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(Circulation. 2005;111:996-1005.)
© 2005 American Heart Association, Inc.

Heart Failure

Interleukin-6 Causes Myocardial Failure and Skeletal Muscle Atrophy in Rats

Sofie P.M. Janssen; Ghislaine Gayan-Ramirez, PhD; An Van Den Bergh; Paul Herijgers, MD, PhD; Karen Maes; Erik Verbeken, MD, PhD; Marc Decramer, MD, PhD

From the Laboratory of Pneumology (S.P.M.J., G.G.-R., K.M., M.D.), Respiratory Muscle Research Unit; the Center for Experimental Surgery and Anesthesiology (A.V.D.B., P.H.), Cardiovascular Research Unit; and the Department of Pathology (E.V.), Katholieke Universiteit Leuven, Leuven, Belgium.

Reprint requests to Prof Marc Decramer, MD, PhD, Respiratory Division, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail Marc.Decramer{at}uz.kuleuven.ac.be

Received June 21, 2004; revision received October 7, 2004; accepted November 12, 2004.

	Abstract

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Background— The impact of interleukin (IL)-6 on skeletal muscle function remains the subject of controversy.

Methods and Results— The effects of 7-day subcutaneous administration of recombinant human IL-6 were examined at 3 doses, 50, 100, or 250 µg · kg^–1 · d^–1, in rats. Skeletal muscle mass decreased dose-dependently (with increasing dose: in the diaphragm, –10%, P=NS; –15%, P=0.0561; and –15% P<0.05; and in the gastrocnemius, –9%, P=NS; –9%, P=NS; and –18%, P<0.005) because of decreases in cross-sectional area of all fiber types without alterations in diaphragm contractile properties. Cardiovascular variables showed a dose-dependent heart dilatation (for end-diastolic volume: control, 78 µL; moderate dose, 123 µL; and high dose, 137 µL, P<0.001), reduced end-systolic pressure (control, 113 mm Hg; moderate dose, 87 mm Hg; and high dose, 90 mm Hg; P=0.037), and decreased myocardial contractility (for preload recruitable stroke work: control, 79 mm Hg; moderate dose, 67 mm Hg; and high dose, 48 mm Hg; P<0.001). Lung edema was confirmed by an increased wet-to-dry ratio (control, 4.2; moderate dose, 4.6; and high dose, 4.5; P<0.001) and microscopy findings. These cardiovascular alterations led to decreases in organ blood flow, particularly in the diaphragm (control, 0.56 mL · min^–1 · g^–1; moderate dose, 0.21 mL · min^–1 · g^–1; and high dose, 0.23 mL · min^–1 · g^–1; P=0.037). In vitro recombinant human IL-6 administration did not cause any alterations in diaphragm force or endurance capacity.

Conclusions— IL-6 clearly caused ventilatory and peripheral skeletal muscle atrophy, even after short-term administration. Blood flow redistribution, resulting from the myocardial failure induced by IL-6, was likely responsible for this muscle atrophy, because IL-6 did not exert any direct effect on the diaphragm.

Key Words: interleukins • cardiomyopathy • blood flow • contractility • muscles

	Introduction

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Serum levels of interleukin (IL)-6 are elevated in various conditions such as heart failure,¹ sepsis,² burns,³ acute respiratory distress syndrome, pneumonia,⁴ and exacerbations of chronic obstructive pulmonary disease,⁵ in all of which clinically significant skeletal muscle weakness is known to be present.^6–11 However, whether this muscle weakness is caused by IL-6 remains unclear. Indeed, on one hand, IL-6–transgenic mice exhibited skeletal muscle atrophy,¹² which was fully reversed by administration of IL-6 receptor antagonists.¹³ By contrast, IL-6 failed to augment muscle proteolysis in vitro¹⁴ and even facilitated differentiation of cultured rat myoblasts.¹⁵

