) hypertrophy (Moore-Gillon & Cameron 1985). Diastolic dysfunction of the right ventricle after chronic hypoxia exposure has also been shown (Larsen et al. 2006). Moreover, in a previous study, we showed that mice exposed to chronic alveolar hypoxia also develop diastolic dysfunction of the LV (Larsen et al. 2006). In previous work, we have demonstrated a role for the pro-inflammatory cytokine interleukin (IL)-18 in the development of diastolic dysfunction after chronic exposure to hypoxia (Larsen et al. 2008), suggesting inflammation as a link between alveolar hypoxia and diastolic dysfunction. In an experimental model, we showed that mice exposed to hypoxia developed diastolic dysfunction (Larsen et al. 2006) and that these animals had markedly increased circulating levels of IL-18 (Larsen et al. 2008). IL-18 is one of the effectors of the innate immune system (Fantuzzi & Dinarello 1999). Other studies support the notion that IL18 might be involved in diastolic dysfunction (Woldbaek et al. 2005, Yu et al. 2009). A naturally occurring soluble inhibitor of IL-18, the IL-18-binding protein (IL-18BP), has been identified, and its ability to inhibit IL-18 has been documented (Kim et al. 2000). In this study, we hypothesized that inhibition of IL-18 in vivo using IL-18BP may attenuate the development of left ventricular diastolic dysfunction induced by exposure to hypoxia.
Materials and methods Animals Male C57Bl/6 mice were obtained from Taconic (Skensved, Denmark) at 6 weeks of age and were acclimated for 7 days before start of any experiments. Animals were housed on standard bedding under day/ night cycles of 12/12 h at 21 °C. Food and water were given ad libidum. All experiments were approved by the Norwegian National Animal Welfare Committee (project ID 1385 and 4432) and conform to the Guide for the Use and the Care of Laboratory Animals published by the US National Institutes of Health (NIH publication No.85-23, revised 1996).
Materials Recombinant human IL-18BP isoform a (rhIL-18BPa) was purchased from R&D systems (Minneapolis, MN, USA) and reconstituted in PBS containing 0.1% serum albumin according to the manufacturer’s recommendations. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 was obtained from LIST
· IL-18 neutralization improves diastolic function
Biological Laboratories (Campbell, CA, USA). Reconstitution in PBS 0.1% serum albumin was achieved by heating at 37 °C with intermittent vortexing according to manufacturer’s recommendation.
Experimental procedures For all invasive procedures anaesthesia was induced and maintained by inhalation of isofluran (Zuurbier et al. 2002). Blood samples for ELISA analysis were drawn from the inferior vena cava under general anaesthesia and collected on ice into lithium–heparin coated tubes (Puls-Norge, Oslo, Norway) before centrifugation at 4000 g at 4 °C for 20 min. Plasma was stored at 70 °C. Heart and lungs were excised after dislocation of the neck in anaesthetized mice. The atria were removed and the right ventricle, LV and septum were separated, weighed and immediately snap frozen in liquid nitrogen.
Determination of administration dose and administration frequency of IL-18BP To study pharmacokinetics of the IL-18BP and to establish an administration protocol, two separate sets of experiments were carried out. In the first set of experiments, mice (n = 24) received IL-18BP (12.5 lg per mouse) or vehicle solution only. Plasma levels were determined by ELISA after 6, 10 or 24 h. In the second set of experiments, mice (n = 16) received an initial injection of IL-18BP 1 mg kg1 i.p. followed by i.p. injections of IL-18BP 0.5 mg kg1 every 48 h. Control animals received PBS only. Plasma concentrations of IL-18BP were determined at 2, 4 or 8 days after the initial injections.
Biological effect of IL-18BP In a separate set of experiments, the ability of IL18BP to inhibit IL-18 in vivo was investigated using LPS as previously described (Faggioni et al. 2001). Briefly, mice (n = 12) were injected with LPS (15 mg kg1 i.p.). Twenty-four hours prior to LPS administration, the animals received either IL-18BP (0.5 mg kg1) or vehicle solution only. Control animals received two injections of vehicle solution only. Plasma was collected 8 h after LPS injection and levels of IFN-c and IL-18 were determined by ELISA.
Chronic hypoxia model In the chronic hypoxia model, mice were either exposed to hypoxia (10% O2 in normobaric conditions) or to ambient air (21% O2) (Larsen et al.
