Inhibition of sarcoplasmic reticular function by chronic interleukin-6 exposure via iNOS in adult ventricular myocytes
1 Department of Pharmacology and Toxicology
2 Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA
3 Department of Physiology, Loyola University Medical Center, Stritch School of Medicine, Maywood, IL 60153, USA
Abstract
Interleukin (IL)-6 has been shown to decrease cardiac contractility via a nitric oxide synthase (NOS)-dependent pathway during acute exposure. We previously reported that IL-6 decreases contractility and increases inducible NOS (iNOS) in adult rat ventricular myocytes (ARVM) after 2 h exposure. The goal of this study was to investigate the cellular mechanism underlying this chronic IL-6-induced negative inotropy and the role of iNOS. Pretreatment for 2 h with 10 ng ml–1 IL-6 decreased the kinetics of cell shortening (CS) and contractile responsiveness to Ca2+o ([Ca2+]o from 0 to 2 mM) in ARVM. We first examined whether IL-6 reduced Ca2+ influx via L-type Ca2+-channel current (ICa,L). Whole-cell ICa,L in ARVM was measured under conditions similar to those used for CS measurements, and it was found to be unaltered by IL-6. The sarcoplasmic reticular (SR) function was then assessed by examining postrest potentiation (PRP) and caffeine responsiveness of CS. Results showed that treatment with IL-6 for 2 h significantly decreased PRP, which was concomitant with a decrease in the phosphorylation of phospholamban. Following removal of IL-6, PRP and responsiveness to 10 mM caffeine were also reduced. Meanwhile, the IL-6-induced increase in nitric oxide (NO) production after 2 h (but not 1 h) was abolished by NG-monomethyl-L-arginine (L-NMMA) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective inhibitor of iNOS). Furthermore, IL-6-elicited suppressions of PRP and responsiveness to caffeine and Ca2+o were abolished by L-NMMA and AMT. Thus, these results suggest that activation of iNOS mediates IL-6-induced inhibition of SR function in ARVM during chronic exposure.
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Introduction
Interleukin (IL)-6, a pro-inflammatory cytokine, is produced during the acute phase of the immune response by immune and nonimmune cells (for review, see Akira et al. 1990). After binding to its receptor, IL-6 elicits numerous biological effects, including antibody induction, haematopoiesis, thrombocytopoiesis and acute-phase protein synthesis (for reviews, see Akira et al. 1990; Kishimoto et al. 1995). Recently, significant increases in serum levels of IL-6 and its mRNA expression and protein production in cardiac tissues have been reported in patients with chronic cardiac failure (Satoh et al. 1996; Steele et al. 1996; Roig et al. 1998; Cesari et al. 2003), myocarditis (Satoh et al. 1996), myocardial infarction (Ikeda et al. 1992; Guillen et al. 1995; Neumann et al. 1995), endotoxaemia (Hack et al. 1989), and the injury associated with ischaemia/reperfusion (Sawa et al. 1998) and cardiopulmonary bypass (Wan et al. 1996). In patients undergoing cardiopulmonary bypass, the increase in plasma IL-6 levels in the coronary sinus begins 5 min after aortic declamping, peaks at 1 h, and is sustained for at least 2 h (Wan et al. 1996). Therefore, IL-6 has been associated with the severity of several cardiovascular pathophysiological states (Cesari et al. 2003).
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In vitro studies on cardiac contractile function have shown that exposure to IL-6 for 2–3 min decreased contractility in papillary muscle isolated from hamster heart (Finkel et al. 1992). Studies with single myocytes also showed that IL-6 suppressed peak systolic [Ca2+]i and cell shortening (CS) within 5 min in chick embryonic cardiac myocytes (Kinugawa et al. 1994) and in adult guinea-pig ventricular myocytes (Sugishita et al. 1999). This acute negative inotropic effect of IL-6, accompanied by an increase in cell cGMP production (Kinugawa et al. 1994), was blocked by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase (NOS) (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999). Thus, acute IL-6-induced suppression of cardiac contractility and [Ca2+]i have been suggested to be mediated by a NO-dependent pathway via activation of NOS (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999), probably a constitutive endothelial isoform (eNOS) (Kinugawa et al. 1994). In contrast, studies on chronic cardiac effects of IL-6 showed that after 24 h incubation, IL-6 also increased cell cGMP, which was blocked by L-NMMA but not by EGTA, suggesting the involvement of a Ca2+-independent NOS (Kinugawa et al. 1994). However, the subtype of NOS that is involved in chronic cardiac effect of IL-6 has not been defined.
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As mentioned above, an in vivo study showed that elevated IL-6 levels were observed 1–2 h after aortic declamping in cardiopulmonary bypass (Wan et al. 1996). The direct cardiac effect of IL-6 during this period of time remains undefined. We recently reported that de novo synthesis and activation of iNOS induced by IL-6 can be detected in adult rat ventricular myocytes (ARVM) as early as 2 h after exposure (Yu et al. 2003). This earlier study also demonstrated that the IL-6-elicited iNOS activation and decrease in postrest potentiation (PRP) of contraction in ARVM are mediated by activation of Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3, the upstream mediators of IL-6 signalling (Yu et al. 2003). However, whether iNOS is the downstream mediator of IL-6-induced negative inotropy remains undefined.
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In the present study, we examined the effect of chronic exposure (for 2–24 h) of ARVM to IL-6 on contractility, contractile responsiveness to Ca2+o, L-type Ca2+-channel current (ICa,L), and sarcoplasmic reticular (SR) function. SR function was assessed using two protocols: PRP and caffeine-induced contraction (Bassani et al. 1993, 1994; Bers et al. 1998). We also examined the role of iNOS in IL-6-induced changes in SR function. We found that IL-6 decreased contractility and Ca2+o responsiveness of ARVM, primarily resulting from suppression of SR function accompanied by reduction in phosphorylation of phospholamban (PLB), a SR Ca2+-pump regulatory protein. This chronic cardiac effect of IL-6 was sustained after removal of IL-6. We also showed that inhibition of iNOS and NO production blocked the IL-6 negative inotropic action. A preliminary report of some of these results has been presented in an abstract (Yu et al. 2000).
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Methods
Ventricular myocyte preparation
Single ventricular myocytes were isolated from hearts of adult male Sprague-Dawley rats (250–300 g) using enzymatic dissociation as previously described (Liu & Schreur, 1995). Briefly, rats were deeply anaesthetized with ether or isoflurane (Vedco, St Joseph, MO, USA) followed by a thoracotomy. Hearts were rapidly excised and perfused at 37°C via the aorta with a control buffer solution, followed by enzymic and mechanical dissociation. Isolated ARVM were resuspended in culture medium containing antibiotic-free, bicarbonate-buffered culture medium 199 (60%; Gibco, Grand Island, NY, USA) with 36% Earle's balanced salt solution composed of (mM): 116 NaCl, 4.7 KCl, 0.9 NaH2PO4, 0.8 MgSO4, 26 NaHCO3, and 5.6 glucose and 4% fetal bovine serum (Gibco) (pH 7.40 in 5% CO2/95% air at 37°C). After incubation for 3–4 h to allow recovery, ARVM were plated in culture dishes containing serum-free L-arginine-containing medium, incubated overnight, and treated with designated concentrations (1–30 ng ml–1) of IL-6 for various periods of time (1–24 h). After the designated exposure duration, quiescent rod-shaped ARVM with clear striations were used for electrophysiological and CS measurements. All experiments were performed at 37°C. The use of animals was carried out under a protocol approved by the Animal Care and Use Committee at the University of Arkansas for Medical Sciences.
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Measurement of ICa,L
ICa,L of ARVM was measured in normal Tyrode solution, or Na+- and K+-free solutions with 2 mM Ca2+, using conventional whole-cell patch techniques (Hamill et al. 1981) with a patch-clamp amplifier (Axopatch 200 A, Axon Instruments, Union City, CA, USA) as previously described (Liu et al. 1997). Briefly, patch electrodes were filled with pipette solution and had a tip resistance of 1–3 M. The pipette solution consisted of (mM): 100 CsOH, 70 aspartate, 11 CsCl, 15 tetraethylammonium chloride, 2 MgC12, 5 MgATP, 5 EGTA, 0.1 CaC12, 5 pyruvic acid, 5.6 glucose, 5 Tris2-phosphocreatine, 0.4 Li4-GTP, 10 Hepes/Tris (pH adjusted to 7.20 with CsOH). The bath solution contained (mM): 145 N-methyl-D-glucamine chloride, 0.8 MgC12, 2 CaC12, 2 4-aminopyridine, 10 Hepes/Tris (pH adjusted to 7.40 with CsOH). ICa,L was elicited by 250 ms voltage pulses to potentials between –60 and +60 mV from a holding potential of –80 mV in 10 mV increments at 0.2 Hz. In some experiments, ICa,L was elicited by 25 ms voltage pulses and measured in normal Tyrode solution when myocytes were voltage clamped at –40 mV before switching to the Na+- and K+-free bath solution. The magnitude of ICa,L was determined by the difference between the holding current and the peak amplitude in response to 25 ms pulses, or by the difference between the peak amplitude and the level at the end of 250 ms pulses. Series resistance was 3–6 M and electronically compensated (90%), while the recorded currents were filtered at 1–2 kHz and sampled at 5 kHz using PClamp 8.0 software (Axon Instruments). The density of ICa,L in each myocyte was normalized to membrane capacitance (Cm), which was calculated from uncompensated capacity current transients recorded in response to 5 mV hyperpolarizing pulses from the holding potential.
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Contractile function or CS
Unloaded CS of ARVM was elicited in normal Tyrode solution and measured as previously described (Liu et al. 2002). Briefly, CS was elicited by field stimulation (2 ms duration, 1.5-fold threshold voltage) at 0.5 Hz in normal Tyrode solution containing (mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 0.8 MgCl2, 10 Hepes/Tris and 5.6 glucose (pH 7.40 at 37°C). CS was monitored with a video edge-motion detector system (Crescent Electronics, Sandy, UT, USA). The voltage signal was calibrated to determine actual motion (micrometres). Data of CS were not expressed as percentages of original cell length because the degree of CS is significantly affected by the degree of cell attachment to the dish, which varies from cell to cell, and from dish to dish.
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The measured parameters of contractile function in single ARVM included the peak magnitude of CS and maximum rates of contraction ((+dL/dt)max) and relaxation ((–dL/dt)max). The responsiveness of CS to Ca2+o was assessed by monitoring the change in CS in response to various [Ca2+]o (0–2 mM). The magnitude of CS in different [Ca2+]o relative to that observed in 0.5 mM [Ca2+]o was plotted as a function of [Ca2+]o. The SR function of ARVM was estimated by measurements of PRP and by caffeine-induced CS (Bassani et al. 1993; Bers et al. 1998). PRP was measured as the first contraction after a rest interval between 5 and 120 s, and the amplitude of PRP relative to that of the steady-state CS before the rest was plotted as a function of rest interval. Similarly, 10 mM caffeine was applied for 10 s following a rest interval between 15 and 60 s in the absence of electrical stimulation. The amplitude of caffeine-induced CS was normalized to that of the steady-state CS before electrical cessation.
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NO production
NO production was estimated by measuring total production of nitrate and nitrite (NOx) in culture medium and cell lysate, as previously described (Yu et al. 2003). Briefly, after treatment with 10 ng ml–1 IL-6 for 2, 4 and 24 h, cells were collected after centrifugation at 100 g for 10 min. The supernatant (culture media) was removed and kept at –20°C until the NOx assay. Cell pellets were resuspended in phosphate-buffered saline (PBS), half of which was used to determine protein content (see below). The other half of the cell suspension was sonicated and centrifuged at 10 000 g, and the supernatant was centrifuged at 100 000 g for 15 min at 4°C. The final supernatant was filtered via a 10 kDa molecular weight cut-off filter at 12 000 g for 30 min, and kept at –20°C until the NOx assay. The concentration of NOx in culture media and cell lysate was determined using a colourimetric assay kit (Cayman Co., Ann Arbor, MI, USA) according to the manufacturer's instructions. The total NOx concentration was calculated according to a standard curve of sodium nitrate and expressed in nanomoles per milligram of cell protein. Results of treated groups were normalized to each time-control.