On the other hand, systolic ventricular dysfunction is known to induce selective diaphragm atrophy.⁶ Hence, it appears possible that the aforementioned effects of IL-6 on skeletal muscle would be caused by an effect on the myocardium, because IL-6 or members of its family are known to induce ventricular hypertrophy and to reduce basic hemodynamic variables. Indeed, concentric hypertrophy and decreases in ventricular volume were observed in IL-6⁺/IL-6 receptor⁺–transgenic mice.¹⁶ Moreover, in vivo administration of cardiotrophin-1, a cytokine belonging to the same family as IL-6, induced increases in ventricular-to-body weight ratio, associated with reduced mean arterial pressure and systemic vascular resistance.^17,18 Furthermore, the occurrence of morphological changes in the myocardium and decreased myocardial contractility are also well supported by several in vitro experiments after administration of the aforementioned cytokines. First, cardiotrophin-1 was shown to induce a clear hypertrophic response in neonatal rat cardiac myocytes.¹⁹ Second, a direct negative inotropic effect on isolated hamster papillary muscle was exerted by IL-6, likely related to upregulation of myocardial nitric oxide synthase.²⁰ In addition, IL-6 was shown to downregulate sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2) activity in isolated rat cardiac myocytes.²¹ Finally, administration of IL-6 to rat cardiac myocyte cultures resulted in reduced expression of -myosin heavy chain (MHC), ß-myosin heavy chain, and cardiac -actin.²² The present study was designed to reconcile the aforementioned observations by determining whether IL-6 causes myocardial dysfunction, leading to skeletal muscle atrophy.

	Methods

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A detailed version of this section is available in the online-only Data Supplement.

Experimental Protocol
The ethics committee of the medical faculty of the Katholieke Universiteit Leuven, Leuven, Belgium, approved the present study. Six series of experiments were conducted on 139 male Wistar rats, 8 to 10 weeks old and weighing 350 to 450 g. In the first series, 16 rats received a 7-day continuous infusions of a low (50 µg · kg^–1 · d^–1, n=8) or moderate (100 µg · kg^–1 · d^–1, n=8) dose of recombinant human (rh) IL-6. Eight control rats received phosphate-buffered saline (PBS) infusions. In a second series, 15 rats received a high dose (250 µg · kg^–1 · d^–1, n=9) of rhIL-6 or PBS (control, n=6). In both experiments, body and tissue mass, diaphragm in vitro contractile properties, and morphometry of the diaphragm and gastrocnemius were performed.

In a third series of experiments, hemodynamic measurements were performed in 31 rats combined either with determination of body and tissue mass, lung histology and water content, and blood sampling (control, n=4; 100 µg · kg^–1 · d^–1 rhIL-6, n=8; or 250 µg · kg^–1 · d^–1 rhIL-6, n=5) or with examination of heart histology (control, n=4; 100 µg · kg^–1 · d^–1 rhIL-6, n=6; or 250 µg · kg^–1 · d^–1 rhIL-6, n=4).

In a fourth series, organ and muscle blood flows were assessed with the use of colored microspheres in 17 rats (control, n=4; 100 µg · kg^–1 · d^–1 rhIL-6, n=6; or 250 µg · kg^–1 · d^–1 rhIL-6, n=7). In a fifth series, food intake was measured in 24 rats (control, 100 µg · kg^–1 · d^–1 rhIL-6, and 250 µg · kg^–1 · d^–1 rhIL-6; n=8 per group), whereas 18 rats were used to assess activity levels (control, 100 µg · kg^–1 · d^–1 rhIL-6, and 250 µg · kg^–1 · d^–1 rhIL-6; n=6 per group). Serum rhIL-6 and endogenous rat IL-6 were measured in the moderate- and high-dose groups of the third and fifth experimental series. Finally, in a sixth series, diaphragm force was assessed in vitro by adding rhIL-6 or Krebs’ solution to the tissue bath (n=5 per group).

Pump Implantation
Rats were anesthetized intraperitoneally with sodium pentobarbital (60 mg/kg; Nembutal, Sanofi). Subsequently, Alzet miniosmotic pumps (model 2001, Iffa Credo) were implanted subcutaneously. Pumps implanted in experimental animals were filled with sterile PBS (Cambrex Bio Science Verviers), rhIL-6, and rat serum (1%, vol/vol). Control rats received an identical volume of PBS, human albumin (0.1% wt/vol; ICN Biomedicals, Inc), and rat serum.

