We have recently reported that tumour necrosis factor-α (TNF-α) increases oxidative stress and apoptosis in cardiomyocytes by upregulating p38 mitogen-activated protein (MAP) kinase (MAPK) phosphorylation. Interleukin-10 (IL-10) blocked these effects of TNF-α by upregulating extracellular signal-regulated kinase 1/2 (ERK 1/2) MAPK phosphorylation. However, the precise site of this IL-10 action is still unknown, and this is investigated in the present study.
Cardiomyocytes isolated from adult Sprague–Dawley rats were exposed to TNF-α (10 ng/mL), IL-10 (10 ng/mL), and IL-10+TNF-α (ratio 1) for 4 h. Hydrogen peroxide and antioxidant trolox were used as positive controls. Exposure to TNF-α resulted in an increase in the production of reactive oxygen species, the number of apoptotic cells, caspase-3 activation, and poly-ADP ribose polymerase (PARP) cleavage. Increased oxidative stress by using hydrogen peroxide also caused apoptosis. The changes due to TNF-α were associated with an increase in the inhibitor of κB kinase (IKK) and nuclear factor kappa-B (NFκB) phosphorylation. IL-10 by itself had no effect, but it prevented the above mentioned TNF-α-induced changes. Trolox also mitigated TNF-α induced changes. Pre-exposure of cells to an IKK inhibitor (PS-1145) prevented TNF-α-induced caspase-3 and PARP cleavage. Inhibition of ERK 1/2 MAPK with PD98059 attenuated the protective role of IL-10 against TNF-α-induced activation of IKK and NFκB as well as cardiomyocyte apoptosis.
The present study shows that TNF-α-induced activation of the NFκB pathway plays a critical role in cardiomyocyte apoptosis. IL-10 prevents TNF-α-induced NFκB activation and pro-apoptotic changes in cardiomyocytes by inhibiting IKK phosphorylation through the activation of ERK 1/2 MAPK.
Tumour necrosis factor-α (TNF-α), a pro-inflammatory cytokine, is elevated in many pathogenic conditions including cardiovascular injury and disease states.1–3 The anti-inflammatory cytokine interleukin-10 (IL-10) inhibits the production of various pro-inflammatory cytokines including TNF-α.4 IL-10 has also been shown to act as an antagonist to TNF-α by inhibiting TNF-α-induced oxidative stress.5,6 Sustained long-term over-expression of TNF-α provokes the induction of cardiomyocyte apoptosis,7 which contributes to the pathophysiology of several heart diseases, including dilated cardiomyopathy, myocardial infarction, and heart failure.8,9 Surprisingly, anti-TNF-α therapy had no benefit or even had harmful effects in patients with heart failure.10,11 Thus there is a need to study how TNF-α influences downstream cell-signalling pathways leading to cardiomyocyte apoptosis and how IL-10 mitigates the harmful effects of TNF-α. Identification of the intermediary steps involved in the TNF-α-induced cardiomyocyte apoptosis can offer potential therapeutic targets to abrogate TNF-α-induced cardiovascular diseases.
Nuclear factor kappa-B (NFκB) is a transcription factor that regulates gene transcription of many pro-inflammatory cytokines.12 Thus suppression of NFκB activity can be a potential mechanism for regulating inflammatory responses. Several studies have identified NFκB as the key regulator of TNF-α gene activation.13 The NFκB family of transcription factors exists in the cytoplasm of unstimulated cells as homo- or heterodimers complexed with the inhibitory-κB (IκB) proteins. On stimulation, phosphorylation of IκB by inhibitory-κB kinase (IKK) triggers its degradation and the activation of NFκB. The activation and translocation of NFκB to the nucleus is followed by transcription of various pro-inflammatory genes including TNF-α.14,15 The anti-inflammatory actions of IL-10 inhibit the production of pro-inflammatory cytokines in human monocytes through the suppression of NFκB activation.16 In rat alveolar macrophages and lung tissue, it has been reported that IL-10 and IL-13 inhibit nuclear localization of NFκB by preserving the expression of IκB protein.17
In a recent study, we have reported that TNF-α induces cardiomyocyte apoptosis by upregulating p38 phosphorylation. IL-10 prevented this effect of TNF-α by upregulating extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation.6 However, the precise site of action of ERK 1/2 in the TNF-α signalling pathway is still unknown. The current study investigated (i) the role of NFκB pathway in TNF-α-induced cardiomyocyte apoptosis, and (ii) how IL-10 regulated the NFκB pathway to suppress TNF-α-induced-cardiomyocyte apoptosis.