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Western blot analysis
Preparation of cell lysate of ventricular myocytes and Western blot analyses were carried out as previously described (Liu & McHowat, 1998). Briefly, control and treated myocytes (0.5 x 106 cells per group) were lysed in 300 μl of ice-cold lysis buffer containing (mM): 20 Hepes (pH 7.6), 250 sucrose, 2 dithiothreitol, 2 EDTA, 2 EGTA, 10 sodium orthovanadate, 2 phenylmethylsulphonyl fluoride, 20 μg ml–1 leupeptin and 10 μg ml–1 aprotinin. The concentration of total protein was determined using a Bradford assay (Bio-Rad, Hercules, CA, USA). Forty micrograms of each protein sample was then electrophoretically separated on 8% SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes (Bio-Rad). Non-specific binding sites of membranes were blocked by incubation in Tris buffer solution containing 0.05% Tween-20 and 5% nonfat milk for 1 h at room temperature. The membrane was then probed with polyclonal anti-phospho-PLB antibodies (1:1000, Cayman Co.) overnight at 4°C, followed by treatment with horseradish-peroxidase-conjugated secondary antibodies. Immunoblots were detected using enhanced chemiluminescent kits (SuperSignal; Pierce, Rockford, IL, USA) and analysed with a densitometer (Bio-Rad). After being stripped, the membranes were reprobed with monoclonal anti-PLB antibodies (1:2000 dilution; Upstate Biotechnology, NY, USA). The phosphorylation level of PLB was presented as the relative density of phospho-PLB to total PLB protein in each sample.
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Chemicals
Recombinant rat IL-6 was purchased from Pepro Tech Inc. (Rocky Hill, NJ, USA); the nitrate/nitrite colourimetric assay kit was obtained from Cayman Co. L-NMMA and lipopolysaccharide (LPS) were purchased from Sigma (St Louis, MO, USA). 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) was purchased from Alexis (San Diego, CA, USA).
Statistics
Data of ICa,L and CS were collected from ARVM isolated from 3–5 hearts. The data for NOx production were averaged from duplicate or triplicate measurements taken from 4–6 hearts. Values are presented as means ± S.E.M. Statistical significance (P < 0.05) was evaluated by Student's t test, or one-way ANOVA with Duncan's multiple range test when more than two conditions were compared.
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Results
Effect of chronic exposure to IL-6 on contractility and contractile responsiveness to Ca2+o
After treatment with 10 ng ml–1 IL-6 for 2 or 24 h, myocytes were removed from the incubator, placed on the microscope stage and continuously superfused with normal Tyrode solution in the presence (treatment) or absence (pretreatment) of the same concentration of IL-6. The basal CS of ARVM was elicited at 0.5 Hz and remained stable for at least 1 h in both conditions. When ARVM were superfused with IL-6-containing solution, the kinetics of steady-state CS of IL-6-treated ARVM, i.e. (+dL/dt)max and (–dL/dt)max, were found to be decreased by 45 and 35%, respectively, when compared with time-controls (Table 1). Interestingly, when myocytes were superfused in the absence of IL-6 (pretreated group) following IL-6 incubation, the (+dL/dt)max and (–dL/dt)max of steady-state CS were still decreased by 37 and 28%, respectively (Table 1). Thus, the results suggest that the contractile function of IL-6-pretreated ARVM remains suppressed at least 1 h after removal of IL-6.
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The inotropic response of CS to Ca2+o was then examined in IL-6-pretreated (2 h) and time-control ARVM by monitoring CS in Tyrode solution containing various [Ca2+]o in the absence of IL-6. After contraction reached a steady state at 1 mM [Ca2+]o (in normal Tyrode solution), the solution was randomly switched to solutions containing [Ca2+]o between 0 and 2 mM. Figure 1A shows representative traces of steady-state CS from a time-control and an IL-6-pretreated (2 h) ARVM in 1, 1.5 and 2 mM [Ca2+]o solutions. When [Ca2+]o was increased, the enhanced amplitude of CS in IL-6-pretreated ARVM appeared to be smaller than that in the time-control. When [Ca2+]o was reduced to 0 mM, CS was terminated in both groups. Because ARVM started to contract when [Ca2+]o was resumed to 0.5 mM, Ca2+o responsiveness of CS in ARVM was presented as relative amplitude (normalized to that in 0.5 mM [Ca2+]o) as a function of [Ca2+]o. Results are shown in Fig. 1B in which CS responsiveness to Ca2+o in IL-6-pretreated ARVM was significantly decreased when compared with time-controls. In contrast to the decreased maximal Ca2+o responsiveness, IL-6 had no significant effect on the EC50 value for [Ca2+]o (1.2 mM) when data were curve-fit with a Hill equation using a Hill coefficient of 2. These data suggest that the IL-6-induced decrease in responsiveness to Ca2+o could result from a decrease in Ca2+ influx via ICa,L and/or SR Ca2+ release and uptake.
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Myocytes in the time-control and IL-6-pretreated groups were exposed to various [Ca2+]o (0–2 mM). A, representative traces of steady-state cell shortening (CS) in response to [Ca2+]o between 1 and 2 mM were obtained from a time-control cell and an IL-6-pretreated ARVM (10 ng ml–1 for 2 h). B, the magnitude of CS in each [Ca2+]o relative to that in 0.5 mM [Ca2+]o was plotted as a function of [Ca2+]o in time-control and IL-6-pretreated ARVM. Data represent means ± S.E.M. *P < 0.05, compared with the control group (ANOVA). Continuous lines are curves fit by the Hill equation using a Hill coefficient of 2.
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Effect of chronic exposure to IL-6 on ICa,L
We then examined whether IL-6-induced negative inotropy and Ca2+o responsiveness result from a decrease in ICa,L. After incubation for 2 h with 10 ng ml–1 IL-6, ICa,L was first measured in normal Tyrode solution (Fig. 2A) when cells were voltage clamped at –40 mV and K+ currents were largely diminished. After construction of the current–voltage (I–V) relationship of ICa,L (Fig. 2C), single 250 ms voltage pulses to +10 mV from the holding potential at 1 Hz were used to monitor ICa,L while the external solution was switched to Na+- and K+-free solution containing 2 mM [Ca2+]o. After ICa,L reached a new steady state, the holding potential was changed to –80 mV (Fig. 2B), and the I–V relationship of peak ICa,L was generated using the pulse protocols illustrated in the inset of Fig. 2D. Results show that the maximum current density and voltage dependency of peak ICa,L measured in both solutions were not significantly altered after 2 h pretreatment with IL-6. The maximum current density of ICa,L measured at +10 mV in the Na+- and K+-free solution was –17.3 ± 1.4 pA pF–1 (n = 10) in time-controls and –15.5 ± 0.7 pA pF–1 (n = 19) in IL-6-pretreated ARVM. Thus, these data suggest that the IL-6-induced negative inotropy does not result from a decrease in ICa,L, thereby leading to the hypothesis that IL-6 suppresses SR function.
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A and B, peak L-type Ca2+-channel current (ICa,L) in a control (left) and IL-6-pretreated (10 ng ml–1 for 2 h, right) ARVM in response to a voltage pulse to +10 mV were recorded in normal Tyrode solution and in Na+- and K+-free bath solution containing 2 mM Ca2+, respectively. The current–voltage (I–V) relationships of peak ICa,L in time-control and IL-6 pretreated ARVM were constructed using voltage-pulse protocols from a holding potential (Vh) of –40 mV in normal Tyrode solution (C) or –80 mV in Na+- and K+-free solution containing 2 mM Ca2+ (D). Membrane capacitance (Cm): A, 158 pF; and B, 207 pF. Dashed lines in A and B represent the zero current level. The positive capacitance transient in each trace of A and B was truncated. Data in C and D represent means ± S.E.M.
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Effect of chronic exposure to IL-6 on PRP
The effect of IL-6 on PRP of CS in ARVM was first examined during continuous perfusion with Tyrode solution containing 10 ng ml–1 IL-6 after 2 h incubation with IL-6 in culture medium. Figure 3A shows representative traces of PRP following a 60 s rest interval (PRP60) in a time-control (left panel) and an IL-6-treated ARVM (right panel). The IL-6-treated myocyte displayed a smaller PRP than the time-control. Figure 3B shows combined data from 10 experiments, which demonstrate that the PRP (measured following rest intervals of 30 s) in IL-6-treated ARVM was significantly decreased (by 25%) after 2 h incubation in the continued presence of IL 6.
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PRP was measured as the amplitude of the first contraction following different rest-intervals (5–120 s) relative to that of the prerest steady-state CS. A, representative traces of steady-state CS and PRP following a 60 s rest interval in a time-control ARVM (left) and an IL-6-treated myocyte (right, 10 ng ml–1 for 2 h); the latter was superfused with Tyrode solution containing 10 ng ml–1 IL-6. Combined data of PRP in IL-6-treated (B, measured in continuous presence of IL-6) or IL-6-pretreated (C, measured after removal of IL-6) ARVM were compared with time-controls. Data represent means ± S.E.M., n = 7–23. *P < 0.05, compared with the time-control (ANOVA).
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We then examined whether the inhibition of PRP induced by chronic exposure to IL 6 could be reversed after removal of IL 6. Measurements of PRP in IL-6-pretreated (10 ng ml–1 for 2 h) ARVM were completed within 30 min after beginning perfusion with normal Tyrode solution. PRPs of CS in IL-6-pretreated ARVM were decreased to almost the same degree as observed in continuously IL-6-treated ARVM (Fig. 3C versus B). Such reductions in PRP induced by pretreatment with IL-6 were both concentration and time dependent. For example, pretreatment with 10 and 30 ng ml–1 IL-6 caused 15 ± 5 and 39 ± 6% decreases in PRP after a 30 s rest interval (PRP30), respectively (n = 23, P < 0.05). Lower concentrations (e.g. 1 and 3 ng ml–1) had no significant effect. In addition, the IL-6-induced suppression of PRP30 was 15 ± 5% (n = 19, P < 0.05), 21 ± 7% (n = 23, P < 0.05) and 27 ± 4% (n = 10, P < 0.05) after pretreatment with 10 ng ml–1 IL-6 for 2, 4 and 24 h, respectively. Since the IL-6-induced inhibition of CS and PRPs was sustained at least 1 h after removal of IL-6, the results of CS measurements presented in the following sections were obtained from IL-6-pretreated ARVM.
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Furthermore, to confirm that inhibition of PRP after IL-6 pretreatment was caused specifically by IL-6, anti-IL-6 antibodies (100 ng ml–1, 10-fold of IL-6 concentration) were added to the culture medium 15–30 min prior to and during 2 h treatment with IL-6. Only the PRP30 and PRP60 of CS were examined under these conditions. Figure 4 shows that anti-IL-6 antibodies completely blocked the IL-6-induced inhibition of PRP, while alone they had no effect. Taken together, these results indicate that IL-6 reduces contractility of ARVM by suppressing SR function, not Ca2+ influx via ICa,L.
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ARVM were incubated with 100 ng ml–1 anti-IL-6 antibodies 15–30 min before and during 2 h treatment with 10 ng ml–1 IL-6. Anti-IL-6 antibodies prevented the decrease in PRP (after 30 or 60 s rest) induced by IL-6, while alone they had no effect. Data represent means ± S.E.M., n = 6–7. *P < 0.05 compared with the time-control.