Diaphragm In Vitro Contractile Properties
Animals were anesthetized intraperitoneally with sodium pentobarbital, tracheotomized (14-gauge Insyte catheter, BD), and ventilated at a constant rate of 60 cycles/min (Harvard Apparatus Co). Subsequently, 2 small strips of both costal diaphragm regions were dissected and placed at their optimal length. Contractile properties were further studied at 37°C.^23,24 The whole diaphragm, heart, right scalenus medius, gastrocnemius, soleus, extensor digitorum longus, and plantaris muscles were weighed after being trimmed and dried in the first experiment. Similar measurements were performed in the second and third experiments, together with the weighing of lung and liver.

Morphometry of the Diaphragm and Gastrocnemius
Gastrocnemius and costal diaphragm samples were frozen in isopentane cooled with LN₂.²³ Serial 10-µm cross sections were then stained for myofibrillar ATPase at pH 4.3 or 4.5 to identify the different fiber types.²⁵ Morphometric examination was carried out microscopically (Leitz Laborlux S) at x20 magnification with a digital video camera (model VC-2512, Sanyo B/W CCD camera) connected to a computerized image analysis system (Quantimet 500, Leica, Cambridge Ltd) to determine fiber diameter, fiber proportion, cross-sectional area (CSA), and residual interstitial area.²³

Diaphragm In Vitro IL-6 Administration
Contractile properties and endurance of untreated diaphragm strips were studied at 37°C^23,24 after 30 minutes’ incubation in Krebs’ solution containing 12 ng rhIL-6/mL or Krebs’ solution alone.

Cardiovascular Functional Parameters
Rats were anesthetized intraperitoneally with urethane (600 mg/kg) and -chloralose (160 mg/kg), tracheotomized, and ventilated with a pressure-controlled respirator (end-inspiratory pressure, 12 mm Hg; end-expiratory pressure, 2 mm Hg; Hugo Sachs Elektronik). The right carotid artery was exposed, and a microtip pressure-conductance combination catheter (SPR-847 catheter connected to an ARIA-1 setup, Millar Instruments) was advanced into the left ventricle to measure basal hemodynamic parameters by generating pressure-volume loops. Contractility parameters were obtained by decreasing left ventricular preload, performed by temporary occlusion of the inferior vena cava. Volume calibrations of the measured conductance were performed at the end of each experiment with known blood volume.

Heart Histology
A winged needle (21-gauge, BD Valu-Set) was inserted into the left ventricle. Simultaneously a small hole was made in the right atrium, and 2 mL of 0.1 mol/L cadmium chloride was injected into the left ventricle to arrest the heart in dilatation. After perfusion with 0.9% NaCl, fixation was performed with 4% formaldehyde. Excised hearts were then divided in 4 transverse slices and embedded in paraffin. Serial 7-µm slices were stained with hematoxylin and eosin (H&E). The right ventricular cavity was measured by using a microscope digital video camera connected to a computerized image analysis system. Values were expressed as a percentage of total heart surface area limited by the outer margin of the ventricular wall.

Blood Flow Measurements
Anesthesia and tracheotomy were performed in the same way as for the hemodynamic measurements. Subsequently, 2 PE50 catheters (Intramedic, Clay Adams) were inserted into the left ventricle and femoral artery to determine blood flow of the right and left ventricles, diaphragm, right gastrocnemius, right and left kidneys, spleen, jejunum, and colon by injecting 15-µm yellow microspheres into the left ventricle (400 µL; Dye-Trak, Triton Technology).²⁶ Simultaneously, heart rate and arterial and ventricular pressures were recorded. Blood flow was divided by tissue weight and expressed as milliliters per minute per gram wet weight.

Lung Histology and Water Content
Left lungs were fixed in 4% formaldehyde. Subsequently, 5-µm paraffin slices were stained with H&E and examined by a pathologist. The right lobes were used to determine water content by measuring wet-to-dry ratio after desiccation for 3 days at 80°C.

Serum Levels
Blood was sampled after cleavage of the vena cava and collected in EDTA tubes (BD Vacutainer systems). Serum rhIL-6 and rat IL-6 were measured by sandwich ELISA (IL-6 Flexia kit: sensitivity 3 pg/mL; rat IL-6 ELISA kit: sensitivity <8 pg/mL; Biosource Europe).