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2006). Duration of hypoxia exposure was 2 weeks. We have previously described the development of elevated pulmonary artery pressure (PAP), RV hypertrophy and left ventricular diastolic dysfunction in this model (Larsen et al. 2006). Half of the animals, both in the hypoxia and the normoxia group, received IL18BP according to the protocol described in the preliminary experiments. The other half received equal volumes of vehicle solution only. After 2 weeks of hypoxia exposure, echocardiography, magnetic resonance imaging (MRI) or cardiac catheterization was performed. For all in vivo measurements of cardiac function, experiments were initiated with n = 8 in each group. The size of the groups was determined to achieve sufficient power and to minimize the number of animals used for the experiments. Some animals died, mainly due to bleeding during the procedure. All group sizes are given in figure legends of the figure corresponding to each experiment. The animals in the various groups were randomly killed, and we did not select one group first. Blood and organs were collected as described in Experimental procedures.
Doppler echocardiography Echocardiography was performed with VEVO 2100 (VisualSonics, Toronto, ON, Canada), which is designed to investigate rodents. Animals exposed to 2 weeks of hypoxia, with or without IL-18BP treatment, as well as their respective controls, were examined as previously described (Larsen et al. 2006). Briefly, animals were anaesthetized and placed in a supine position. An indirect measurement of the PAP was provided by pulmonary artery blood flow and pulmonary artery acceleration time (PAAT). To assess left ventricular diastolic function, mitral flow and tissue velocities were recorded using pulsed-wave Doppler mode. It is recognized that high heart rates in mice might result in fusion of the E and A waves (Collins et al. 2003, Stypmann et al. 2009). Decreased heart rates can be obtained by increasing the amount of anaesthesia. However, we wished to keep the animals as lightly sedated as possible to avoid cardiodepressive effects, as has been recommended by others (Lairez et al. 2013). Hence, for the analysis of maximal mitral inflow and mitral deceleration velocity, 10 of 32 animals had to be excluded due to fused E and A waves.
Magnetic resonance imaging Magnetic resonance imaging experiments were performed on a 9.4T pre-clinical MR system (Agilent Technologies, Palo Alto, CA, USA) with high-performance gradient and RF coils dedicated to mouse 494
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imaging (Espe et al. 2013). Anaesthesia was induced in an anaesthesia chamber with a mixture of O2 and 4% isoflurane and further maintained by administration of a mixture of O2 and 1.5–2.0% isoflurane in freely breathing animals. Heart rate, body temperature and respiration rate were constantly monitored during experiments, and the level of anaesthesia was regulated to keep the heart and respiration rate as stable as practically possible. Animal temperature was maintained by heated air. Magnetic resonance imaging cine data sets were acquired using a motion compensated gradient echo sequence. Left ventricular short-axis slices (7–10) were acquired, covering the heart from base to apex. Central imaging parameters: echo/repetition time = 2.50/3.67 ms; field of view = 25.6 9 25.6 mm; acquisition matrix 128 9 128; slice thickness = 1.00 mm; flip angle 15°; and averages = 3. Acquisition was prospectively gated from ECG and respiration. The total experiment duration was 75 min per animal. MRI data were analysed using MATLAB (The MathWorks, Natick, MA, USA) with the operator blinded to animal groups. While hypoxiainduced LV diastolic dysfunction was primarily described by intraventricular pressure measurements and echocardiography, MRI provided valuable information about RV morphology.
Cardiac catheterization Animals exposed to 2 weeks of hypoxia, with or without IL-18BP treatment, as well as their respective controls, were anaesthetized and placed in a supine position. The investigator was blinded to the experimental groups. A catheter with an optical transducer tip (Samba Prelim 420; Samba Sensors, V€ astra Fr€ olunda, Sweden) was introduced into the right or LV via the surgically exposed right jugular vein or right carotid artery, respectively, and data from 10 consecutive beats were recorded and analysed using DIADEM software (National Instruments, Austin, TX, USA). Right ventricular systolic pressure (RVSP), right ventricular end-diastolic pressure (RVEDP), left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP) and positive/minimum derivative of the pressure curve were registered. The time constant of isovolumetric relaxation (s) was calculated using a custom made script fitting the pressure curve during the isovolumetric relaxation phase to a monoexponential function described by the following formula: P = P0expt/s, where P is left ventricular diastolic pressure, t is time and P0 is pressure at the time point for dP/dtmin. The isovolumetric relaxation phase started at the time point for dP/dtmin and ended when pressure was declined by 1/e.