Materials and methods
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All animal-experiment protocols were approved by the University of Manitoba Animal Care Committee following the guidelines established by the Canadian Council on Animal Care.
Cardiomyocytes were isolated from normal adult male Sprague–Dawley rats (250–300 g) using a previously described procedure.5 Hearts were excised and mounted on a modified Langendorff perfusion apparatus. The perfusate (modified Krebs buffer) contained 110 mM NaCl, 2.6 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, and 11 mM Glucose (pH 7.4). The Ca2+ free perfusion was then switched to recirculating mode with the buffer containing 25 µM calcium, 0.1% w/v collagenase, and 0.25% w/v bovine serum albumin, for 20 min. The collagenase-digested ventricles were chopped into small pieces and gently passed through pipettes with progressively smaller tip diameters and with an increasing concentration of CaCl2. The suspension was filtered through a nylon mesh (200 µM) and was allowed to settle for 10 min. The supernatant was discarded and the cell pellet was resuspended in medium M199 containing CaCl2 (1.8 mM). Cardiomyocytes (1 × 105 per dish) were plated on 4% serum-coated polystyrene tissue culture dishes. Plated cells were incubated in serum-free culture medium M199 supplemented with antibiotics (streptomycin/penicillin, 100 µg/mL) at 37°C under a 5% CO2−95% air atmosphere. Two hours after plating, the culture medium was changed to remove unattached dead cells, and the viable cardiomyocytes were incubated overnight.
Treatment of cardiomyocytes
After initial incubation of 24 h, >90% of cardiomyocytes were viable and these cells were treated with one of the following: H2O2 (100 µM), TNF-α (10 ng/mL), IL-10 (10 ng/mL), and combination of IL-10 + TNF-α (ratio 1) for 4 h. This dose and treatment protocol is based on our earlier studies.6 To investigate the involvement of oxidative stress in TNF-α-induced changes in NFκB pathway and caspase-3 activation, cells were treated with 20 µmol/L of trolox (a water-soluble antioxidant) for 30 min and then incubated with the combination of trolox and TNF-α (10 ng/mL) for 4 h. In order to study the physiological role of NFκB pathway and ERK 1/2 MAPK (mitogen-activated protein kinase) in the TNF-α and IL-10 regulation of cardiomyocyte apoptosis, cells were pre-treated with 25 µM of PS-1145 (IKK inhibitor) or 25 µM of PD-98059 (ERK inhibitor) or with vehicle solution (DMSO at a concentration of 0.1%) for 15 min.
The concentration and time of exposure for PS-1145 were chosen following the completion of a separate pilot study (data not shown), and for PD-98059, they were chosen from our previous study.6
Measurement of reactive oxygen species
The level of oxidative stress was monitored by the measurement of reactive oxygen species (ROS).18 Cardiomyocytes from different treatment groups in the culture dishes were washed with phosphate-buffered saline (PBS) and incubated with 10 µM solution of fluorescent probe, 5-(6)-chloromethyl-2′7′-dihydroflourescein diacetate probe (DCFDA) (Molecular Probes, Eugene, Oregon, USA) at 37°C for 30 min in a humidified chamber. Fluorescent images of 100 cells from multiple fields per dish were recorded with the Olympus BX 51 fluorescent microscope. From each perfused heart, at least 10 dishes (35 mm × 10 mm) were prepared; the study was done in duplicate for each treatment group. Fluorescence intensity was measured using digital image-processing software (Image Pro Plus). Experiment was repeated six times (n = 6) and statistical analysis was done.