Food Intake
Free-fed animals were individually housed in metabolic cages at 23°C with a 14-hour light/10-hour dark cycle. Ad libitum food intake and body mass were recorded daily. Water was available ad libitum.

Activity Level
Behavioral responses were tested for 16 hours on day 7 (8 hours under light conditions and 8 hours in the dark) with an infrared camera (Agema Thermovision 570) coupled to a digitizer board. Animals were represented by bright points on a dark background from which movements consequently could be deduced.²⁷ Values were expressed as centimeters of displacement per hour.

Statistics
Hemodynamics and blood flow variables were analyzed with Statistica 6 software. All other statistical analyses were performed with SAS version 6.12 (SAS Institute Inc). Data from 2 different diaphragm strips obtained from each rat were averaged. Differences between means of rats treated with rhIL-6 and control rats were assessed with a 1-way ANOVA or Kruskal-Wallis test for the first, third, fourth, and fifth experiments (with appropriate Gabriel, Scheffé, or Fisher post hoc comparison) or a Wilcoxon or unpaired t test for the second and sixth experiments. Pearson’s correlation analysis was performed to assess relationships. A 2-sided probability value <0.05 was considered significant. Data are expressed as mean±SD.

	Results

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Body and Organ Weights
Body weight (Table 1) was similar between controls and experimental groups at the time of pump implantation. Only for the high-dose rhIL-6 group did body weight decrease significantly, by 8%, at the time of dissection (P<0.005). The low rhIL-6 dose had no effect on tissue weights. In rats treated with the moderate rhIL-6 dose, only diaphragm weight tended to decrease by 15% (P=0.0561). Heart weight increased significantly by 9% (P<0.05). For rats treated with the high rhIL-6 dose, the mass of all muscles studied decreased significantly by 15% (P<0.05).

View this table: [in this window] [in a new window]   TABLE 1. Body and Organ Weights

Diaphragm In Vitro Contractile Properties
Twitch (P_t) and tetanic (P_o) tension were similar among all groups (pooled values of 465±84 and 2407±418 g/cm², respectively), although P_t/P_o decreased significantly in the high-dose group (0.196±0.007 versus 0.209±0.013 in control rats, P<0.05). Time to peak tension was identical in all groups (pooled value of 20±2 ms), whereas half-maximal relaxation time tended to decrease in the high-dose group (22±3 versus 23±3 ms in controls, P=0.0584). In the force-frequency curve, diaphragm force was comparable in all groups (Figure 1, upper panel). Furthermore, during the low-frequency fatigue run, no differences in force decline (pooled values of 56±5%; Figure 1, lower panel) were observed among the different groups.

View larger version (22K): [in this window] [in a new window]   Figure 1. Diaphragm force-frequency (top) and endurance (bottom) data in low- (open squares), moderate- (open triangles), and high- (open circles) dose groups of animals compared with controls (solid circles). Force is expressed in g/cm2; frequency in Hz.

Morphometry
Fiber proportion and residual interstitial area remained unchanged in both the diaphragm and gastrocnemius at all doses. Also, no changes in fiber dimensions were observed in the low- and moderate-dose groups. In the high-dose group, the diameter of all fiber types of the diaphragm decreased significantly, by 14%, compared with controls (type I, 30±3 versus 35±4 µm; type IIa, 35±1 versus 40±4 µm; and type IIx/b, 49±4 versus 58±7 µm; all P<0.05). The CSA of type IIa fibers decreased significantly by 19% (799±93 versus 987±158 µm² in control rats, P<0.01), whereas that of type IIx/b and type I fibers tended to decrease by 24% (1587±302 versus 2078±518 µm² in controls, P=0.0518) and 17%, respectively (Figure 2).

View larger version (66K): [in this window] [in a new window]   Figure 2. A, CSA of different fiber types (I, solid bars; IIa, open bars; IIx/b, gray bars) in diaphragm expressed as percentage difference from respective controls in rats treated with low (left), moderate (middle), or high (right) doses of rhIL-6. **P<0.01, +P=0.0518. B, Representative diaphragm ATPase staining (pH 4.3) of control (left) and high-dose (right) rhIL-6 animal. Top panels correspond to x200 magnification, bar=35 µm; bottom panels, to x400 magnification, bar=17.5 µm. Note decreases in CSA of type IIa (white) and type IIx/b fibers (gray) in high-dose rhIL-6–treated animal. Abbreviations are as defined in text.