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Real-time PCR
Enzyme-linked immunosorbent assay
RNA from right and LV was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Extracted RNA was quantified by spectrophotometry (ND-1000 Saveen Werner; Nanodrop, Malm€ o, Sweden), and RNA integrity was evaluated by agarose gel electrophoresis (Bioanalyser 2100; Agilent Technologies). All samples had a RNA integrity number (RIN) higher than 7.5. Quantitative real-time PCR was performed using TaqMan technology (7900 HT real-time PCR; Applied Biosystems, Foster City, CA, USA). All samples were reverse transcribed using iScript Reverse Transcription kit (Bio-Rad laboratories, Hercules, CA, USA). Gene-specific TaqMan probes (Applied Biosystems) for collagen I (Mm00483888_m1) and collagen III (Mm00802331_m1), brain natriuretic peptide (BNP, Mm00435304_g1), Acta1 (a skeletal actin, Mm00808218_g1) and b myosin heavy chain (b-MCH, Mm00600555_m1). All samples were run in duplicates and quantification was done by comparing the average cycle of threshold (Ct) values to a standard curve.
Commercially available ELISA kits (R&D systems) were used following manufacturer’s instructions to determine the plasma concentrations of human IL18BP, mouse IL-18 and mouse IFN-c (catalogue number 7625 and DY485). ELISA kit used for the detection of mouse IL-18BP was obtained from USCN Life Sciences Inc (Wuhan, China).
Western blot analysis Homogenates were made from left ventricular tissue of mice exposed to 2 weeks of hypoxia and were analysed by SDS page and Western blotting as previously described (Larsen et al. 2006). The following antibodies were used for detection: phospholamban total anti-mouse (#MA3-922), sarcoplasmatic reticulum calcium ATPase anti-mouse (#MA3-910) (AH diagnostic, Oslo, Norway), phospholamban ser16 anti-rabbit (#A010-12) (Badrilla, Leeds, UK) and NCX anti-rabbit [a kind gift from Thomas MJ (Thomas et al. 2003)]. For analysis of the amount of collagen III, left ventricular tissue was homogenized in extraction buffer containing 10 mmol L1 cacodylic acid pH 5.0, 0.15 mol L1 NaCl, 1 lmol L1 ZnCl2, 20 mmol L1, CaCl2, 1.5 mmol L1, NaN3, 0.01% (v/v) Triton X-100 and protease inhibitor. Samples were incubated for 12 h at 4 °C. After centrifugation at 1200 g, supernatants were collected and separated into aliquots. Collagen III anti-rabbit (#Ab 82354) (Abcam, Cambridge, MA, USA) was used for detection. Immunoreactive bands were detected using an enhanced chemiluminescence kit (Amersham, Oakville, ON, Canada) according to manufacturer’s protocol. Blots were detected using a high-resolution video camera system LAS-3000 mini CCD (Fuji Photo Film Europe, D€ usseldorf, Germany) and the staining on the blots was quantified with the IMAGE GAUGE software (Version 2.54; Fuji Photo Film, Japan).
Statistics All statistical analyses were performed using SIGMASversion 12.0 (Systat Software Inc., San Jose, CA, USA). To evaluate the effect of IL-18 neutralization during chronic hypoxia exposure, two-way ANOVA was performed, correcting for multiple comparison using Student–Newman–Keuls’ all pairwise procedures. The two-way ANOVA implementation in SIGMASTAT automatically handles unequal group sizes using a general linear model approach which constructs hypothesis tests using the marginal sums of squares. Student’s t-test was used when comparing two groups only. A P value lower than 0.05 was considered statistically significant. TAT
Results Determination of administration dose and administration frequency of IL-18BP To determine the administration dose and frequency, two sets of experiments were carried out. The first set of experiments showed elevated plasma levels of IL18BP 6 h after administration of 0.5 mg kg1 (Fig. 1a). Very low levels of IL-18BP were measured in plasma of control animals and in animals treated with PBS (Fig. 1a). Previous studies have shown significant biological effects of 0.5 mg kg1 rhIL-18 Pa in vivo (Plater-Zyberk et al. 2001, Wang et al. 2006), and we therefore decided to use this dose in the following experiments. ELISA analysis 6, 10 and 24 h after injection of IL-18BP (0.5 mg kg1) revealed a time-dependent decrease in plasma levels, but the levels were still high 24 h after administration. These findings were used to calculate plasma half-life of IL18BP in vivo in mice, and we found a t1/2 value of 72 h. This relatively high value of t1/2 is seen with peptides linked with antibody Fc domain, as is the case for IL-18BP (Lee et al. 2008). Based on these findings we designed an administration protocol consisting of a bolus dose of 1 mg kg1 followed by injections of 0.5 mg kg1 every 48 h. ELISA performed 2, 4 and 8 days after the primary injection
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Biological effect of IL-18BP Figure 2 shows circulating levels of IFN-c and IL-18 measured 8 h after LPS injection in mice that had received i.p. injections with either 0.5 mg kg1 of IL18BP or vehicle. Decreased levels of IFN-c in the IL18BP group compared with controls receiving vehicle showed that IL-18BP is a potent inhibitor of IL-18 produced in vivo (259 102 vs. 1081 284 pg mL1, P < 0.05, Fig. 2a). The IL-18 concentration in plasma was markedly upregulated by LPS as expected (6396 530 vs. 291 105 pg mL1, P < 0.001, Fig. 2b). A reduction was observed with IL-18BP pretreatment (4067 624 vs. 6396 530 pg mL1, P < 0.01).