Effect of chronic exposure to IL-6 on contractile responsiveness to caffeine
After stable CS of myocytes was obtained in the control solution, electrical stimulation was terminated for 15–60 s to reload the SR Ca2+. ARVM were then exposed for 10 s to 10 mM caffeine to deplete SR Ca2+ content before resuming electrical stimulation. Figure 5A shows representative traces of responsiveness of CS to caffeine following a 15 s rest interval in a 2 h time-control (left panel) and an IL-6-pretreated ARVM (right panel). The amplitude of caffeine-induced CS in IL-6-pretreated ARVM was smaller than that in the time-control, suggesting less available SR Ca2+. After being normalized to the amplitude of steady-state CS, the relative amplitude of caffeine-induced CS in each group was plotted as a function of the rest interval. Figure 5B and C shows that the relative amplitude of caffeine-induced CS in time-controls (from at least four experiments) was slightly increased when the rest interval was prolonged from 15 to 60 s, suggesting no significant SR Ca2+ leak after Ca2+ reloading during the rest period in ARVM. The combined data in Fig. 5B also show that the relative amplitude of caffeine-induced CS was significantly decreased and lost the time-dependent Ca2+ reload after 2 h pretreatment with 10 ng ml–1 IL-6. After 24 h pretreatment, a similar time-dependent SR Ca2+ loading was observed in time-controls, and the decrease in the caffeine-induced CS appeared to increase (Fig. 5C). These results suggest that pretreatment with IL-6 attenuated SR Ca2+ content, probably by decreasing Ca2+ uptake via the SR Ca2+ pump. This is consistent with the IL-6-induced decrease in PRP.
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Caffeine (10 mM), depleting SR Ca2+ store, was used to assess available SR Ca2+ content in ARVM. A, representative traces of caffeine-induced contraction following a 15 s rest interval in a time-control (left) and an IL-6-preteated ARVM (10 ng ml–1 for 2 h, right). The relative amplitude of caffeine-induced CS to that of the steady-state CS following a rest interval was plotted as a function of the rest interval (15–60 s) in time-controls and ARVM pretreated with 10 ng ml–1 IL-6 for 2 h (B) and 24 h (C). Data represent means ± S.E.M., n = 4–10. *P < 0.05, compared with the time-control (ANOVA).
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Effect of chronic exposure to IL-6 on phosphorylation of PLB
The dephosphorylation of PLB decreases the affinity of the SR Ca2+ pump (SERCA2a) for Ca2+, and phosphorylation of PLB relieves this inhibitory effect (Koss & Kranias, 1996). Thus, the phosphorylation level of PLB in ARVM was examined after exposure to 10 ng ml–1 IL-6. Figure 6A shows representative immunoblots from time-control and IL-6-treated ARVM detected with anti-phospho-PLB (upper panel) and anti-PLB antibodies (lower panel). When cell lysates were treated at 37°C for 5 min before electrophoresis, both pentameric (26 kDa) and monomeric (6 kDa) forms of PLB were detectable in time-controls, in which the abundance and phosphorylation of pentamers were predominant. Treatment of ARVM for 5 min with 10 nM isoproterenol (ISO), serving as a positive control, resulted in a marked increase in the phosphorylation of both pentamers and monomers of PLB (right lanes in Fig. 6A). By contrast, IL-6 significantly decreased the phosphorylation of PLB pentamers to 63 ± 16 and 74 ± 12% of time-control levels after 2 and 24 h, respectively, in four experiments (Fig. 6B, P < 0.05). The phosphorylation level of the PLB monomers was 17 ± 2 and 38 ± 14% of the time-controls after 2 and 24 h exposure to IL-6, respectively (Fig. 6C, n = 3, P < 0.05). When cell lysates were treated for 5 min at 95°C, the low molecular mass of PLB was the only form detected. However, under this condition, the change in phosphorylation of PLB induced by IL-6 was less consistent or evident than that observed in oligomers. Nevertheless, these data support the suggestion that IL-6 suppresses SR function by decreasing the Ca2+ uptake via SR Ca2+ pumps.
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Protein samples (40 μg) extracted from time-controls (C) and IL-6-treated ARVM (I) were loaded in each lane. A, immunoblots of phosphorylated (at Ser16, upper panel) and total (lower panel) PLB in time-control and IL-6-treated ARVM. A 5 μg protein extract from ARVM was treated with 10 nM isoproterenol (ISO) for 5 min and used as a positive control. The phosphorylation level (p-PLB/PLB) of pentamers (B, n = 4) and monomers (C, n = 3) of PLB in IL-6-treated ARVM was normalized to that of each time-control. Data represent means ± S.E.M. *P < 0.05, compared with each time-control.
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IL-6-elicited induction of NO production via activation of iNOS
We previously showed IL-6-induced iNOS protein expression and NOx production after 2 h exposure (Yu et al. 2003). In this study, we examined whether NOx production results specifically from IL-6-elicited activation of iNOS. Figure 7A shows that total NOx concentrations in culture media were not significantly altered at 1 h (1.08 ± 0.04-fold of the control level, n = 5, P > 0.05) but increased to 2.31-fold of the time-control level (n = 14, P < 0.05) after 2 h incubation with 10 ng ml–1 IL-6. Similarly, the NOx concentrations in the culture media after 4 and 24 h incubation with IL-6 were 2.72- (n = 9, P < 0.05) and 2.91-fold (n = 7, P < 0.05) of the time-control level, respectively. Note that there was no significant difference in the concentrations of NOx in the culture media of 2, 4 and 24 h time-controls (i.e. in the absence of IL-6); values were 1.55 ± 0.23 (n = 14), 1.16 ± 0.15 (n = 9), 1.47 ± 0.17 (n = 7) nmol (mg protein)–1, respectively. Incubation with 10 μg ml–1 LPS, a positive control (Brady et al. 1992), also increased the concentration of NOx in the culture media (Fig. 7A). Figure 7B shows that the concentrations of NOx in cell lysates of IL-6-treated ARVM (for 2, 4 and 24 h) were also increased significantly (e.g. 3.19-, 4.98- and 6.26-fold of each time-control level, P < 0.05). The concentrations of NOx in cell lysates of 2, 4 and 24 h time-controls were 1.67 ± 0.28 (n = 8), 0.92 ± 0.16 (n = 7), and 1.10 ± 0.23 nmol (mg protein)–1 (n = 7), respectively.
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Total concentrations of nitrite and nitrate (NOx) in culture media (A) and cell lysate (B) were measured in IL-6-treated (10 ng ml–1 for 2, 4 and 24 h, open bars) samples and normalized to those of each time-control (dashed lines). Lipopolysaccharide (LPS) (10 μg ml–1, filled bars) served as a positive control. Data represent means ± S.E.M.; numbers in parentheses are numbers of experiments. *P < 0.05, compared with the time-controls.
To determine the involvement of iNOS, we examined the effect of AMT (a specific and potent iNOS inhibitor) on the IL-6-induced increase in NOx production. Figure 8A shows that treatment with 50 nM AMT or 10 μM L-NMMA alone had no effect on NOx production in ARVM. By contrast, when L-NMMA or AMT was added to the culture media 15–30 min before and during incubation with IL-6, each inhibitor completely blocked the IL-6-induced increase in NOx production. Similar results were observed when ARVM were incubated with L-NMMA or AMT during 4 and 24 h treatments with IL-6 (Fig. 8B and C). Moreover, L-NMMA and AMT completely blocked the IL-6-induced increases in NOx concentration in cell lysate obtained from myocytes incubated with IL-6 for 2, 4 and 24 h (data not shown). These data show that iNOS was the primary isoform of NOS responsible for the production of NOx induced by IL-6 in ARVM. Thus, we examined whether IL-6-induced NO production is responsible for its inhibitory effect on SR function in ARVM.
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Myocytes were incubated with 10 μMNG-monomethyl-L-arginine (L-NMMA) or 50 nM 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) in the absence and presence of 10 ng ml–1 IL-6 for 2 (A), 4 (B) and 24 h (C). The NO production in each treat group was normalized to that of time-controls (dashed lines). Data represent means ± S.E.M.; numbers in parentheses are numbers of experiments. *P < 0.05 compared with the control group.
Effects of inhibition of NO production on IL-6-induced suppression of PRP, responsiveness to caffeine and Ca2+o and phosphorylation of PLB
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Experiments described in Figs 1, 3 and 5 were repeated in the presence of 10 μM L-NMMA or 50 nM AMT. Figure 9A shows that L-NMMA and AMT inhibited the IL-6-induced decrease in PRP30 of CS in ARVM after 2 h pretreatment. Similarly, the IL-6-elicited suppression of caffeine-induced CS was abolished in the presence of L-NMMA or AMT (Fig. 9B). Note that L-NMMA or AMT alone had no significant effect on PRP30 or caffeine-induced CS. Figure 9C shows that L-NMMA completely blocked the IL-6-induced inhibition of the contractile response to 1.5 mM [Ca2+]o, whereas alone it had no significant effect on Ca2+o responsiveness. Moreover, L-NMMA diminished the IL-6-induced decrease in responsiveness of CS in 2 mM [Ca2+]o, and similar results were observed in ARVM pretreated with IL-6 for 4 and 24 h (data not shown). Figure 9D shows that after 2 h treatment with IL-6, L-NMMA abolished the IL-6-induced decrease in phosphorylation of PLB with no effect on total PLB protein expression. The same results were obtained in three out of four experiments. Taken together, these data suggest that after 2 h exposure, the IL-6-induced inhibition of cardiac SR function is mediated by enhanced NO production via iNOS activation.
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The PRP after a 30 s rest (A), responsiveness to 10 mM caffeine (B) and 1.5 mM [Ca2+]o (C), and phosphorylation of PLB (D) were measured in ARVM incubated with or without 10 μML-NMMA or 50 nM AMT in the absence and presence of 2 h pretreatment with 10 ng ml–1 IL 6. Immunoblots of PLB with low molecular mass were obtained from cell lysates of the time-control (C), IL-6-, L-NMMA-, and IL-6 plus L-NMMA-treated myocytes treated at 95°C prior to electrophoresis. The same results were observed in another two experiments. Data represent means ± S.E.M. from 5 experiments. *P < 0.05, compared with the time-controls.
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Discussion
IL 6 has been associated with many chronic cardiac diseases (Kanda & Takahashi, 2004), including myocardial failure (Wollert & Drexler, 2001; Janssen et al. 2005), and the severity of several cardiovascular pathophysiological states (Cesari et al. 2003). However, studies on the cellular mechanisms for the cardiac effects of IL-6 are limited. While the acute negative inotropic effect of IL-6 has been studied (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999), the chronic cardiac effect of IL-6 has not been explored. We previously demonstrated that chronic exposure to IL-6 decreases PRP of CS and activates iNOS in ARVM, via activation of JAK2/STAT3 (Yu et al. 2003). The present study further investigated the cellular mechanism for the previously observed chronic inotropic effect of IL-6 in ARVM and examined whether iNOS is the downstream mediator of IL-6-induced negative inotropy. We found for the first time that after 2 h incubation: (1) IL-6 decreases contractility and Ca2+o responsiveness with no significant effect on ICa,L in ARVM; (2) IL-6 reduces PRP, caffeine responsiveness and the phosphorylation of PLB; (3) the negative inotropic effect is sustained for at least 1 h after removal of IL-6; and (4) IL-6 induces production of NO via activation of iNOS, with inhibition of iNOS abolishing IL-6-induced changes in PRP, caffeine- and Ca2+o-responsiveness, and PLB phosphorylation.
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Effective concentrations of IL-6
Under physiological conditions, studies on cytokine releases during exercise reported that plasma IL-6 levels rise to 5 pg ml–1 after eccentric exercise (Toft et al. 2002) and reach 94 pg ml–1, primarily derived from working skeletal muscle, immediately after a marathon race (Ostrowski et al. 1998). In contrast, a recent study using microdialysis techniques showed that repetitive low-intensitiy exercise causes an increase in IL-6 over 2.0 ng ml–1 in the interstitium of human skeletal muscle without an increase in the plasma level (Rosendal et al. 2005). Under pathological conditions, elevated serum IL-6 levels were reported to be 2.5 ng ml–1 in patients with stunned myocardium (Wan et al. 1996; Werdan, 1998). The increased IL-6 acts concentration dependently on a variety of targets in autocrine, paracrine or/and endocrine manners (Kishimoto et al. 1995; Wollert & Drexler, 2001; Pedersen et al. 2003). Moreover, IL-6 mRNA levels were shown to be greater in the heart than other tissues following stimulation with LPS (Saito et al. 2000). Therefore, although concentrations (1–30 ng ml–1) of IL-6 used in this study are higher than the reported values, it is conceivable that IL-6 concentration in the interstitium of ventricular muscle reaches 10 ng ml–1 or higher under pathophysiological conditions, regardless of sources of IL-6 production. Our data showing IL-6-induced concentration- and time-dependent negative inotropic effects are consistent with its pathophysiological role in aforementioned cardiac disorders.