The diameter of gastrocnemius internal fiber types I and IIx/b tended to decrease by 9% in the high-dose group (type I, 49±5 versus 54±2 µm in controls, P=0.0731; type IIx/b, 50±6 versus 55±3 µm in control rats; P=0.0855), whereas that of internal type IIa fibers remained unchanged. For the external part, type IIx/b diameter tended to decrease by 5% (60±2 versus 64.0±4 µm in control animals, P=0.0747). The CSA of fiber types I and IIa also tended to decrease by 15% and 14%, respectively (type I, 2184±384 versus 2575±283 µm² in control rats, P=0.0677; type IIa, 2036±371 versus 2384±170 µm² in controls, P=0.0875), whereas the CSA of both internal and external IIx/b fiber types did not change (Figure 3).

View larger version (76K): [in this window] [in a new window]   Figure 3. A, CSA of different fiber types (internal part: I, solid bar; IIa, open bar; IIx/b, gray bar; external part: IIx/b, hatched bar) in gastrocnemius expressed as percentage difference from respective controls in rats treated with high doses of rhIL-6. +P<0.0875. B, Representative gastrocnemius ATPase staining (pH 4.5, internal part) of control (left) and high-dose–treated (right) rhIL-6 animal. Top panels correspond to x200 magnification, bar=35 µm; bottom panels, to x400 magnification, bar=17.5 µm. Note decreases in CSA of type I fibers (black) and type IIa fibers (white) in high-dose rhIL-6–treated animal. Abbreviations are as defined in text.

Diaphragm In Vitro IL-6 Administration
Addition of rhIL-6 to the tissue bath did not affect diaphragm P_t (366±75 versus 381±50 g/cm²) or P_o (1965±457 versus 1975±165 g/cm²), P_t/P_o (0.19±0.03 versus 0.19±0.01), time to peak tension (22.8±1.0 versus 20.8±1.5 ms), or half-maximal relaxation time (22.4±1.8 versus 27.2±5.0 ms) when compared with control strips. During determination of the force-frequency curve and the low-frequency fatigue run, generated forces were similar in both groups.

Cardiovascular Functional Parameters
End-diastolic volume (P<0.005) and stroke volume (P<0.005) under steady-state conditions increased significantly in a dose-dependent manner (Table 2), causing a remarkable shift to the right of the pressure-volume loops (Figure 4, upper panels). End-diastolic pressure was somewhat higher in the treated groups, but this difference failed to reach statistical significance. Heart rate, ejection fraction, maximal change of pressure over time (dP/dt), and , the time constant of relaxation, were not affected by treatment. End-systolic pressure (P<0.05) and arterial elastance (P<0.001) decreased significantly with both the moderate and high dose.

View this table: [in this window] [in a new window]   TABLE 2. Hemodynamic Variables

View larger version (29K): [in this window] [in a new window]   Figure 4. Pressure-volume (top) and stroke work–volume (bottom) relations of left ventricle in control (left), moderate- (middle), and high- (right) dose rhIL-6 groups in 3 representative animals. Pressure is expressed in mm Hg, volume in µL, and stroke work in mm Hg · µL. Dashed line in top panels corresponds to end-systolic pressure–volume relation of left ventricle. Slope of this line denotes end-systolic elastance. In bottom panels, slope corresponds to preload recruitable stroke work. Note dose-dependent decrease in end-systolic elastance, increase in end-diastolic volume, and decrease in preload recruitable stroke work after IL-6 administration. Abbreviations are as defined in text.

Preload recruitable stroke work and end-systolic elastance decreased significantly in a dose-dependent manner (P<0.001; Figure 4, lower panels). From these parameters, it is clear that despite the reduced contractility, cardiac output (P<0.005) increased because of larger stroke volumes combined with reduced afterload.

Correlations: Muscle Weight and Cardiovascular Functional Parameters
Positive correlations were found between diaphragm weight and end-systolic elastance, preload recruitable stroke work, and maximal elastance (0.50<r<0.63, 0.012<P<0.056). Negative correlations were present between diaphragm weight and end-systolic volume, end-diastolic volume, and stroke work (–0.62<r<–0.51, 0.013<P<0.052). All other skeletal muscles showed similar relationships.