IL-18 neutralization attenuates the development of left ventricular diastolic dysfunction We have previously shown left ventricular diastolic dysfunction in animals exposed to hypoxia (Larsen et al.
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2006). Intraventricular pressure measurements confirmed diastolic dysfunction in the present experiments, as shown by a prolongation of the time constant of isovolumetric relaxation s (6.0 0.4 ms vs. 4.8 0.3 ms, P < 0.05, Fig. 3a) and longer isovolumetric relaxation time (IVRT, 7.9 0.4 vs. 5.6 0.3 ms, Fig. 3b) in animals exposed to 2 weeks of hypoxia compared with normoxic controls. Interestingly, in mice treated with IL-18BP during hypoxia exposure, s and IVRT values were significantly shortened compared to the hypoxia vehicle group (4.6 0.3 vs. 6.0 0.4 ms, Fig. 3a, and 5.5 0.2 vs. 7.9 0.4, Fig. 3b, both P < 0.05). A trend towards increased LVEDP in the hypoxia PBS group was also found (6.4 0.8 vs. 4.3 0.7 mmHg, P = 0.10, Fig. 3c). Following treatment with IL-18BP, LVEDP values were similar to control values (4.2 0.6 vs. 4.3 0.7 mmHg, Fig. 3c). No significant differences were found in dP/dtmax relative to peak systolic pressure or heart rate between the four groups (Fig. 3d,e). Representative haemodynamic traces and echocardiographic images are shown in Figure 4. Diastolic parameters analysed by echocardiography are shown in Table 1, and body and heart weights are shown in Table 2.
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Figure 1 Plasma concentrations of IL-18BP. (a) Plasma concentration of recombinant human IL-18BP measured 6, 10 and 24 h (h) after i.p. injections (n = 4 in each group). (b) Plasma concentration of IL-18BP after i.p. injections of IL-18BP (filled symbols) or PBS only (open symbols) every 2. Day (d) (n = 4 in each group).
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Figure 2 Plasma concentration of IFN-c and IL-18. Plasma concentrations of interferon (IFN)-c (a) and endogenous produced mouse interleukin (IL)-18 (b) in mice receiving lipopolysaccharide (LPS). Twenty-four hour prior to LPS injections, the animals received injections with either PBS only (LPS + vehicle, n = 6) or recombinant human IL-18BP (LPS + IL-18BP, n = 6). For mice receiving vehicle only n = 4. *P < 0.05 vs. vehicle only, #P < 0.05 vs. LPS + vehicle.
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Figure 3 In vivo diastolic function. Mice exposed to hypoxia had increased s (a) and isovolumetric relaxation time (IVRT) (b) compared with control animals, indicating diastolic dysfunction. In the hypoxia IL-18BP group, s (a) and IVRT (b) were normalized to control values. End-diastolic pressure, maximum negative decay of pressure curve relative to peak systolic pressure and heart rate are shown in panel c, d and e respectively. For all panels, n = 7 in control vehicle group, n = 2 in control IL18BP group, n = 5 in the hypoxia vehicle group and n = 8 in the hypoxia IL-18BP group. *P < 0.05 vs. control vehicle, # P < 0.05 vs. hypoxia vehicle
Levels of Ca2+-handling proteins To investigate whether improved diastolic function after IL-18 neutralization could be explained by enhanced relaxation due to an effect on phosphory lation levels of phospholamban in the LV, Western blotting was performed, investigating levels of phosphorylated phospholamban (P-PLB) at serine16 residue relative to total phospholamban levels. Our group has previously identified this as a possible mechanism for diastolic dysfunction during hypoxia (Larsen et al. 2006). We found that levels of serine16 P-PLB were decreased to 55% in the hypoxia group compared with the normoxia group (55 9 vs. 100 8%, P = 0.01, Fig. 5a). A complete normalization was found in the hypoxia group treated with IL-18BP, as the level of P-PLB in the hypoxia IL-18BP group was not significantly different from the control vehicle group (94 9 vs. 100 8%, Fig. 5a). Hypoxia exposure did not induce changes in the levels of SERCA2 protein (110 5 vs. 100 4%, Fig. 5c) or in NCX protein (117 2 vs. 100 6%, Fig. 5e). In animals exposed to hypoxia, there were no significant changes in the level of SERCA2 or NCX between animals receiving IL-18BP and animals receiving vehicle only (111 7 vs. 110 5%, and 119 10 vs. 117 2% respectively, Fig. 5c,e).