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Irreversible chronic cardiac effect of IL-6
The present study shows that after 2 h incubation, IL-6-induced inhibitory effects on CS kinetics, PRP and Ca2+o- and caffeine-responsiveness persist for at least 1 h after removal of the cytokine. These results suggest that once IL-6 initiates its signalling cascade, such as JAK2/STAT3 (Yu et al. 2003), the agonist–receptor complex is no longer required for its downstream inotropic effect (as would be expected if enhanced iNOS expression is involved). This is consistent with clinical observations, which show that an early transient increase in cytokines (1–4 h) elicits long-term alterations in cardiac function. By contrast, it is not clear whether the acute negative inotropic effect of IL-6 is reversible. Although recovery of muscle contraction was complete within 40 min after removal of IL-6 from acute exposure (Finkel et al. 1992), it has never been addressed in studies using isolated cardiac myocytes (Kinugawa et al. 1994; Sugishita et al. 1999). If the acute inotropic effect of IL-6 is reversible as reported in isolated hamster papillary muscle (Finkel et al. 1992), it would suggest that the cellular mechanisms involved in acute and chronic inotropic effects of IL-6 are different, such as activation of different isoforms of NOS as suggested by studies in chick embryonic cardiomyocytes (Kinugawa et al. 1994). Thus, the present study was designed to examine whether this sustained chronic cardiac effect of IL-6 is mediated by iNOS.
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Cardiac inotropic effect of IL-6 after chronic exposure
The present study showed that IL-6 acts chronically in ARVM to reduce (+dL/dt)max, an effect that could result from a decrease in Ca2+ influx, Ca2+ release from SR or/and myofilament Ca2+ sensitivity. We showed that the suppression of contractile responsiveness to Ca2+o after 2 h pretreatment with IL 6 is probably not due to reductions in Ca2+ influx via ICa,L. In addition, our indirect evidence showing no change in the EC50 of responsiveness for Ca2+o suggests that IL-6 does not alter the Ca2+ sensitivity of myofilaments. Thus, these results suggest that IL-6 suppresses SR function. We also showed that IL-6 reduces (–dL/dt)max; this could result primarily from decreasing Ca2+ uptake via the SR Ca2+ pump, though sarcolemmal Na+–Ca2+ exchange also contributes to relaxation (Bassani et al. 1994). Our data showing IL-6-induced reductions in PRP and caffeine responsiveness in combination with findings suggesting no significant SR leak support the hypothesis that IL-6 suppresses SR Ca2+ uptake. Moreover, the phosphorylation of PLB is reduced by IL-6 treatment, which is opposite to the effect of ISO. This further supports our hypothesis. Whether IL-6 also suppresses Ca2+ release via SR Ca2+ channels and Ca2+ extrusion via sarcolemmal Na+–Ca2+ exchangers require further investigations.
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Role of iNOS activation in IL-6-elicited cardiac effect
Studies in chick embryonic cardiomyocytes suggested that different isoforms of NOS are involved in the acute (<10 min) and chronic (24 h) inotropic effects of IL-6 (Kinugawa et al. 1994). However, the identification of specific NOS isoforms has not been explored. We have previously shown that IL-6 induces iNOS protein expression 2 h after exposure, and that this action can be blocked by a JAK2 inhibitor (Yu et al. 2003). The present study showed that IL-6 increases NO production after treatments of 2 h (Fig. 7), supporting our previous findings of the increased iNOS protein expression. Inhibition of this increase in NO production by AMT (Fig. 8) is consistent with the involvement of iNOS in the chronic cardiac effects of IL-6. These results suggest that iNOS activation is a downstream event of the JAK2/STAT3 signalling of IL-6.
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Studies of protein or/and mRNA expression of constitutive NOS in rat and mouse hearts showed that endothelial NOS (eNOS) is the only isoform expressed in normal adult ventricular myocytes (Balligand et al. 1995; Vandecasteele et al. 1999). Meanwhile, neural NOS (nNOS) was found on SR membrane vesicles prepared from rabbit and mouse hearts (Xu et al. 1999) and cardiac mitochondria (Kanai et al. 2001). Studies using genetically engineered mice showed that nNOS and eNOS independently modulate basal cardiac contractile function in opposite fashion (Barouch et al. 2002; Khan et al. 2003). However, the inotropic role of constitutive NOS remains controversial (Massion & Balligand, 2003), probably because of concern regarding adaptive changes of genetically engineered mice during development and/or low physiological activities (French et al. 2001). Our results showing no significant effect of NOS inhibitors on the basal NO level (Fig. 8) suggest that constitutive NOS activity is low and/or the change in NO is beyond the detection limit. By contrast, we showed time-dependent increases in NO production induced by chronic exposure to IL-6, which was blocked by AMT, in accord with our previous findings of IL-6-induced iNOS expression (Yu et al. 2003). Moreover, it is well known that iNOS expression is not detectable under physiological conditions (Gallo et al. 1998), and the level of NO produced via iNOS is greater than that elicited by constitutive NOS after stimulation by cytokines.
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It is also well known that the cardiac effects of NO are complex and somewhat controversial, probably due to the distinct concentration-dependent sensitivities of its targets and distinguishable signalling mechanisms. NO is known to target the L-type Ca2+ channel and SR in cardiac myocytes through signalling pathways such as cGMP, and/or by directly modifying the proteins (such as by S-nitrosylation/oxidation) (Campbell et al. 1996; Hare, 2003; Massion et al. 2003). Studies in adult ferret ventricular myocytes showed that SIN-1, a NO donor, induces biphasic and bimodal changes in basal ICa,L (Campbell et al. 1996), which were not observed in frog (Mery et al. 1993), guinea-pig (Wahler & Dollinger, 1995) or rat (Abi-Gerges et al. 2001) ventricular myocytes. We also observed no significant change in basal ICa,L in ARVM upon acute exposure to 0.1 mM SIN-1 (S. J. Liu, unpublished data). Similarly, SIN-1 (up to 0.1 mM) has no significant effect on contractile function of papillary muscle isolated from adult rat (Wyeth et al. 1996). Although we found that cumulative NO production in cell lysates and in culture medium is 2.5- to 3-fold of the basal level after 2 h of IL-6 treatment, it is not surprising that ICa,L is not significantly altered in our experimental conditions. However, the possibility for alteration in ICa,L during longer exposure (e.g. >24 h) to IL-6 cannot be excluded.
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For the aforementioned reasons, reported effects of NO on SR function, including ryanodine-sensitive Ca2+ release channel and SR Ca2+ pump, are also diverse (Hare, 2003; Massion et al. 2003). For example, NO inhibits SR Ca2+ ATPase (Ishii et al. 1998) and the ryanodine-sensitive channel (Meszáros et al. 1996) in skeletal muscle, whereas at low concentration it has no effect on PRP in rat papillary muscle (Prabhu et al. 1999). At high concentrations NO increases Ca2+ leak from SR in skeletal muscle (Pouvreau et al. 2004) and cardiac ryanodine-sensitive channel (Xu et al. 1998). Although free cytosolic Ca2+ was not measured in the present study, the lack of significant changes in the diastole state during rest intervals in IL-6-treated ARVM (Fig. 6A) suggests that the Ca2+ leak in the presence of increased NO was insignificant. Moreover, our data showing reductions in maximum shortening and relaxation rates, PRP and caffeine-responsiveness, and PLB phosphorylation induced by IL-6, which were abolished by NOS inhibitors (Fig. 9) support the possibility that NO reduces SR function by acting on the SR Ca2+ pump and/or via PLB. Although local NO produced by different NOS isoforms has been suggested to contribute differently to changes in the function of each target (Barouch et al. 2002; Khan et al. 2003), the larger amounts of NO produced by iNOS are probably more diffuse rather than localized. Our data suggest that such increased NO after 2 h exposure to IL-6 has a profound effect on SR with little detectable action on the L-type Ca2+ channel in ARVM. Somewhat in contrast, nNOS has been suggested to stimulate SR Ca2+ release (Barouch et al. 2002; Khan et al. 2003). It is also possible that a large increase in NO that occurs following iNOS activation elicits an inhibitory action that decreases the stimulatory effect of nNOS on SR.
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Our data also showed that IL-6 reduces PLB phosphorylation at Ser16, where is primarily phosphorylated by cAMP-dependent protein kinase A (PKA) (Kuschel et al. 1999). This IL-6 effect is also NO-dependent because the reduction of PLB phosphorylation is inhibited by L-NMMA (Fig. 9D). A study in ARVM using SIN-1 showed that NO reduces PLB phosphorylation and cAMP production induced by ISO in a cGMP-independent manner (Stojanovic et al. 2001). This NO-induced decrease in PLB phosphorylation is supported by a recent study showing that SNAP, an NO donor, inhibits basal and forskolin-stimulated cAMP production by blocking adenylyl cyclase, effects independent of guanylyl cyclase activity (Ostrom et al. 2004). Thus, it is likely that the IL-6-induced reduction in PLB phosphorylation observed in the present study results from NO-elicited inhibition of basal cAMP/PKA activities. Another possibility is that NO activates protein phosphatase type 2A (PP2A), thereby enhancing dephosphorylation of PLB, as suggested by a study in neonatal cardiomyocytes which showed tumour necrosis factor (TNF)-, known to activate iNOS, decreases PLB phosphorylation by activating PP2A (Yokoyama et al. 1999). Taken together, the concentration-dependent sensitivities of target proteins associated with E–C coupling (such as SR and L-type Ca2+ channels) to NO could be distinct and determine the ultimate effect on contractile function under physiological and pathological conditions. Cellular mechanisms for NO-associated alterations in these target proteins have been postulated to be mediated via cGMP-dependent or/and cGMP-independent (including direct S-nitrosylation of target proteins) mechanisms. Whether IL-6-induced alterations in SR function result solely from NO-dependent mechanism is still unknown and requires further investigation.
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In summary, we have shown that exposure to IL-6 for two or more hours results in a negative inotropy probably by inhibiting SR Ca2+ reuptake in concomitance with increases in NO in ARVM. The IL-6-induced decrease in PLB phosphorylation is also in accord with its reduction of SR function. The increase in NO production results primarily from expression/activation of iNOS that is mediated via IL-6-induced JAK2/STAT3 activation as shown previously (Yu et al. 2003). Inhibition of iNOS/NO abolishes the IL-6-induced decrease in SR function. Therefore, this leads to our conclusion that IL-6 suppresses SR function via iNOS after chronic exposure. Moreover, such cardiac effects of IL-6 are sustained even after removal of IL-6. This suggests that the transient elevation of IL-6 observed in plasma or ventricular muscle of patients with many cardiac diseases can cause prolonged effects on contractility. An in vivo study in transgenic mice showed that cardiac-specific overexpression of iNOS causes an increase in NOx in hearts (2.5-fold above the wild-type controls) but no severe cardiac dysfunction (Heger et al. 2002). One interpretation for this seeming inconsistency is that the increase in plasma and heart NOx in the transgenic models is moderate so the associated systemic adaptation during development in these transgenic mice results in less detrimental functional consequences. A recent in vivo study reported that IL-6 administration causes heart failure in a dose-dependent manner (Janssen et al. 2005). Thus, IL-6 might play an important role in the pathogenesis of associated chronic heart diseases. Treatment to antagonize the induction and activation of iNOS in the early stage of IL-6-associated heart dysfunction might be beneficial.