Western blot analysis
Phosphorylated and total IKKα, IKKβ, and NFκB levels, as well as caspase-3 and poly-ADP ribose polymerase (PARP) cleavage were measured by western blotting using specific antibody kits (Cell Signaling Technology). Whole-cell protein extracts prepared from control and treated cardiomyocytes in different groups were suspended in PBS containing protease inhibitor cocktail. The protein samples (30 µg) were then subjected to 12% SDS–PAGE and transferred to PVDF (Roche Diagnostics, USA) membrane. Proteins bound to the membrane were detected with BM chemiluminiscence blotting substrate (POD) kit (Roche Diagnostics, USA). Bands were visualized with Fluor S-MultiImager MAX System (Bio-Rad Laboratories, Canada) and quantified by image analysis software (Quantity One, Bio-Rad Laboratories, Canada).
Caspase-3 and PARP cleavage were studied by western blotting (as described earlier), and caspase-3 activity was measured by fluorescence spectrophotometric assay using the fluoro-chrome 7-amino-4-methyl coumarin (AMC) and substrate DEVD-AMC, provided in the CaspACE™ assay system (Promega Corp., Madison, WI, USA). The assay was performed in 96-well polystyrene plate (Becton Dickinson, USA). From each sample, 100 µg of protein was added to the reaction wells, together with 32 µL of caspase assay buffer, 2 µL DMSO, 10 µL of dithiothreitol, and 2 µL of DEVD-AMC substrate (dissolved in DMSO). The reaction mixture was incubated at 30°C for 60 min and thereafter analysed for AMC fluorescence in a plate reader (Spectra Max Gemini) at excitation and emission wavelengths of 360 and 460 nm, respectively. The fluorescence data given in the figures were corrected by subtracting background values.
Cardiomyocyte apoptosis was also studied after staining the cells with Hoescht 33258. For this, the cardiomyocytes from different groups in culture dishes were washed three times with PBS and incubated with Hoescht 33258 (1 µg/mL) for 10 min in a humidified chamber, protected from light, at 37°C. After staining, the culture plates were examined using fluorescent microscope (Olympus BX 51). Cardiomyocytes were observed for apoptosis. A total of six different fields per dish were counted to quantify the number of cells containing fragmented nuclei. From each perfused heart, at least 10 dishes (35 mm × 10 mm) were prepared; the study was done in duplicate for each treatment group. Experiment was repeated four times (n = 4) and statistical analysis was done.
Protein and statistical analysis
Total protein concentration was determined using bovine serum albumin as a standard.19 Data are expressed as the mean ± SEM. Groups were compared by one-way ANOVA, and Bonferroni’s test was performed to identify differences between groups. A value of P < 0.05 was considered significant.
Oxidative stress studies
The production of intracellular reactive oxygen species (ROS) was significantly increased in TNF-α-exposed cardiomyocytes. By itself, IL-10 treatment of the cardiomyocytes did not show any effect on ROS levels (Figure 1A). However, TNF-α-induced increase in ROS production was significantly mitigated by IL-10 treatment. Exposure to H2O2 (positive control) significantly increased the ROS production in cardiomyocytes as measured in terms of green fluorescence intensity (Figure 1A).
Cardiomyocyte apoptosis was studied by fluorescent microscopy after staining the cells with Hoescht 33258. In the control group, the cells were rod-shaped, binucleated, and the nuclei had normal appearance (Figure 1B). Exposure to TNF-α resulted in nuclear fragmentation and the number of apoptotic cells significantly increased. Treatment with IL-10 alone did not show any significant change, whereas TNF-α-induced increase in nuclear fragmentation in cardiomyocytes was significantly prevented by IL-10 treatment. Hydrogen peroxide treatment also increased the number of apoptotic cells (Figure 1B).