Effect of IL-18 neutralization on expression of genes encoding extracellular matrix proteins in the left ventricle Expression of collagen I mRNA was not significantly altered, neither by hypoxia nor by treatment with IL18BP (100 11% in control vehicle, 118 12% in hypoxia vehicle and 108 5% in hypoxia IL-18BP, Fig. 6a). However, the expression of collagen III
mRNA was slightly, but significantly upregulated in the hypoxia vehicle group compared with the control vehicle group (132 4% in hypoxia vehicle vs. 100 9% in control vehicle, P < 0.05, Fig. 6b). Within the time frame examined, we did not find any significant increase in the amount of collagen III protein, neither following hypoxia exposure, nor after IL-18BP treatment (100 14% in control vehicle, 94 16% in hypoxia vehicle, and 113 11% in hypoxia IL-18BP, Fig. 6c).
IL-18 neutralization partly prevents development of right ventricular hypertrophy Two weeks of hypoxia exposure increased RV weight (RVW) normalized to tibia length (RVW/TL) by 35% compared with control animals (1.5 0.1 vs. 1.1 0.1 mg mm1, P < 0.001, Fig. 7b), indicating RV hypertrophy. This was supported by an increased thickness of right ventricle free wall measured by MRI (0.59 0.06 vs. 0.45 0.02 mm, P < 0.001, Fig. 7a). The reduction in RVW/TL did not reach statistical significance in the IL-18BP-treated mice exposed to hypoxia compared with mice exposed to hypoxia receiving vehicle solution (1.4 0.1 vs. 1.5 0.1 mg mm1). However, the IL-18BP treated group had a significantly reduced wall thickness on MRI compared with the hypoxia PBS group (0.51 0.02 vs. 0.59 0.06 mm, Fig. 7a).
IL-18 neutralization and hypoxia-induced pulmonary hypertension Several parameters were measured to investigate the effect of IL-18 neutralization on pulmonary hypertension. In mice exposed to 2 weeks of hypoxia, RVSP was increased by 46% compared with control animals
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12376
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(a)
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Figure 4 Hemodynamic traces and echocardiographic images. Hemodynamic traces, images from flow Doppler and tissue Doppler are shown for one representative animal in each group. Red line indicates the phase of the curve used for s analysis. LVSP, left ventricular peak systolic pressure; dP/dtmin, time point for maximum decay of pressure curve. White arrows indicate maximal tissue velocity.
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(45.0 2.0 vs. 29.3 0.7 mmHg, P < 0.001, Fig. 7c) and PAAT was decreased to 70% (15.2 0.7 vs. 21.9 0.9 ms, P < 0.001, Fig. 7d). These results show that hypoxia exposure induces pulmonary hypertension. The trend towards normalization of RVSP and PAAT in hypoxic animals receiving IL18BP compared with hypoxic animals receiving vehicle only did not reach statistical significance (41.4 1.3 mmHg in hypoxia IL-18BP vs. 45.0 2.0 mmHg in hypoxia vehicle and 16.8 0.6 ms in hypoxia IL-18BP vs. 15.2 0.7 ms in hypoxia vehicle, P = 0.07 and P = 0.16 respectively, Fig. 7c,d). 498
Molecular changes in the right ventricle We also investigated expression of genes known to be regulated in myocardial hypertrophy. Results are shown in Table 3. Although several genes were found to be regulated in animals exposed to hypoxia, only a-skeletal actin was differentially regulated in animals treated with IL-18BP.
Endogenous mouse plasma IL-18BP To investigate whether the injected human IL-18BP interfered with the in vivo production of IL-18BP, we
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Table 1 Cardiac function and morphology Control vehicle n=8 Cardiac dimensions measured by MRI RV-free wall ED (mm) IVS ES (mm) LV-free wall ED (mm) RV-free wall ES (mm) IVS ES (mm) LV-free wall ES (mm) Ejection fraction (%) Echocardiography: (tissue) Doppler Heart rate Maximum mitral velocity (mm s1) Diastolic tissue velocity (m s1) E/E0 Mitral flow deceleration (m s2) Cardiac output (mL min1)
Control IL-18BP n=8
Hypoxia vehicle n=8
Hypoxia IL-18BP n=8
0.46 1.10 1.06 0.59 1.33 1.50 73.56
0.02 0.06 0.05 0.02 0.10 0.08 2.49
0.40 1.13 0.95 0.53 1.39 1.52 67.66
0.02 0.04 0.04 0.03 0.05 0.08 2.33
0.59 1.23 1.05 0.70 1.40 1.38 65.77
0.02 *,*** 0.05 0.02 0.03*,*** 0.07 0.06 1.49
0.51 1.13 1.05 0.66 1.26 1.64 64.17
0.02 *,***,** 0.03 0.03 0.04*,*** 0.02 0.07 2.57
454.5 586.8 16.1 39.6 24.7 27.2
13.3 29.6 1.4 4.4 2.8 2.8
453.5 632.0 14.9 43.2 23.6 25.7
8.0 22.9 0.7 2.4 2.4 1.8
475.8 559.1 13.5 42.6 26.0 18.1
20.0 42.3 0.9 3.7 2.4 1.4
521.8 531.5 16.1 34.6 22.5 25.2
20.5 11.0 1.2 2.8 1.4 2.2
RV, right ventricle; LV, left ventricle; IVS, interventricular septum; ES, end systole; ED, end diastole; MRI, magnetic resonance imaging. *P < 0.05 vs. control vehicle, **P < 0.05 vs. hypoxia vehicle, ***P < 0.05 vs. control IL-18BP.