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Yu X-W, Kennedy RH & Liu SJ (2003). JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes. J Biol Chem 278, 16304–16308., 百拇医药(Xin-Wen Yu, Qian Chen, Ri)
2 Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA
3 Department of Physiology, Loyola University Medical Center, Stritch School of Medicine, Maywood, IL 60153, USA
Abstract
Interleukin (IL)-6 has been shown to decrease cardiac contractility via a nitric oxide synthase (NOS)-dependent pathway during acute exposure. We previously reported that IL-6 decreases contractility and increases inducible NOS (iNOS) in adult rat ventricular myocytes (ARVM) after 2 h exposure. The goal of this study was to investigate the cellular mechanism underlying this chronic IL-6-induced negative inotropy and the role of iNOS. Pretreatment for 2 h with 10 ng ml–1 IL-6 decreased the kinetics of cell shortening (CS) and contractile responsiveness to Ca2+o ([Ca2+]o from 0 to 2 mM) in ARVM. We first examined whether IL-6 reduced Ca2+ influx via L-type Ca2+-channel current (ICa,L). Whole-cell ICa,L in ARVM was measured under conditions similar to those used for CS measurements, and it was found to be unaltered by IL-6. The sarcoplasmic reticular (SR) function was then assessed by examining postrest potentiation (PRP) and caffeine responsiveness of CS. Results showed that treatment with IL-6 for 2 h significantly decreased PRP, which was concomitant with a decrease in the phosphorylation of phospholamban. Following removal of IL-6, PRP and responsiveness to 10 mM caffeine were also reduced. Meanwhile, the IL-6-induced increase in nitric oxide (NO) production after 2 h (but not 1 h) was abolished by NG-monomethyl-L-arginine (L-NMMA) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective inhibitor of iNOS). Furthermore, IL-6-elicited suppressions of PRP and responsiveness to caffeine and Ca2+o were abolished by L-NMMA and AMT. Thus, these results suggest that activation of iNOS mediates IL-6-induced inhibition of SR function in ARVM during chronic exposure.
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Introduction
Interleukin (IL)-6, a pro-inflammatory cytokine, is produced during the acute phase of the immune response by immune and nonimmune cells (for review, see Akira et al. 1990). After binding to its receptor, IL-6 elicits numerous biological effects, including antibody induction, haematopoiesis, thrombocytopoiesis and acute-phase protein synthesis (for reviews, see Akira et al. 1990; Kishimoto et al. 1995). Recently, significant increases in serum levels of IL-6 and its mRNA expression and protein production in cardiac tissues have been reported in patients with chronic cardiac failure (Satoh et al. 1996; Steele et al. 1996; Roig et al. 1998; Cesari et al. 2003), myocarditis (Satoh et al. 1996), myocardial infarction (Ikeda et al. 1992; Guillen et al. 1995; Neumann et al. 1995), endotoxaemia (Hack et al. 1989), and the injury associated with ischaemia/reperfusion (Sawa et al. 1998) and cardiopulmonary bypass (Wan et al. 1996). In patients undergoing cardiopulmonary bypass, the increase in plasma IL-6 levels in the coronary sinus begins 5 min after aortic declamping, peaks at 1 h, and is sustained for at least 2 h (Wan et al. 1996). Therefore, IL-6 has been associated with the severity of several cardiovascular pathophysiological states (Cesari et al. 2003).
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In vitro studies on cardiac contractile function have shown that exposure to IL-6 for 2–3 min decreased contractility in papillary muscle isolated from hamster heart (Finkel et al. 1992). Studies with single myocytes also showed that IL-6 suppressed peak systolic [Ca2+]i and cell shortening (CS) within 5 min in chick embryonic cardiac myocytes (Kinugawa et al. 1994) and in adult guinea-pig ventricular myocytes (Sugishita et al. 1999). This acute negative inotropic effect of IL-6, accompanied by an increase in cell cGMP production (Kinugawa et al. 1994), was blocked by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase (NOS) (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999). Thus, acute IL-6-induced suppression of cardiac contractility and [Ca2+]i have been suggested to be mediated by a NO-dependent pathway via activation of NOS (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999), probably a constitutive endothelial isoform (eNOS) (Kinugawa et al. 1994). In contrast, studies on chronic cardiac effects of IL-6 showed that after 24 h incubation, IL-6 also increased cell cGMP, which was blocked by L-NMMA but not by EGTA, suggesting the involvement of a Ca2+-independent NOS (Kinugawa et al. 1994). However, the subtype of NOS that is involved in chronic cardiac effect of IL-6 has not been defined.
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As mentioned above, an in vivo study showed that elevated IL-6 levels were observed 1–2 h after aortic declamping in cardiopulmonary bypass (Wan et al. 1996). The direct cardiac effect of IL-6 during this period of time remains undefined. We recently reported that de novo synthesis and activation of iNOS induced by IL-6 can be detected in adult rat ventricular myocytes (ARVM) as early as 2 h after exposure (Yu et al. 2003). This earlier study also demonstrated that the IL-6-elicited iNOS activation and decrease in postrest potentiation (PRP) of contraction in ARVM are mediated by activation of Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3, the upstream mediators of IL-6 signalling (Yu et al. 2003). However, whether iNOS is the downstream mediator of IL-6-induced negative inotropy remains undefined.
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In the present study, we examined the effect of chronic exposure (for 2–24 h) of ARVM to IL-6 on contractility, contractile responsiveness to Ca2+o, L-type Ca2+-channel current (ICa,L), and sarcoplasmic reticular (SR) function. SR function was assessed using two protocols: PRP and caffeine-induced contraction (Bassani et al. 1993, 1994; Bers et al. 1998). We also examined the role of iNOS in IL-6-induced changes in SR function. We found that IL-6 decreased contractility and Ca2+o responsiveness of ARVM, primarily resulting from suppression of SR function accompanied by reduction in phosphorylation of phospholamban (PLB), a SR Ca2+-pump regulatory protein. This chronic cardiac effect of IL-6 was sustained after removal of IL-6. We also showed that inhibition of iNOS and NO production blocked the IL-6 negative inotropic action. A preliminary report of some of these results has been presented in an abstract (Yu et al. 2000).
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Methods
Ventricular myocyte preparation
Single ventricular myocytes were isolated from hearts of adult male Sprague-Dawley rats (250–300 g) using enzymatic dissociation as previously described (Liu & Schreur, 1995). Briefly, rats were deeply anaesthetized with ether or isoflurane (Vedco, St Joseph, MO, USA) followed by a thoracotomy. Hearts were rapidly excised and perfused at 37°C via the aorta with a control buffer solution, followed by enzymic and mechanical dissociation. Isolated ARVM were resuspended in culture medium containing antibiotic-free, bicarbonate-buffered culture medium 199 (60%; Gibco, Grand Island, NY, USA) with 36% Earle's balanced salt solution composed of (mM): 116 NaCl, 4.7 KCl, 0.9 NaH2PO4, 0.8 MgSO4, 26 NaHCO3, and 5.6 glucose and 4% fetal bovine serum (Gibco) (pH 7.40 in 5% CO2/95% air at 37°C). After incubation for 3–4 h to allow recovery, ARVM were plated in culture dishes containing serum-free L-arginine-containing medium, incubated overnight, and treated with designated concentrations (1–30 ng ml–1) of IL-6 for various periods of time (1–24 h). After the designated exposure duration, quiescent rod-shaped ARVM with clear striations were used for electrophysiological and CS measurements. All experiments were performed at 37°C. The use of animals was carried out under a protocol approved by the Animal Care and Use Committee at the University of Arkansas for Medical Sciences.
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Measurement of ICa,L
ICa,L of ARVM was measured in normal Tyrode solution, or Na+- and K+-free solutions with 2 mM Ca2+, using conventional whole-cell patch techniques (Hamill et al. 1981) with a patch-clamp amplifier (Axopatch 200 A, Axon Instruments, Union City, CA, USA) as previously described (Liu et al. 1997). Briefly, patch electrodes were filled with pipette solution and had a tip resistance of 1–3 M. The pipette solution consisted of (mM): 100 CsOH, 70 aspartate, 11 CsCl, 15 tetraethylammonium chloride, 2 MgC12, 5 MgATP, 5 EGTA, 0.1 CaC12, 5 pyruvic acid, 5.6 glucose, 5 Tris2-phosphocreatine, 0.4 Li4-GTP, 10 Hepes/Tris (pH adjusted to 7.20 with CsOH). The bath solution contained (mM): 145 N-methyl-D-glucamine chloride, 0.8 MgC12, 2 CaC12, 2 4-aminopyridine, 10 Hepes/Tris (pH adjusted to 7.40 with CsOH). ICa,L was elicited by 250 ms voltage pulses to potentials between –60 and +60 mV from a holding potential of –80 mV in 10 mV increments at 0.2 Hz. In some experiments, ICa,L was elicited by 25 ms voltage pulses and measured in normal Tyrode solution when myocytes were voltage clamped at –40 mV before switching to the Na+- and K+-free bath solution. The magnitude of ICa,L was determined by the difference between the holding current and the peak amplitude in response to 25 ms pulses, or by the difference between the peak amplitude and the level at the end of 250 ms pulses. Series resistance was 3–6 M and electronically compensated (90%), while the recorded currents were filtered at 1–2 kHz and sampled at 5 kHz using PClamp 8.0 software (Axon Instruments). The density of ICa,L in each myocyte was normalized to membrane capacitance (Cm), which was calculated from uncompensated capacity current transients recorded in response to 5 mV hyperpolarizing pulses from the holding potential.
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Contractile function or CS
Unloaded CS of ARVM was elicited in normal Tyrode solution and measured as previously described (Liu et al. 2002). Briefly, CS was elicited by field stimulation (2 ms duration, 1.5-fold threshold voltage) at 0.5 Hz in normal Tyrode solution containing (mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 0.8 MgCl2, 10 Hepes/Tris and 5.6 glucose (pH 7.40 at 37°C). CS was monitored with a video edge-motion detector system (Crescent Electronics, Sandy, UT, USA). The voltage signal was calibrated to determine actual motion (micrometres). Data of CS were not expressed as percentages of original cell length because the degree of CS is significantly affected by the degree of cell attachment to the dish, which varies from cell to cell, and from dish to dish.
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The measured parameters of contractile function in single ARVM included the peak magnitude of CS and maximum rates of contraction ((+dL/dt)max) and relaxation ((–dL/dt)max). The responsiveness of CS to Ca2+o was assessed by monitoring the change in CS in response to various [Ca2+]o (0–2 mM). The magnitude of CS in different [Ca2+]o relative to that observed in 0.5 mM [Ca2+]o was plotted as a function of [Ca2+]o. The SR function of ARVM was estimated by measurements of PRP and by caffeine-induced CS (Bassani et al. 1993; Bers et al. 1998). PRP was measured as the first contraction after a rest interval between 5 and 120 s, and the amplitude of PRP relative to that of the steady-state CS before the rest was plotted as a function of rest interval. Similarly, 10 mM caffeine was applied for 10 s following a rest interval between 15 and 60 s in the absence of electrical stimulation. The amplitude of caffeine-induced CS was normalized to that of the steady-state CS before electrical cessation.
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NO production
NO production was estimated by measuring total production of nitrate and nitrite (NOx) in culture medium and cell lysate, as previously described (Yu et al. 2003). Briefly, after treatment with 10 ng ml–1 IL-6 for 2, 4 and 24 h, cells were collected after centrifugation at 100 g for 10 min. The supernatant (culture media) was removed and kept at –20°C until the NOx assay. Cell pellets were resuspended in phosphate-buffered saline (PBS), half of which was used to determine protein content (see below). The other half of the cell suspension was sonicated and centrifuged at 10 000 g, and the supernatant was centrifuged at 100 000 g for 15 min at 4°C. The final supernatant was filtered via a 10 kDa molecular weight cut-off filter at 12 000 g for 30 min, and kept at –20°C until the NOx assay. The concentration of NOx in culture media and cell lysate was determined using a colourimetric assay kit (Cayman Co., Ann Arbor, MI, USA) according to the manufacturer's instructions. The total NOx concentration was calculated according to a standard curve of sodium nitrate and expressed in nanomoles per milligram of cell protein. Results of treated groups were normalized to each time-control.