Blood Flow Analysis
All measured organ blood flows, except for the kidney, tended to decrease after rhIL-6 treatment. This was also observed for the gastrocnemius muscle (control, 0.12±0.05 mL · min^–1 · g^–1; moderate dose, 0.10±0.05 mL · min^–1 · g^–1; and high dose, 0.08±0.02 mL · min^–1 · g^–1). However, this tendency reached statistical significance only for the diaphragm (Figure 5) and jejunum (control, 1.89±0.51 mL · min^–1 · g^–1; moderate dose, 1.33±0.47 mL · min^–1 · g^–1; and high dose, 1.10±0.24 mL · min^–1 · g^–1; P=0.021).

View larger version (36K): [in this window] [in a new window]   Figure 5. Diaphragm blood flow, expressed in mL · min–1 · g–1, in control (Con) rats (solid bar) or rats treated with moderate (Mod; open bar) or high (gray bar) doses of rhIL-6. *P<0.05. Abbreviations are as defined in text.

Heart Histology
Microscopic examination of diastolic right ventricular cavity dimensions revealed significant dose-dependent increases in surface when compared with those of control rats (15.4±3.4 µm² in controls versus 28.6±5.2 µm² in the moderate-dose and 31.9±8.1 µm² in the high-dose groups; P<0.01; Figure 6).

View larger version (63K): [in this window] [in a new window]   Figure 6. A, Right ventricle surface, expressed as percentage of total heart surface in control (Con) rats (solid bar) or rats treated with moderate (Mod; open bar) or high (gray bar) doses of rhIL-6. **P<0.01. B, Heart H&E staining of representative control (left), moderate-dose (middle), and high-dose (right) rhIL-6–treated animals. Panels correspond to x8 magnification, bar=875 µm. Note dose-dependent dilatation of right ventricular (RV) cavity surface in both treated groups when expressed as percentage of total surface. Left ventricular (LV) dilatation did not occur. Other abbreviations are as defined in text.

Lung Histology and Water Content
Left lung microscopic sections of treated animals all showed congestion and interstitial edema, whereas alveolar edema was seen in only some sections (Figure 7). Neither alveolar septal thickening nor focal edema was present in control rats. There were no signs of inflammatory infiltrates in any of the 3 groups. The wet-to-dry ratio of the right lung was significantly higher in moderate- (4.6±0.10) and high- (4.5±0.05) dose groups than in controls (4.2±0.13, P<0.001).

View larger version (131K): [in this window] [in a new window]   Figure 7. Left lung H&E staining of representative control (left) and high-dose rhIL-6–treated (right) animals. Top panels correspond to x200 magnification, bar=50 µm; bottom panels, to x650 magnification, bar=15 µm. Note alveolar edema, identified as pale eosinophilic fluid within groups of alveoli (arrow in B), and congestion or interstitial edema, marked by thickened septa engorged by red blood cells (arrow in D), in micrographs from high-dose group. None of these characteristics were present in control rats. Abbreviations are as defined in text.

Serum Levels
rhIL-6 serum levels averaged 765±529 pg/mL in the moderate-dose group and 12±10 ng/mL in the high-dose group. Conversely, endogenous IL-6 concentrations did not differ among groups and were negligible (mean value of 23±53 pg/mL).

Food Intake
Food intake decreased in all groups immediately after pump implantation (control, –26%; moderate dose, –53%; and high dose, –45%; P<0.001). However, at day 1, food intake started to increase in all groups, still yielding a significantly smaller food intake in both treated groups (P<0.005). Conversely, from day 5 onward, food intake increased more in treated rats, so that by the end of treatment, food intake was again similar among all groups (control, 26.10±7.21 g; moderate dose, 25.92±2.65 g; and high dose, 22.88±2.68 g). Body weight decreased significantly after pump implantation in the high-dose group until the end of treatment (control, 412±27 g; moderate dose, 398±20 g; and high dose, 382±10 g; P<0.05).