Table 2 Organ weights Control vehicle n = 16 Body weight (g) RVW (mg) RVW/TL (mg mm1) LVW/TL (mg mm1) LW/TL (mg mm1)
27.05 19.57 1.15 4.97 8.09
0.52 0.69 0.04 0.12 0.25
Control IL-18BP n = 16 27.03 20.26 1.19 5.21 8.65
0.56 0.76 0.04 0.14 0.24
Hypoxia vehicle n = 16 25.08 27.36 1.62 4.76 11.10
0.51*,** 1.02*,** 0.06*,** 0.13 0.25*,**
Hypoxia IL-18BP n = 16 25.22 25.44 1.50 4.76 10.94
0.27*,** 1.1*,** 0.06*,** 0.12 0.33*,**
RVW, right ventricular weight; TL, tibia length; LVW, left ventricular weight; LW, lung weight. *P < 0.05 vs. control vehicle, **P < 0.05 vs. control IL-18BP.
measured endogenous mouse IL-18BP in animals exposed to hypoxia for 2 weeks. No significant changes were induced, neither by hypoxia, nor by IL18BP injections (488 57 pg mL1 in control vehicle, 404 57 in pg mL1 control IL-18BP, 488 47 pg mL1 in hypoxia vehicle and 506 66 pg mL1 in hypoxia IL-18BP, Fig. 8). These results show that the concentration of the injected human IL-18BP was about 20-fold higher than the levels of endogenous mouse IL-18BP during hypoxia.
Discussion The present study shows that in vivo injection of IL18BP is an efficient method for IL-18 neutralization. Moreover, inhibition of IL-18 during hypoxia exposure attenuates development of left ventricular
diastolic dysfunction. We demonstrate that the dephosphorylation of phospholamban previously observed after hypoxia exposure (Larsen et al. 2006) can be prevented by IL-18 neutralization, which may explain the observed improvement in diastolic function. Moreover, IL-18 neutralization inhibits the development of RV hypertrophy. Diastolic dysfunction secondary to lung disease has received relatively little attention, and currently, no available treatment exists to prevent the development of diastolic dysfunction secondary to pulmonary disease. It has been shown that exposure to hypoxia can lead to diastolic dysfunction of both ventricles in healthy human beings (Huez et al. 2005). Furthermore, although hypoxia leads to RV hypertrophy, systolic function is generally preserved (Huez et al. 2005, Kolb & Hassoun 2012). Development of diastolic dysfunction of both right and LV in the experimental
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Figure 5 Calcium-handling proteins. Two weeks of hypoxia exposure induced a decrease in serine16 phosphorylated phospholamban (PLN) which was normalized to control levels in the hypoxia IL-18BP group (panel a and d, n = 4 in each group). Protein levels of sarcoplasmatic reticulum calcium ATPase 2 (SERCA2, panel b and e, n = 4 in each group) and sodium–calcium exchanger (NCX, panel c and f, n = 4 in each group) and were not significantly changed, neither by hypoxia nor by IL-18BP treatment. Results are shown as per cent of control vehicle. Vinculin was used as loading control. *P < 0.05 vs. control vehicle.
mouse model of hypoxia has previously been described by us and these animals also have increased circulating levels of IL-18 (Larsen et al. 2008). We here show that specific neutralization of IL-18 during hypoxia exposure can partly prevent development of diastolic dysfunction, as shown by normalization of the time constant of isovolumic relaxation (s). s is considered to be one of the most reliable parameters 500
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Figure 6 Collagen content in the left ventricle (LV). Hypoxia exposure did not induce any significant changes in left ventricular gene expression of collagen I (a, n = 8 in each group) or collagen III (b, n = 8 in each group), neither did IL-18BP treatment. The amount of collagen III protein in the LV was not altered (panel c, n = 4 in each group), Vinculin was used as loading control (panel d). *P < 0.05 vs. control vehicle.