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Western blot analysis
Preparation of cell lysate of ventricular myocytes and Western blot analyses were carried out as previously described (Liu & McHowat, 1998). Briefly, control and treated myocytes (0.5 x 106 cells per group) were lysed in 300 μl of ice-cold lysis buffer containing (mM): 20 Hepes (pH 7.6), 250 sucrose, 2 dithiothreitol, 2 EDTA, 2 EGTA, 10 sodium orthovanadate, 2 phenylmethylsulphonyl fluoride, 20 μg ml–1 leupeptin and 10 μg ml–1 aprotinin. The concentration of total protein was determined using a Bradford assay (Bio-Rad, Hercules, CA, USA). Forty micrograms of each protein sample was then electrophoretically separated on 8% SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes (Bio-Rad). Non-specific binding sites of membranes were blocked by incubation in Tris buffer solution containing 0.05% Tween-20 and 5% nonfat milk for 1 h at room temperature. The membrane was then probed with polyclonal anti-phospho-PLB antibodies (1:1000, Cayman Co.) overnight at 4°C, followed by treatment with horseradish-peroxidase-conjugated secondary antibodies. Immunoblots were detected using enhanced chemiluminescent kits (SuperSignal; Pierce, Rockford, IL, USA) and analysed with a densitometer (Bio-Rad). After being stripped, the membranes were reprobed with monoclonal anti-PLB antibodies (1:2000 dilution; Upstate Biotechnology, NY, USA). The phosphorylation level of PLB was presented as the relative density of phospho-PLB to total PLB protein in each sample.
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Chemicals
Recombinant rat IL-6 was purchased from Pepro Tech Inc. (Rocky Hill, NJ, USA); the nitrate/nitrite colourimetric assay kit was obtained from Cayman Co. L-NMMA and lipopolysaccharide (LPS) were purchased from Sigma (St Louis, MO, USA). 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) was purchased from Alexis (San Diego, CA, USA).
Statistics
Data of ICa,L and CS were collected from ARVM isolated from 3–5 hearts. The data for NOx production were averaged from duplicate or triplicate measurements taken from 4–6 hearts. Values are presented as means ± S.E.M. Statistical significance (P < 0.05) was evaluated by Student's t test, or one-way ANOVA with Duncan's multiple range test when more than two conditions were compared.
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Results
Effect of chronic exposure to IL-6 on contractility and contractile responsiveness to Ca2+o
After treatment with 10 ng ml–1 IL-6 for 2 or 24 h, myocytes were removed from the incubator, placed on the microscope stage and continuously superfused with normal Tyrode solution in the presence (treatment) or absence (pretreatment) of the same concentration of IL-6. The basal CS of ARVM was elicited at 0.5 Hz and remained stable for at least 1 h in both conditions. When ARVM were superfused with IL-6-containing solution, the kinetics of steady-state CS of IL-6-treated ARVM, i.e. (+dL/dt)max and (–dL/dt)max, were found to be decreased by 45 and 35%, respectively, when compared with time-controls (Table 1). Interestingly, when myocytes were superfused in the absence of IL-6 (pretreated group) following IL-6 incubation, the (+dL/dt)max and (–dL/dt)max of steady-state CS were still decreased by 37 and 28%, respectively (Table 1). Thus, the results suggest that the contractile function of IL-6-pretreated ARVM remains suppressed at least 1 h after removal of IL-6.
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The inotropic response of CS to Ca2+o was then examined in IL-6-pretreated (2 h) and time-control ARVM by monitoring CS in Tyrode solution containing various [Ca2+]o in the absence of IL-6. After contraction reached a steady state at 1 mM [Ca2+]o (in normal Tyrode solution), the solution was randomly switched to solutions containing [Ca2+]o between 0 and 2 mM. Figure 1A shows representative traces of steady-state CS from a time-control and an IL-6-pretreated (2 h) ARVM in 1, 1.5 and 2 mM [Ca2+]o solutions. When [Ca2+]o was increased, the enhanced amplitude of CS in IL-6-pretreated ARVM appeared to be smaller than that in the time-control. When [Ca2+]o was reduced to 0 mM, CS was terminated in both groups. Because ARVM started to contract when [Ca2+]o was resumed to 0.5 mM, Ca2+o responsiveness of CS in ARVM was presented as relative amplitude (normalized to that in 0.5 mM [Ca2+]o) as a function of [Ca2+]o. Results are shown in Fig. 1B in which CS responsiveness to Ca2+o in IL-6-pretreated ARVM was significantly decreased when compared with time-controls. In contrast to the decreased maximal Ca2+o responsiveness, IL-6 had no significant effect on the EC50 value for [Ca2+]o (1.2 mM) when data were curve-fit with a Hill equation using a Hill coefficient of 2. These data suggest that the IL-6-induced decrease in responsiveness to Ca2+o could result from a decrease in Ca2+ influx via ICa,L and/or SR Ca2+ release and uptake.
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Myocytes in the time-control and IL-6-pretreated groups were exposed to various [Ca2+]o (0–2 mM). A, representative traces of steady-state cell shortening (CS) in response to [Ca2+]o between 1 and 2 mM were obtained from a time-control cell and an IL-6-pretreated ARVM (10 ng ml–1 for 2 h). B, the magnitude of CS in each [Ca2+]o relative to that in 0.5 mM [Ca2+]o was plotted as a function of [Ca2+]o in time-control and IL-6-pretreated ARVM. Data represent means ± S.E.M. *P < 0.05, compared with the control group (ANOVA). Continuous lines are curves fit by the Hill equation using a Hill coefficient of 2.
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Effect of chronic exposure to IL-6 on ICa,L
We then examined whether IL-6-induced negative inotropy and Ca2+o responsiveness result from a decrease in ICa,L. After incubation for 2 h with 10 ng ml–1 IL-6, ICa,L was first measured in normal Tyrode solution (Fig. 2A) when cells were voltage clamped at –40 mV and K+ currents were largely diminished. After construction of the current–voltage (I–V) relationship of ICa,L (Fig. 2C), single 250 ms voltage pulses to +10 mV from the holding potential at 1 Hz were used to monitor ICa,L while the external solution was switched to Na+- and K+-free solution containing 2 mM [Ca2+]o. After ICa,L reached a new steady state, the holding potential was changed to –80 mV (Fig. 2B), and the I–V relationship of peak ICa,L was generated using the pulse protocols illustrated in the inset of Fig. 2D. Results show that the maximum current density and voltage dependency of peak ICa,L measured in both solutions were not significantly altered after 2 h pretreatment with IL-6. The maximum current density of ICa,L measured at +10 mV in the Na+- and K+-free solution was –17.3 ± 1.4 pA pF–1 (n = 10) in time-controls and –15.5 ± 0.7 pA pF–1 (n = 19) in IL-6-pretreated ARVM. Thus, these data suggest that the IL-6-induced negative inotropy does not result from a decrease in ICa,L, thereby leading to the hypothesis that IL-6 suppresses SR function.
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A and B, peak L-type Ca2+-channel current (ICa,L) in a control (left) and IL-6-pretreated (10 ng ml–1 for 2 h, right) ARVM in response to a voltage pulse to +10 mV were recorded in normal Tyrode solution and in Na+- and K+-free bath solution containing 2 mM Ca2+, respectively. The current–voltage (I–V) relationships of peak ICa,L in time-control and IL-6 pretreated ARVM were constructed using voltage-pulse protocols from a holding potential (Vh) of –40 mV in normal Tyrode solution (C) or –80 mV in Na+- and K+-free solution containing 2 mM Ca2+ (D). Membrane capacitance (Cm): A, 158 pF; and B, 207 pF. Dashed lines in A and B represent the zero current level. The positive capacitance transient in each trace of A and B was truncated. Data in C and D represent means ± S.E.M.
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Effect of chronic exposure to IL-6 on PRP
The effect of IL-6 on PRP of CS in ARVM was first examined during continuous perfusion with Tyrode solution containing 10 ng ml–1 IL-6 after 2 h incubation with IL-6 in culture medium. Figure 3A shows representative traces of PRP following a 60 s rest interval (PRP60) in a time-control (left panel) and an IL-6-treated ARVM (right panel). The IL-6-treated myocyte displayed a smaller PRP than the time-control. Figure 3B shows combined data from 10 experiments, which demonstrate that the PRP (measured following rest intervals of 30 s) in IL-6-treated ARVM was significantly decreased (by 25%) after 2 h incubation in the continued presence of IL 6.
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PRP was measured as the amplitude of the first contraction following different rest-intervals (5–120 s) relative to that of the prerest steady-state CS. A, representative traces of steady-state CS and PRP following a 60 s rest interval in a time-control ARVM (left) and an IL-6-treated myocyte (right, 10 ng ml–1 for 2 h); the latter was superfused with Tyrode solution containing 10 ng ml–1 IL-6. Combined data of PRP in IL-6-treated (B, measured in continuous presence of IL-6) or IL-6-pretreated (C, measured after removal of IL-6) ARVM were compared with time-controls. Data represent means ± S.E.M., n = 7–23. *P < 0.05, compared with the time-control (ANOVA).
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We then examined whether the inhibition of PRP induced by chronic exposure to IL 6 could be reversed after removal of IL 6. Measurements of PRP in IL-6-pretreated (10 ng ml–1 for 2 h) ARVM were completed within 30 min after beginning perfusion with normal Tyrode solution. PRPs of CS in IL-6-pretreated ARVM were decreased to almost the same degree as observed in continuously IL-6-treated ARVM (Fig. 3C versus B). Such reductions in PRP induced by pretreatment with IL-6 were both concentration and time dependent. For example, pretreatment with 10 and 30 ng ml–1 IL-6 caused 15 ± 5 and 39 ± 6% decreases in PRP after a 30 s rest interval (PRP30), respectively (n = 23, P < 0.05). Lower concentrations (e.g. 1 and 3 ng ml–1) had no significant effect. In addition, the IL-6-induced suppression of PRP30 was 15 ± 5% (n = 19, P < 0.05), 21 ± 7% (n = 23, P < 0.05) and 27 ± 4% (n = 10, P < 0.05) after pretreatment with 10 ng ml–1 IL-6 for 2, 4 and 24 h, respectively. Since the IL-6-induced inhibition of CS and PRPs was sustained at least 1 h after removal of IL-6, the results of CS measurements presented in the following sections were obtained from IL-6-pretreated ARVM.
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Furthermore, to confirm that inhibition of PRP after IL-6 pretreatment was caused specifically by IL-6, anti-IL-6 antibodies (100 ng ml–1, 10-fold of IL-6 concentration) were added to the culture medium 15–30 min prior to and during 2 h treatment with IL-6. Only the PRP30 and PRP60 of CS were examined under these conditions. Figure 4 shows that anti-IL-6 antibodies completely blocked the IL-6-induced inhibition of PRP, while alone they had no effect. Taken together, these results indicate that IL-6 reduces contractility of ARVM by suppressing SR function, not Ca2+ influx via ICa,L.
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ARVM were incubated with 100 ng ml–1 anti-IL-6 antibodies 15–30 min before and during 2 h treatment with 10 ng ml–1 IL-6. Anti-IL-6 antibodies prevented the decrease in PRP (after 30 or 60 s rest) induced by IL-6, while alone they had no effect. Data represent means ± S.E.M., n = 6–7. *P < 0.05 compared with the time-control.