Activity Level
Displacement distance was similar between treated and control rats, both during the day (control, 1286±451 cm/h; moderate dose, 1063±414 cm/h; and high dose, 1165±314 cm/h) and the night (control, 710±521 cm/h; moderate dose, 537±363 cm/h; and high dose, 541±256 cm/h).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present data show that 7-day administration of rhIL-6 to rats resulted in a dose-dependent respiratory and peripheral skeletal muscle atrophy without alterations in diaphragm contractile properties. It also caused a dose-dependent myocardial contractile deterioration, as was evident from alterations in invasive cardiovascular functional parameters, increased lung wet-to-dry ratios, and lung microscopic examination. It appears likely that the observed diaphragm and peripheral muscle atrophy predominantly resulted from blood flow redistribution, because a direct effect of rhIL-6 on the diaphragm was absent.

In the present study, contractility was expressed in grams per square centimeter, meaning that all generated forces were corrected for potential reductions in CSA. Because the observed diaphragm atrophy occurred with unchanged contractile properties, there was, in essence, less muscle with the quality of the remaining muscle unchanged. However, the diaphragm’s absolute force generation is expected to be reduced proportionately with respect to reductions in diaphragm weight.

The effects of IL-6 on myocardial function have been well documented in vitro. Reversible negative inotropic effects on isolated hamster papillary muscles,²⁰ downregulation of SERCA2 in neonatal rat ventricular myocytes,²¹ reduced RNA expression of both cardiac myosin heavy chain isoforms, and a loss of cardiac actin in rat cardiac myocytes²² have been reported. Conversely, although in vivo experiments showed that IL-6 overexpression in mice caused skeletal muscle atrophy, a direct negative effect of IL-6 on skeletal muscle cells had never been observed.^12,13

There is abundant evidence that cardiac failure induces ventilatory and peripheral skeletal muscle atrophy. However, in studies concurrently addressing peripheral and respiratory muscle strength in heart failure patients, the impairment in respiratory muscle contractility was proportionally greater^28–30 or existed even in the absence of peripheral muscle dysfunction.²⁹ Ventricular dysfunction induced by experimental coronary ligation in rats confirmed the aforementioned findings; ie, moderate ventricular dysfunction affected the diaphragm only, whereas severe heart failure also affected other respiratory and peripheral muscles.⁶

It appears plausible from the present study that skeletal muscle atrophy results from the observed cardiac failure induced by rhIL-6, because the pattern of muscle atrophy observed in the present study was similar to that observed in studies of myocardial failure.^31,32 Furthermore, systolic ventricular dysfunction in rats in which alterations in hemodynamic variables resulted in a pattern of heart failure similar to that in the present study led to an identical pattern of muscle atrophy, also without changes in diaphragm contractile properties.⁶ Finally, in the present study, reductions in ventilatory and peripheral muscle weight were related to reduced myocardial contractility.

Other factors might be implicated in the observed skeletal muscle atrophy but probably contributed to a lesser extent. Indeed, short-term nutritional deprivation would be expected to result in selective type IIx/b atrophy in the diaphragm,³³ which is in contrast with the generalized atrophy observed herein. Furthermore, deconditioning was unlikely to be responsible as well, because activity levels were similar among all groups. A direct effect of rhIL-6 to the diaphragm could also be excluded, because no changes in diaphragmatic contractility were observed after in vitro rhIL-6 administration. Conversely, skeletal muscle insulin resistance could not be ruled out in our study and might have contributed somewhat to the observed skeletal muscle atrophy, because it is known that IL-6 administration in mice results in hepatic insulin resistance by inhibiting the insulin-dependent insulin receptor signal transduction at serum concentrations of 112 pg/mL. However, at that concentration, it did not affect the signal transduction pathway in skeletal muscles.³⁴

The pattern of hemodynamic alterations observed in the present study is remarkable. Indeed, a dose-dependent heart dilatation was observed, as was evident from increases in end-diastolic volume (moderate dose, 59%; high dose, 77%), leading to functioning at lower end-systolic pressures (–23% and –20%, respectively). Significantly higher stroke volumes (59% and 75%) are compatible with unaltered ejection fraction. However, load-independent contractility parameters decreased significantly; eg, preload recruitable stroke work decreased in a dose-dependent fashion (–16% and –39%). Because peripheral resistance decreased, stroke volume and cardiac output increased despite the reduced cardiac contractility. Although paradoxical at first, this phenomenon was associated with reductions in diaphragm blood flow of 60% in both treated groups. In addition, decreases in systolic arterial pressure as observed in the present study are known to decrease diaphragmatic blood flow.³⁵ This decrease is likely to be responsible for the diaphragm atrophy observed. A similar trend was present for blood flow to peripheral muscles, though less pronounced. This finding is consistent with the fact that only a tendency to atrophy was observed in the gastrocnemius. Nevertheless, because blood flow reductions were also observed in all measured organs except kidneys, increases in other tissues are expected.