of isovolumic relaxation as it is considered independent of load and heart rate. This is not the case for the maximal rate of pressure decay (dP/dtmax) which is considered dependent on the prevailing load of the intact circulation. To our knowledge, this is the first study showing that inhibition of a specific cytokine can improve diastolic function due to alveolar hypoxia, and our findings are in agreement with other studies which have shown that administration of IL18 can cause and aggravate diastolic dysfunction (Woldbaek et al. 2005, Yu et al. 2009, Xing et al. 2010). Improved cardiac function following IL-18 neutralization has been shown in a model of endotoxaemia by a reduction in LVEDP, which does not reflect diastolic function per se (Raeburn et al. 2002). We sought to determine the mechanisms behind the positive effect of IL-18 neutralization on diastolic function. Several theories have been raised to explain the mechanisms causing diastolic dysfunction in chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (Schena et al. 1996, Tutar et al. 1999, Ozer et al. 2001). In a previous study
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Figure 7 Right ventricular (RV) hypertrophy and pulmonary hypertension. Hypoxia exposure induced RV hypertrophy and pulmonary hypertension, as shown by increased RV wall thickness (panel a, n = 8 in each group), increased RV weight (panel b, n = 16 in each group), increased right ventricular systolic pressure (RVSP, panel c, n = 8 in each group) and shortened pulmonary acceleration time (PAAT, panel d, n = 8 in each group). *P < 0.05 vs. control vehicle, $ P < 0.05 vs. control IL-18BP, #P < 0.05 vs. hypoxia vehicle.
carried out by our group, decreased level of P-PLB was suggested to be responsible for the observed impairment in relaxation (Larsen et al. 2006). Phospholamban is tightly coupled to SERCA at the sarcoplasmatic reticulum membrane and plays a crucial role in regulating calcium reuptake during diastole (MacLennan & Kranias 2003). When phospholamban is phosphorylated at the serine16 residue, conformational changes in the protein lead to a relief of the
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Hypoxia
Figure 8 Endogenous mouse plasma IL-18BP. Circulating levels of endogenous mouse IL-18BP in mice exposed to 2 weeks of normoxia (control) or hypoxia and receiving injections with either human IL-18BP or vehicle, n = 4 in each group.
inhibitory effect of phospholamban on SERCA activity. A relationship between circulating IL-18 and phosphorylation levels of phospholamban has already been suggested (Woldbaek et al. 2003, Larsen et al. 2008). Here, we show that phosphorylation levels of phospholamban were completely normalized when circulating IL-18 was inhibited by IL-18BP treatment. Hence, our finding indicates that IL-18 is directly involved in regulation of phosphorylation levels of phospholamban and that this response can be inhibited by neutralizing the cytokine. To our knowledge, this physiological effect of specific anti-inflammatory treatment has not previously been reported. As our group has already demonstrated that the hypoxiainduced decline in P-PLB is related to an IL-18 induced increase in protein phosphatase 2A activity, IL-18BP treatment may reverse that increase. Phospholamban phosphorylation may also be regulated by beta-adrenergic stimulation, but we have previously
Table 3 Markers of hypertrophy
BNP/rpl32 Acta1/rpl32 b-MHC/rpl32
Control vehicle n = 16
Control IL-18BP n = 16
Hypoxia vehicle n = 16
Hypoxia IL-18BP n = 16
1.00 0.16 1.00 0.19 1.00 0.13
1.93 0.38 1.69 0.65 1.17 0.17
5.25 1.32*,** 3.32 0.57*,** 1.33 0.16
7.09 1.4*,** 6.19 1.16*,**,*** 1.06 0.18
BNP, brain natriuretic peptide; RPL32, ribosomal protein L32; Acta1, alpha sceletal actin; b-MCH, b-myosin heavy chain. All data are normalized to the expression of ribosomal protein L32 (RPL32) and are shown as percent of control vehicle. *P < 0.05 vs. control vehicle, **P < 0.05 vs. control IL-18BP, ***P < 0.05 vs. hypoxia vehicle.