Effect of chronic exposure to IL-6 on contractile responsiveness to caffeine
After stable CS of myocytes was obtained in the control solution, electrical stimulation was terminated for 15–60 s to reload the SR Ca2+. ARVM were then exposed for 10 s to 10 mM caffeine to deplete SR Ca2+ content before resuming electrical stimulation. Figure 5A shows representative traces of responsiveness of CS to caffeine following a 15 s rest interval in a 2 h time-control (left panel) and an IL-6-pretreated ARVM (right panel). The amplitude of caffeine-induced CS in IL-6-pretreated ARVM was smaller than that in the time-control, suggesting less available SR Ca2+. After being normalized to the amplitude of steady-state CS, the relative amplitude of caffeine-induced CS in each group was plotted as a function of the rest interval. Figure 5B and C shows that the relative amplitude of caffeine-induced CS in time-controls (from at least four experiments) was slightly increased when the rest interval was prolonged from 15 to 60 s, suggesting no significant SR Ca2+ leak after Ca2+ reloading during the rest period in ARVM. The combined data in Fig. 5B also show that the relative amplitude of caffeine-induced CS was significantly decreased and lost the time-dependent Ca2+ reload after 2 h pretreatment with 10 ng ml–1 IL-6. After 24 h pretreatment, a similar time-dependent SR Ca2+ loading was observed in time-controls, and the decrease in the caffeine-induced CS appeared to increase (Fig. 5C). These results suggest that pretreatment with IL-6 attenuated SR Ca2+ content, probably by decreasing Ca2+ uptake via the SR Ca2+ pump. This is consistent with the IL-6-induced decrease in PRP.
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Caffeine (10 mM), depleting SR Ca2+ store, was used to assess available SR Ca2+ content in ARVM. A, representative traces of caffeine-induced contraction following a 15 s rest interval in a time-control (left) and an IL-6-preteated ARVM (10 ng ml–1 for 2 h, right). The relative amplitude of caffeine-induced CS to that of the steady-state CS following a rest interval was plotted as a function of the rest interval (15–60 s) in time-controls and ARVM pretreated with 10 ng ml–1 IL-6 for 2 h (B) and 24 h (C). Data represent means ± S.E.M., n = 4–10. *P < 0.05, compared with the time-control (ANOVA).
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Effect of chronic exposure to IL-6 on phosphorylation of PLB
The dephosphorylation of PLB decreases the affinity of the SR Ca2+ pump (SERCA2a) for Ca2+, and phosphorylation of PLB relieves this inhibitory effect (Koss & Kranias, 1996). Thus, the phosphorylation level of PLB in ARVM was examined after exposure to 10 ng ml–1 IL-6. Figure 6A shows representative immunoblots from time-control and IL-6-treated ARVM detected with anti-phospho-PLB (upper panel) and anti-PLB antibodies (lower panel). When cell lysates were treated at 37°C for 5 min before electrophoresis, both pentameric (26 kDa) and monomeric (6 kDa) forms of PLB were detectable in time-controls, in which the abundance and phosphorylation of pentamers were predominant. Treatment of ARVM for 5 min with 10 nM isoproterenol (ISO), serving as a positive control, resulted in a marked increase in the phosphorylation of both pentamers and monomers of PLB (right lanes in Fig. 6A). By contrast, IL-6 significantly decreased the phosphorylation of PLB pentamers to 63 ± 16 and 74 ± 12% of time-control levels after 2 and 24 h, respectively, in four experiments (Fig. 6B, P < 0.05). The phosphorylation level of the PLB monomers was 17 ± 2 and 38 ± 14% of the time-controls after 2 and 24 h exposure to IL-6, respectively (Fig. 6C, n = 3, P < 0.05). When cell lysates were treated for 5 min at 95°C, the low molecular mass of PLB was the only form detected. However, under this condition, the change in phosphorylation of PLB induced by IL-6 was less consistent or evident than that observed in oligomers. Nevertheless, these data support the suggestion that IL-6 suppresses SR function by decreasing the Ca2+ uptake via SR Ca2+ pumps.
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Protein samples (40 μg) extracted from time-controls (C) and IL-6-treated ARVM (I) were loaded in each lane. A, immunoblots of phosphorylated (at Ser16, upper panel) and total (lower panel) PLB in time-control and IL-6-treated ARVM. A 5 μg protein extract from ARVM was treated with 10 nM isoproterenol (ISO) for 5 min and used as a positive control. The phosphorylation level (p-PLB/PLB) of pentamers (B, n = 4) and monomers (C, n = 3) of PLB in IL-6-treated ARVM was normalized to that of each time-control. Data represent means ± S.E.M. *P < 0.05, compared with each time-control.
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IL-6-elicited induction of NO production via activation of iNOS
We previously showed IL-6-induced iNOS protein expression and NOx production after 2 h exposure (Yu et al. 2003). In this study, we examined whether NOx production results specifically from IL-6-elicited activation of iNOS. Figure 7A shows that total NOx concentrations in culture media were not significantly altered at 1 h (1.08 ± 0.04-fold of the control level, n = 5, P > 0.05) but increased to 2.31-fold of the time-control level (n = 14, P < 0.05) after 2 h incubation with 10 ng ml–1 IL-6. Similarly, the NOx concentrations in the culture media after 4 and 24 h incubation with IL-6 were 2.72- (n = 9, P < 0.05) and 2.91-fold (n = 7, P < 0.05) of the time-control level, respectively. Note that there was no significant difference in the concentrations of NOx in the culture media of 2, 4 and 24 h time-controls (i.e. in the absence of IL-6); values were 1.55 ± 0.23 (n = 14), 1.16 ± 0.15 (n = 9), 1.47 ± 0.17 (n = 7) nmol (mg protein)–1, respectively. Incubation with 10 μg ml–1 LPS, a positive control (Brady et al. 1992), also increased the concentration of NOx in the culture media (Fig. 7A). Figure 7B shows that the concentrations of NOx in cell lysates of IL-6-treated ARVM (for 2, 4 and 24 h) were also increased significantly (e.g. 3.19-, 4.98- and 6.26-fold of each time-control level, P < 0.05). The concentrations of NOx in cell lysates of 2, 4 and 24 h time-controls were 1.67 ± 0.28 (n = 8), 0.92 ± 0.16 (n = 7), and 1.10 ± 0.23 nmol (mg protein)–1 (n = 7), respectively.
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Total concentrations of nitrite and nitrate (NOx) in culture media (A) and cell lysate (B) were measured in IL-6-treated (10 ng ml–1 for 2, 4 and 24 h, open bars) samples and normalized to those of each time-control (dashed lines). Lipopolysaccharide (LPS) (10 μg ml–1, filled bars) served as a positive control. Data represent means ± S.E.M.; numbers in parentheses are numbers of experiments. *P < 0.05, compared with the time-controls.
To determine the involvement of iNOS, we examined the effect of AMT (a specific and potent iNOS inhibitor) on the IL-6-induced increase in NOx production. Figure 8A shows that treatment with 50 nM AMT or 10 μM L-NMMA alone had no effect on NOx production in ARVM. By contrast, when L-NMMA or AMT was added to the culture media 15–30 min before and during incubation with IL-6, each inhibitor completely blocked the IL-6-induced increase in NOx production. Similar results were observed when ARVM were incubated with L-NMMA or AMT during 4 and 24 h treatments with IL-6 (Fig. 8B and C). Moreover, L-NMMA and AMT completely blocked the IL-6-induced increases in NOx concentration in cell lysate obtained from myocytes incubated with IL-6 for 2, 4 and 24 h (data not shown). These data show that iNOS was the primary isoform of NOS responsible for the production of NOx induced by IL-6 in ARVM. Thus, we examined whether IL-6-induced NO production is responsible for its inhibitory effect on SR function in ARVM.
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Myocytes were incubated with 10 μMNG-monomethyl-L-arginine (L-NMMA) or 50 nM 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) in the absence and presence of 10 ng ml–1 IL-6 for 2 (A), 4 (B) and 24 h (C). The NO production in each treat group was normalized to that of time-controls (dashed lines). Data represent means ± S.E.M.; numbers in parentheses are numbers of experiments. *P < 0.05 compared with the control group.
Effects of inhibition of NO production on IL-6-induced suppression of PRP, responsiveness to caffeine and Ca2+o and phosphorylation of PLB
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Experiments described in Figs 1, 3 and 5 were repeated in the presence of 10 μM L-NMMA or 50 nM AMT. Figure 9A shows that L-NMMA and AMT inhibited the IL-6-induced decrease in PRP30 of CS in ARVM after 2 h pretreatment. Similarly, the IL-6-elicited suppression of caffeine-induced CS was abolished in the presence of L-NMMA or AMT (Fig. 9B). Note that L-NMMA or AMT alone had no significant effect on PRP30 or caffeine-induced CS. Figure 9C shows that L-NMMA completely blocked the IL-6-induced inhibition of the contractile response to 1.5 mM [Ca2+]o, whereas alone it had no significant effect on Ca2+o responsiveness. Moreover, L-NMMA diminished the IL-6-induced decrease in responsiveness of CS in 2 mM [Ca2+]o, and similar results were observed in ARVM pretreated with IL-6 for 4 and 24 h (data not shown). Figure 9D shows that after 2 h treatment with IL-6, L-NMMA abolished the IL-6-induced decrease in phosphorylation of PLB with no effect on total PLB protein expression. The same results were obtained in three out of four experiments. Taken together, these data suggest that after 2 h exposure, the IL-6-induced inhibition of cardiac SR function is mediated by enhanced NO production via iNOS activation.
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The PRP after a 30 s rest (A), responsiveness to 10 mM caffeine (B) and 1.5 mM [Ca2+]o (C), and phosphorylation of PLB (D) were measured in ARVM incubated with or without 10 μML-NMMA or 50 nM AMT in the absence and presence of 2 h pretreatment with 10 ng ml–1 IL 6. Immunoblots of PLB with low molecular mass were obtained from cell lysates of the time-control (C), IL-6-, L-NMMA-, and IL-6 plus L-NMMA-treated myocytes treated at 95°C prior to electrophoresis. The same results were observed in another two experiments. Data represent means ± S.E.M. from 5 experiments. *P < 0.05, compared with the time-controls.
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Discussion
IL 6 has been associated with many chronic cardiac diseases (Kanda & Takahashi, 2004), including myocardial failure (Wollert & Drexler, 2001; Janssen et al. 2005), and the severity of several cardiovascular pathophysiological states (Cesari et al. 2003). However, studies on the cellular mechanisms for the cardiac effects of IL-6 are limited. While the acute negative inotropic effect of IL-6 has been studied (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999), the chronic cardiac effect of IL-6 has not been explored. We previously demonstrated that chronic exposure to IL-6 decreases PRP of CS and activates iNOS in ARVM, via activation of JAK2/STAT3 (Yu et al. 2003). The present study further investigated the cellular mechanism for the previously observed chronic inotropic effect of IL-6 in ARVM and examined whether iNOS is the downstream mediator of IL-6-induced negative inotropy. We found for the first time that after 2 h incubation: (1) IL-6 decreases contractility and Ca2+o responsiveness with no significant effect on ICa,L in ARVM; (2) IL-6 reduces PRP, caffeine responsiveness and the phosphorylation of PLB; (3) the negative inotropic effect is sustained for at least 1 h after removal of IL-6; and (4) IL-6 induces production of NO via activation of iNOS, with inhibition of iNOS abolishing IL-6-induced changes in PRP, caffeine- and Ca2+o-responsiveness, and PLB phosphorylation.
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Effective concentrations of IL-6
Under physiological conditions, studies on cytokine releases during exercise reported that plasma IL-6 levels rise to 5 pg ml–1 after eccentric exercise (Toft et al. 2002) and reach 94 pg ml–1, primarily derived from working skeletal muscle, immediately after a marathon race (Ostrowski et al. 1998). In contrast, a recent study using microdialysis techniques showed that repetitive low-intensitiy exercise causes an increase in IL-6 over 2.0 ng ml–1 in the interstitium of human skeletal muscle without an increase in the plasma level (Rosendal et al. 2005). Under pathological conditions, elevated serum IL-6 levels were reported to be 2.5 ng ml–1 in patients with stunned myocardium (Wan et al. 1996; Werdan, 1998). The increased IL-6 acts concentration dependently on a variety of targets in autocrine, paracrine or/and endocrine manners (Kishimoto et al. 1995; Wollert & Drexler, 2001; Pedersen et al. 2003). Moreover, IL-6 mRNA levels were shown to be greater in the heart than other tissues following stimulation with LPS (Saito et al. 2000). Therefore, although concentrations (1–30 ng ml–1) of IL-6 used in this study are higher than the reported values, it is conceivable that IL-6 concentration in the interstitium of ventricular muscle reaches 10 ng ml–1 or higher under pathophysiological conditions, regardless of sources of IL-6 production. Our data showing IL-6-induced concentration- and time-dependent negative inotropic effects are consistent with its pathophysiological role in aforementioned cardiac disorders.