Cardiomyocyte apoptosis was also studied in terms of caspase-3 activation and PARP cleavage. The caspase family of aspartate-specific cysteine proteases is the central executor of apoptosis. The activation of caspase-3 by proteolytic processing of pro-caspase-3 into 17 and 12 kDa subunits serves as an early marker of apoptosis in various cell types. The 17 kDa-cleaved caspase-3 fragment was detected in TNF-α-exposed cardiomyocytes (Figure 2A). Treatment with IL-10 prevented the TNF-α-induced fragmentation of caspase-3. Whereas IL-10 treatment alone did not show any effect on caspase-3 activation (Figure 2A). To confirm the activation of caspase-3, we monitored the cleavage of its substrate, the nuclear enzyme PARP. In TNF-α-treated groups, caspase-3-induced proteolysis of 116 kDa-native PARP molecule was detected in immunoblots as 89 kDa fragment. Treatment with IL-10 prevented the TNF-α-induced cleavage of PARP, whereas IL-10 alone did not show any effect. Antioxidant trolox also prevented the TNF-α-induced caspase-3 activation and PARP cleavage. Trolox by itself had no effect (Figure 2A).
Caspase-3 activity in fluorescence spectrophotometric assay was increased by 107% in TNF-α-exposed cardiomyocytes. There was no difference between control- and IL-10-treated cells with respect to caspase-3 activity. However, the TNF-α-induced increase in caspase-3 activity was completely mitigated by IL-10 treatment. Antioxidant trolox also prevented the TNF-α-induced increase in caspase-3 activity. Trolox by itself had no effect on the baseline caspase-3 activity (Figure 2B).
Phosphorylation of IKKα, IKKβ, and NFκB
Phosphorylation of IKKα, IKKβ, and NFκB was examined in the cardiomyocytes exposed to TNF-α (10 ng/mL), IL-10 (10 ng/mL), and IL-10 + TNF-α (ratio 1). Exposure to TNF-α caused a significant increase (P < 0.001) in IKKα, IKKβ, and NFκB phosporylation compared with the respective control group. Treatment with IL-10 alone did not cause any change in these parameters, whereas TNF-α-induced increase in IKKα, IKKβ, and NFκB phosphorylation was prevented by IL-10 treatment (Figure 3). Antioxidant trolox was used to study the role of TNF-α-induced oxidative stress in the activation of IKKα, IKKβ, and NFκB in cardiomyocytes. Trolox completely prevented the TNF-α-induced changes in these three parameters. Trolox by itself had no effect (Figure 3).
Inhibition of NFκB pathway
There are several pharmacological approaches to study the role of NFκB pathway in cell signalling. These include repression of NFκB transactivation potential and stabilization of inhibitory kappa-B (IkB), and more recently, inhibition of upstream IKK has also been used.20 In order to study the role of NFκB pathway in TNF-α-induced cardiomyocyte apoptosis, we measured the caspase-3 activation and PARP cleavage after inhibiting IKK with PS-1145. To check whether PS-1145 actually inhibited TNF-α-induced IKK phosphorylation, cells were pre-incubated with PS-1145 (25 µM) for 15 min followed by treatment with TNF-α (10 ng/mL) for 4 h and measurement of IKK phosphorylation. Treatment with PS-1145 significantly inhibited TNF-α-induced IKKα and IKKβ phosphorylation (Figure 4A–C). In the presence of IKK inhibitior, TNF-α exposure did not cause cardiomyocyte apoptosis, as no change was observed in caspase-3 activation and PARP cleavage (Figure 5A and B). IL-10 treatment alone as well as along with TNF-α did not show any change in caspase-3 activity and PARP cleavage (Figure 5A and B). When the cells were treated with PS-1145 alone, we did not observe any effect on caspase-3 activation as well as PARP cleavage (data not shown).