All of these hemodynamic changes do not contradict previous findings showing that IL-6 double-transgenic mice suffered from myocyte hypertrophy and decreases in left ventricular volume.¹⁶ In the latter study, hIL-6 serum levels averaged 0.1 to 5 ng/mL, which was less than observed in our high-dose-group of rats (12.1 ng/mL). This low IL-6 concentration might produce hypertrophy, whereas higher concentrations (as in our study) might further affect the myocardium and produce dilatation, which was especially pronounced in the high-dose group. However, transgenic models are not fully comparable because of overexpression during prenatal and postnatal development, which certainly influences long-term morphological findings such as ventricular hypertrophy and remodeling. Furthermore, cardiotrophin-1 administration, at lower doses than those used in our study, also induced myocyte hypertrophy,¹⁷ decreases in mean arterial pressure, and decreases in systemic vascular resistance without changes in left ventricular maximal dP/dt.¹⁸ Those observations are consistent with our results.

The present data may be very clinically significant, because the rhIL-6 serum levels that produced effects in the present study are within the range of those obtained in patients. Even if rats are less susceptible to IL-6 than humans, as demonstrated in human toxicity experiments,³⁶ a serum level of 12 ng/mL was observed with a dose of 250 µg · kg^–1 · d^–1. At 100 µg · kg^–1 · d^–1, at which heart failure was already evident and discrete muscle atrophy was present, a serum level of 765 pg/mL was observed. Both values are in the range of individual serum levels found in patients with sepsis, acute respiratory distress syndrome, and pneumonia.^2,4 Also, in patients suffering from left ventricular dysfunction¹ or after reperfusion of the ischemic heart in cardiopulmonary bypass,³⁷ IL-6 serum levels are known to be elevated. This may consequently lead to further deterioration of cardiac function, because IL-6 is known to be an independent predictor for worsening of heart failure in patients with end-stage heart failure.³⁸ Therefore, anticytokine strategies may be useful during cardiac surgery to improve clinical outcomes, as already shown by the beneficial effects of tumor necrosis factor- and IL-6 removal through hemofiltration in children undergoing cardiopulmonary bypass.³⁹ Although IL-6 serum levels in heart failure patients are not in the range of those observed in the present study,¹ supranormal levels of rhIL-6 as observed herein are clearly relevant to patients with acute respiratory distress syndrome, pneumonia, and sepsis, in which the same pattern of myocardial failure occurs.⁴⁰

We conclude that 7 days of exogenous administration of rhIL-6 to rats caused dose-dependent skeletal muscle atrophy induced by myocardial failure. These effects occurred at serum levels that are likely to be attained in patients. This may be of major clinical relevance to patients with acute respiratory distress syndrome, pneumonia, or sepsis or those undergoing cardiopulmonary bypass.

	Acknowledgments

 
This study was supported by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO), Belgium, grant No. G.0237.01, and FWO Levenslijn grant No. 7.0007.00. Ghislaine Gayan-Ramirez is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. An Van Den Bergh is a researcher funded by a PhD grant of the Institute for the Promotion of Innovation Through Science and Technology in Flanders (IWT-Vlaaderen). The authors sincerely acknowledge Dr Ahmad Kasran for the serum analyses, Leen Luyts for technical assistance, Veerle Leunens for the microsphere analysis, and Toon Leroy for measuring activity levels. The authors also thank Prof Raf Sciot for preparing the histological slides and Kris Van Kuyck and Veerle Reynders for performing the pilot study. Finally, the authors are grateful to the company Ajinomoto (Japan) and particularly Dr Okano for providing the rhIL-6.

	Footnotes

 
The online-only Data Supplement can be found with this article at http://www.circulationaha.org.

	References

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