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Irreversible chronic cardiac effect of IL-6
The present study shows that after 2 h incubation, IL-6-induced inhibitory effects on CS kinetics, PRP and Ca2+o- and caffeine-responsiveness persist for at least 1 h after removal of the cytokine. These results suggest that once IL-6 initiates its signalling cascade, such as JAK2/STAT3 (Yu et al. 2003), the agonist–receptor complex is no longer required for its downstream inotropic effect (as would be expected if enhanced iNOS expression is involved). This is consistent with clinical observations, which show that an early transient increase in cytokines (1–4 h) elicits long-term alterations in cardiac function. By contrast, it is not clear whether the acute negative inotropic effect of IL-6 is reversible. Although recovery of muscle contraction was complete within 40 min after removal of IL-6 from acute exposure (Finkel et al. 1992), it has never been addressed in studies using isolated cardiac myocytes (Kinugawa et al. 1994; Sugishita et al. 1999). If the acute inotropic effect of IL-6 is reversible as reported in isolated hamster papillary muscle (Finkel et al. 1992), it would suggest that the cellular mechanisms involved in acute and chronic inotropic effects of IL-6 are different, such as activation of different isoforms of NOS as suggested by studies in chick embryonic cardiomyocytes (Kinugawa et al. 1994). Thus, the present study was designed to examine whether this sustained chronic cardiac effect of IL-6 is mediated by iNOS.
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Cardiac inotropic effect of IL-6 after chronic exposure
The present study showed that IL-6 acts chronically in ARVM to reduce (+dL/dt)max, an effect that could result from a decrease in Ca2+ influx, Ca2+ release from SR or/and myofilament Ca2+ sensitivity. We showed that the suppression of contractile responsiveness to Ca2+o after 2 h pretreatment with IL 6 is probably not due to reductions in Ca2+ influx via ICa,L. In addition, our indirect evidence showing no change in the EC50 of responsiveness for Ca2+o suggests that IL-6 does not alter the Ca2+ sensitivity of myofilaments. Thus, these results suggest that IL-6 suppresses SR function. We also showed that IL-6 reduces (–dL/dt)max; this could result primarily from decreasing Ca2+ uptake via the SR Ca2+ pump, though sarcolemmal Na+–Ca2+ exchange also contributes to relaxation (Bassani et al. 1994). Our data showing IL-6-induced reductions in PRP and caffeine responsiveness in combination with findings suggesting no significant SR leak support the hypothesis that IL-6 suppresses SR Ca2+ uptake. Moreover, the phosphorylation of PLB is reduced by IL-6 treatment, which is opposite to the effect of ISO. This further supports our hypothesis. Whether IL-6 also suppresses Ca2+ release via SR Ca2+ channels and Ca2+ extrusion via sarcolemmal Na+–Ca2+ exchangers require further investigations.
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Role of iNOS activation in IL-6-elicited cardiac effect
Studies in chick embryonic cardiomyocytes suggested that different isoforms of NOS are involved in the acute (<10 min) and chronic (24 h) inotropic effects of IL-6 (Kinugawa et al. 1994). However, the identification of specific NOS isoforms has not been explored. We have previously shown that IL-6 induces iNOS protein expression 2 h after exposure, and that this action can be blocked by a JAK2 inhibitor (Yu et al. 2003). The present study showed that IL-6 increases NO production after treatments of 2 h (Fig. 7), supporting our previous findings of the increased iNOS protein expression. Inhibition of this increase in NO production by AMT (Fig. 8) is consistent with the involvement of iNOS in the chronic cardiac effects of IL-6. These results suggest that iNOS activation is a downstream event of the JAK2/STAT3 signalling of IL-6.
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Studies of protein or/and mRNA expression of constitutive NOS in rat and mouse hearts showed that endothelial NOS (eNOS) is the only isoform expressed in normal adult ventricular myocytes (Balligand et al. 1995; Vandecasteele et al. 1999). Meanwhile, neural NOS (nNOS) was found on SR membrane vesicles prepared from rabbit and mouse hearts (Xu et al. 1999) and cardiac mitochondria (Kanai et al. 2001). Studies using genetically engineered mice showed that nNOS and eNOS independently modulate basal cardiac contractile function in opposite fashion (Barouch et al. 2002; Khan et al. 2003). However, the inotropic role of constitutive NOS remains controversial (Massion & Balligand, 2003), probably because of concern regarding adaptive changes of genetically engineered mice during development and/or low physiological activities (French et al. 2001). Our results showing no significant effect of NOS inhibitors on the basal NO level (Fig. 8) suggest that constitutive NOS activity is low and/or the change in NO is beyond the detection limit. By contrast, we showed time-dependent increases in NO production induced by chronic exposure to IL-6, which was blocked by AMT, in accord with our previous findings of IL-6-induced iNOS expression (Yu et al. 2003). Moreover, it is well known that iNOS expression is not detectable under physiological conditions (Gallo et al. 1998), and the level of NO produced via iNOS is greater than that elicited by constitutive NOS after stimulation by cytokines.
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It is also well known that the cardiac effects of NO are complex and somewhat controversial, probably due to the distinct concentration-dependent sensitivities of its targets and distinguishable signalling mechanisms. NO is known to target the L-type Ca2+ channel and SR in cardiac myocytes through signalling pathways such as cGMP, and/or by directly modifying the proteins (such as by S-nitrosylation/oxidation) (Campbell et al. 1996; Hare, 2003; Massion et al. 2003). Studies in adult ferret ventricular myocytes showed that SIN-1, a NO donor, induces biphasic and bimodal changes in basal ICa,L (Campbell et al. 1996), which were not observed in frog (Mery et al. 1993), guinea-pig (Wahler & Dollinger, 1995) or rat (Abi-Gerges et al. 2001) ventricular myocytes. We also observed no significant change in basal ICa,L in ARVM upon acute exposure to 0.1 mM SIN-1 (S. J. Liu, unpublished data). Similarly, SIN-1 (up to 0.1 mM) has no significant effect on contractile function of papillary muscle isolated from adult rat (Wyeth et al. 1996). Although we found that cumulative NO production in cell lysates and in culture medium is 2.5- to 3-fold of the basal level after 2 h of IL-6 treatment, it is not surprising that ICa,L is not significantly altered in our experimental conditions. However, the possibility for alteration in ICa,L during longer exposure (e.g. >24 h) to IL-6 cannot be excluded.
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For the aforementioned reasons, reported effects of NO on SR function, including ryanodine-sensitive Ca2+ release channel and SR Ca2+ pump, are also diverse (Hare, 2003; Massion et al. 2003). For example, NO inhibits SR Ca2+ ATPase (Ishii et al. 1998) and the ryanodine-sensitive channel (Meszáros et al. 1996) in skeletal muscle, whereas at low concentration it has no effect on PRP in rat papillary muscle (Prabhu et al. 1999). At high concentrations NO increases Ca2+ leak from SR in skeletal muscle (Pouvreau et al. 2004) and cardiac ryanodine-sensitive channel (Xu et al. 1998). Although free cytosolic Ca2+ was not measured in the present study, the lack of significant changes in the diastole state during rest intervals in IL-6-treated ARVM (Fig. 6A) suggests that the Ca2+ leak in the presence of increased NO was insignificant. Moreover, our data showing reductions in maximum shortening and relaxation rates, PRP and caffeine-responsiveness, and PLB phosphorylation induced by IL-6, which were abolished by NOS inhibitors (Fig. 9) support the possibility that NO reduces SR function by acting on the SR Ca2+ pump and/or via PLB. Although local NO produced by different NOS isoforms has been suggested to contribute differently to changes in the function of each target (Barouch et al. 2002; Khan et al. 2003), the larger amounts of NO produced by iNOS are probably more diffuse rather than localized. Our data suggest that such increased NO after 2 h exposure to IL-6 has a profound effect on SR with little detectable action on the L-type Ca2+ channel in ARVM. Somewhat in contrast, nNOS has been suggested to stimulate SR Ca2+ release (Barouch et al. 2002; Khan et al. 2003). It is also possible that a large increase in NO that occurs following iNOS activation elicits an inhibitory action that decreases the stimulatory effect of nNOS on SR.
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Our data also showed that IL-6 reduces PLB phosphorylation at Ser16, where is primarily phosphorylated by cAMP-dependent protein kinase A (PKA) (Kuschel et al. 1999). This IL-6 effect is also NO-dependent because the reduction of PLB phosphorylation is inhibited by L-NMMA (Fig. 9D). A study in ARVM using SIN-1 showed that NO reduces PLB phosphorylation and cAMP production induced by ISO in a cGMP-independent manner (Stojanovic et al. 2001). This NO-induced decrease in PLB phosphorylation is supported by a recent study showing that SNAP, an NO donor, inhibits basal and forskolin-stimulated cAMP production by blocking adenylyl cyclase, effects independent of guanylyl cyclase activity (Ostrom et al. 2004). Thus, it is likely that the IL-6-induced reduction in PLB phosphorylation observed in the present study results from NO-elicited inhibition of basal cAMP/PKA activities. Another possibility is that NO activates protein phosphatase type 2A (PP2A), thereby enhancing dephosphorylation of PLB, as suggested by a study in neonatal cardiomyocytes which showed tumour necrosis factor (TNF)-, known to activate iNOS, decreases PLB phosphorylation by activating PP2A (Yokoyama et al. 1999). Taken together, the concentration-dependent sensitivities of target proteins associated with E–C coupling (such as SR and L-type Ca2+ channels) to NO could be distinct and determine the ultimate effect on contractile function under physiological and pathological conditions. Cellular mechanisms for NO-associated alterations in these target proteins have been postulated to be mediated via cGMP-dependent or/and cGMP-independent (including direct S-nitrosylation of target proteins) mechanisms. Whether IL-6-induced alterations in SR function result solely from NO-dependent mechanism is still unknown and requires further investigation.
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In summary, we have shown that exposure to IL-6 for two or more hours results in a negative inotropy probably by inhibiting SR Ca2+ reuptake in concomitance with increases in NO in ARVM. The IL-6-induced decrease in PLB phosphorylation is also in accord with its reduction of SR function. The increase in NO production results primarily from expression/activation of iNOS that is mediated via IL-6-induced JAK2/STAT3 activation as shown previously (Yu et al. 2003). Inhibition of iNOS/NO abolishes the IL-6-induced decrease in SR function. Therefore, this leads to our conclusion that IL-6 suppresses SR function via iNOS after chronic exposure. Moreover, such cardiac effects of IL-6 are sustained even after removal of IL-6. This suggests that the transient elevation of IL-6 observed in plasma or ventricular muscle of patients with many cardiac diseases can cause prolonged effects on contractility. An in vivo study in transgenic mice showed that cardiac-specific overexpression of iNOS causes an increase in NOx in hearts (2.5-fold above the wild-type controls) but no severe cardiac dysfunction (Heger et al. 2002). One interpretation for this seeming inconsistency is that the increase in plasma and heart NOx in the transgenic models is moderate so the associated systemic adaptation during development in these transgenic mice results in less detrimental functional consequences. A recent in vivo study reported that IL-6 administration causes heart failure in a dose-dependent manner (Janssen et al. 2005). Thus, IL-6 might play an important role in the pathogenesis of associated chronic heart diseases. Treatment to antagonize the induction and activation of iNOS in the early stage of IL-6-associated heart dysfunction might be beneficial.
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