Inhibition of ERK MAPK
We have previously reported that IL-10 prevented TNF-α-induced cardiomyocyte apoptosis by up-regulating ERK 1/2 phosphorylation.6 In this study, to investigate the involvement of ERK 1/2 MAPK in IL-10 modulation of TNF-α-induced NFκB pathway and caspase-3 activation in cardiomyocytes, we pre-treated the cells for 15 min with PD-98059 (ERK inhibitor) and then studied the effects of TNF-α and IL-10 treatment on caspase-3 activation, PARP cleavage, and IKK as well as NFκB activation. TNF-α-induced increase in caspase-3 activity and PARP cleavage were not blocked by IL-10 in ERK-inhibited cardiomyocytes (Figure 6). Furthermore, IL-10 treatment was not able to prevent TNF-α-induced increase in IKKα, IKKβ, and NFκB phosphorylation in cardiomyocytes pre-treated with PD-98059 (Figure 7). Treatment with the inhibitor alone did not show any effect on IKK, NFκB, caspase-3, and PARP (data not shown).
We have recently reported that IL-10 prevented TNF-α-induced oxidative stress and apoptosis in cardiomyocytes by upregulating ERK 1/2 MAPK phosphorylation.6 The present study examines some of the intermediary steps downstream to TNF-α and IL-10 receptor activation and upstream to the induction of cardiomyocyte apoptosis (Figure 8). Our data show that TNF-α-induced oxidative stress in cardiomyocytes leads to apoptosis by an activation of NFκB pathway. IL-10 prevented the TNF-α-induced apoptosis by downregulating the IKK phosphorylation and in turn NFκB activation. Furthermore, data in this study show that this effect is mediated by IL-10-induced activation of ERK 1/2 MAPK.
Direct exposure of cardiomyocytes to TNF-α caused an increase in oxidative stress as indicated by an increase in ROS signal. In this regard, it has been shown that TNF-α-induced oxidative stress alters redox homeostasis by impairing the membrane permeability, mitochondrial function, and hence cardiac function.21 TNF-α has been shown to increase the oxidative stress in the failing heart in the patients with dilated cardiomyopathy.22 It has also been reported that TNF-α blockade decreases oxidative stress and sympathoexcitation in heart failure in rats.23 Exogenous administration of IL-10 has been shown to protect against TNF-α-induced oxidative stress in other situations. In this regard, IL-10 treatment decreases the severity of acute pancreatitis by attenuating the TNF-α-induced oxidative stress.24 It has been reported that TNF-α-induced increase in ROS production and oxidative stress in cardiomyocytes lead to apoptosis.25 Since cardiomyocytes are terminally differentiated, any loss of cells due to cell death can have critical functional consequences.26,27 Thus, cardiomyocyte apoptosis is implicated in the pathogenesis of heart failure of numerous aetiologies, including myocarditis, ischaemia/reperfusion injury, chronic pressure overload, and congestive heart failure.9,28,29 In the present study, TNF-α caused a significant increase in apoptosis as confirmed by an increase in the caspase-3 activation and PARP cleavage, which is also associated with an increase in the number of apoptotic nuclei.