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shown that alveolar hypoxia exposure does not alter levels of beta-adrenoceptors and their activity in the LV (Larsen et al. 2008). In a previous study we examined the effect of IL-18 on isolated cardiomyocytes and found that IL-18 induced decreased levels of P-PLB (Larsen et al. 2008), supporting the notion that IL-18 inhibition might have beneficial effects on cardiac cells. A study published during progress of our study shows that IL18 can cause cardiac dysfunction by direct effects on the heart and that blocking of IL-18 with IL-18BP prevents the negative effects on cardiac function (Toldo et al. 2014). IL-18BP is considered to exert its effect through IL-18 inhibition, and no off-target effects of IL-18BP have been described. However, we acknowledge that general off-target effects cannot be excluded, including other direct effects of IL-18BP and non-cardiac effects of IL-18 inhibition. Prolongation of the time constant of isovolumetric relaxation reflects slowed relaxation of the cardiomyocytes (Paulus et al. 2007). However, increased stiffness of the ventricle due to extracellular matrix remodelling could also lead to impaired relaxation. Increased fibrosis and remodelling is an important cause of ventricular stiffness, and evidence exists for a link between left ventricular fibrosis and inflammation (Paulus et al. 2007, Wei 2011). Hence, an additional explanation for diastolic dysfunction secondary to hypoxia could be alterations in extracellular matrix, leading to increased myocardial stiffness, affecting the filling phase of diastole. To assess ventricular stiffness measurements of pressure–volume relationships could have been performed. However, we examined extracellular matrix composition by gene expression analysis and determination of protein levels of collagen and did not find alterations indicating altered myocardial stiffness. Apart from a slight increase in collagen III mRNA expression, we could not show any increased amount of collagen at the protein level in animals exposed to hypoxia within the time frame examined. In addition, Larsen et al. (2008) could not observe any sign of fibrosis in this model on histological preparations. Although there is evidence that IL-18 can mediate cardiac fibrosis (Platis et al. 2008, Yu et al. 2009), fibrosis does not seem to play an important role in the development of diastolic dysfunction induced by alveolar hypoxia within the time frame examined in this study. We also found reduced RV hypertrophy after IL18BP treatment. Exposure to hypoxia leads to increased pressure in the pulmonary vasculature which constitutes increased afterload on the right ventricle and stimulates a hypertrophic response of the latter. Diastolic dysfunction has also been shown to be associated with pulmonary hypertension (Tutar et al. 502
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1999). Thus, an effect of IL-18BP on PAP could be a plausible explanation for the observed effects on the heart. Although we did not find a significant reduction of RV systolic pressure in the IL-18BP-treated group compared with the non-treated group, we cannot exclude this mechanism. In addition, IL-18 has been shown to directly affect myocardial hypertrophy (Chandrasekar et al. 2005, Vanderheyden et al. 2005). Findings in those previous studies support the notion that IL-18 may directly affect the hypertrophy of the right ventricle. Our gene expression data did not show a reduction in hypertrophy markers. However, such divergence between hypertrophic growth and foetal gene expression has also been shown by others. Notably, in one study, suppression of hypertrophy in transverse aortic banding led to a further increase in BNP expression (Patrizio et al. 2007). A tendency towards increased expression of hypertrophy markers was present in the control IL-18BP group compared to the control vehicle group. However, we did not observe any increase in RV wall thickness measured by MRI in the control IL-18BP group compared with the control vehicle group. Hence, we do not believe that the control IL-18BP animals have hypertrophy of the right ventricle. Inhibition of IL-18 with IL-18BP could constitute a new therapeutic approach. In our experiments using LPS, we demonstrate the potency of IL-18BP as an inhibitor of inflammation. Indeed, in animals pre-treated with IL-18BP, IFN-c levels were reduced to 20% of levels observed in animals pre-treated with vehicle solution only. Strong effects of IL-18BP on LPSinduced mortality have also been shown by others (Faggioni et al. 2001). Plasma concentrations of IL18BP used in the LPS experiments were similar to the steady-state concentrations we obtained when treating the animals exposed to hypoxia. Hence, we know that the actual plasma concentration is efficient for inhibiting IL-18 in vivo. This is of particular importance, as too large concentrations of IL-18BP may lead to a pooling effect and increased activity of the targeted cytokine, as has been shown to be the case with TNFa and etanercept (Mann et al. 2008). Such effects might explain the lack of effect in heart failure patients treated with etanercept. This should also be considered when designing treatment with IL-18BP. IL-18BP already exists for clinical use and has been tested in patients with rheumatic disease (Tak et al. 2006). IL-18 inhibition might thus offer new possibilities for treatment of diastolic heart failure in hypoxic COPD patients, but beforehand, careful study of dosage of the inhibitor is needed. In conclusion, in this study, we show that neutralization of IL-18 during alveolar hypoxia improves LV diastolic function and partly prevents RV hypertrophy.
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12376
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We thank Almira Hasic, Ulla Enger, Hilde Dishington and Lisbeth Winer for skilful technical work and Marita Martinsen and the rest of the staff at the animal facility for expert animal care. We also thank Finn Olav Levy for useful advice concerning pharmacological calculations.
Conflict of interest None to declare.
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