The pro-apoptotic role of TNF-α has been extensively investigated in a variety of in vitro and in vivo models.16,30,31 Binding of TNF-α to its cell-surface receptors triggers apoptotic signalling through caspase-3 activation and causes apoptosis. Caspase-3 has been identified as a key protease in the execution of apoptosis.32 It is activated by several pathways: one possible route being the release of mitochondrial cytochrome c, formation of apoptosome, and activation of caspase-9, which in turn activates effector caspase-3.33 TNF-α-induced cardiomyocyte apoptosis in the present study was significantly decreased by IL-10 treatment. Pro-apoptotic effects of TNF-α have been shown to be modulated by IL-10 treatment in various cell types. In this regard, in human articular chondrocytes, IL-10 upregulated bcl-2 expression and downregulated TNF-α-induced caspase-3 activity.34 We have recently shown that TNF-α-induced increase in the ratio of pro- and anti-apoptotic (Bax/Bcl-xl) proteins in cardiomyocytes was prevented by IL-10 treatment.6
The NFκB pathway is a key component of the cellular response to a variety of extracellular stimuli. The uniqueness in the response through this pathway to different stimuli may reside in the extent of phosphorylation as well as involvement of other co-factors not well understood at this time. In the current study, we have shown that TNF-α exposure leads to a significant increase in NFκB phosphorylation. It has been shown that TNF-α is responsible for the activation of NFκB pathway in human prostate carcinoma PC-3 cells.35 It is also reported that TNF-α-induced ROS generation leads to apoptosis in human breast MCF-7 cells through the activation of NFκB.36 The major mechanism of NFκB activation involves up-regulation of inhibitor kappa B kinase (IKK), which leads to the degradation of IkB and in turn translocation of NFκB to the nucleus and its activation.37 In the present study, TNF-α exposure leads to a significant increase in IKK phosphorylation. Furthermore, when the cardiomyocytes were pre-exposed to PS-1145, a specific IKK inhibitor, TNF-α-induced increase in cardiomyocyte apoptosis was blocked, as no activation of caspase-3 and PARP cleavage was observed, which implies that NFκB pathway is responsible for TNF-α-induced cardiomyocyte apoptosis. PS-1145 itself had no effect on caspase-3 activation and PARP cleavage.
The present study also shows that IL-10 prevents TNF-α-induced activation of NFκB in cardiomyocytes. In this context, it has been reported that IL-10 inhibits NFκB activation in human monocytes to suppress the synthesis of various pro-inflammatory cytokines.16 This action of IL-10 on NFκB pathway in the monocytes involves the blockade of the release of IκB from the NFκB complex. It has also been shown in human monocytic cell lines THP-1 and U937 that TNF-α-induced phosphorylation of IKK is blocked by IL-10 treatment.38 The present study demonstrates that cardiomyocytes also show a similar interplay between TNF-α and IL-10 with respect to IKK phosphorylation and NFκB activation. Although IL-10 has been found to inhibit the NFκB activation in various cell types, no concrete molecular mechanism has been elucidated so far. Data obtained in the present study suggest the sequence of events shown in Figure 8. Inhibition of ERK 1/2 with PD98059 upregulated IKK phosphorylation, thereby blocking the protective role of IL-10 against TNF-α-induced activation of NFκB pathway and cardiomyocyte apoptosis. Thus, in TNF-α-challenged cardiomyocytes, IL-10 treatment activates the cell-survival pathway by upregulating the ERK 1/2 MAPK, thereby inhibiting the NFκB activation. It should also be noted that ERK 1/2 activation has been reported as the mechanism for cell survival during the activation of different death receptors such as Fas receptor, TNF-R1, and TRAIL-R.39
In conclusion, this study provides the evidence that TNF-α-induced cardiomyocyte apoptosis is mediated by the activation of NFκB pathway. IL-10 is able to mitigate TNF-α-induced NFκB activation and cardiomyocyte apoptosis via the upregulation of ERK 1/2 MAPK and the downregulation of IKK phosphorylation.
Conflict of interest: none declared.
This study was supported by an operating grant from the Canadian Institute of Health Research. P.K.S. is the holder of the Naranjan S. Dhalla Chair in Cardiovascular Research supported by the St Boniface Hospital & Research Foundation. R.C.A. is the recipient of Rudy Falk Clinician Scientist Award. S.D. was supported by a Postdoctoral Fellowship from Manitoba Health Research Council and CIHR/HSFC IMPACT Strategic Training Program Grant in Pulmonary and Cardiovascular Research.