Cardiovascular Research

Cardiovascular Research 2001 51(2):265-274; doi:10.1016/S0008-6363(01)00294-2
© 2001 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Cardiotrophin-1 can protect cardiac myocytes from injury when added both prior to simulated ischaemia and at reoxygenation

Bhawanjit K Brar^a, Anastasis Stephanou^a, Zhihong Liao^a, Rhona M O'Leary^c, Diane Pennica^d, Derek M Yellon^b and David S Latchman^a,*

^aInstitute of Child Health, University College London, London WC1N 1EH, UK
^bHatter Institute of Cardiology, University College Hospitals, London, UK
^cDepartment of Protein Sciences, Genentech Inc, South San Francisco, USA
^dDepartment of Molecular Oncology, Genentech Inc, South San Francisco, USA

^* Corresponding author. Tel.: +44-20-7905-2189; fax: +44-20-7242-8437 d.latchman{at}ich.ucl.ac.uk

Received 15 September 2000; accepted 7 March 2001

	Abstract

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Objective: The cytokine cardiotrophin-1 (CT-1) has previously been shown to protect cultured cardiocytes from cell death induced by serum removal or hypoxia when administered prior to the damaging stimulus. We wished to test whether a similar protective effect could be observed if CT-1 was added after the ischaemic period and to investigate the signalling pathways involved in the protective effect when CT-1 is given prior to or after ischaemia. Methods: We therefore examined the protective effect of CT-1 in cultured rat cardiocytes exposed to simulated ischaemia followed by reoxygenation when CT-1 was administered either prior to simulated ischaemia or at reoxygenation. Results: We show that CT-1 can exert a protective effect against the damaging effects of simulated ischaemia/reoxygenation both when added after the simulated ischaemia at reoxygenation (P<0.05 in trypan blue, TUNEL and annexin V assays) or when added prior to the simulated ischaemia (P<0.05). In both cases, these protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway (P<0.05 in all assays). Conclusion: CT-1 can protect cardiac cells when added either prior to simulated ischaemia or at the time of reoxygenation following simulated ischaemia and these effects are dependent upon its ability to activate the p42/p44 MAPK pathway. Hence CT-1 may have therapeutic potential when added at the time of reperfusion following ischaemic damage.

KEYWORDS Apoptosis; Cell culture/isolation; Cytokines; Hypoxia/anoxia; Myocytes; Cardioplegia; Ischemia; Reperfusion

	1 Introduction

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Cardiotrophin-1 (CT-1) was initially isolated as a factor, which was capable of inducing cardiac myocyte hypertrophy in vitro [1], and was subsequently shown to have a similar effect when injected into adult mice [2].

Subsequent studies established that CT-1 is a member of a family of cytokines including interleukin-6 (IL-6), leukaemia inhibitory factor (LIF) ciliary neurotrophic factor, (CNTF) oncostatin M (OSM) and IL-11 [3,4]. The receptor for each of these factors contains a common component known as gp130 (for review see [5,6]). Although the IL-6, CNTF and OSM receptors contain in addition a specific subunit which is unique to each of these receptors [5,6], LIF and CT-1 and in certain cells, OSM share a common second receptor component, which is known as LIF receptor sub-unit β [3,7]. Following binding to the receptor, members of the IL-6 family activate both the MAP kinase pathway and the Jak/STAT pathway resulting respectively in the phosphorylation of the transcription factors NF-IL6 and STAT-3 allowing them to stimulate transcription [5,8–10].

As well as its potentially damaging hypertrophic effects, CT-1 also has significant protective effects. The apoptotic death of unstressed cardiac myocytes in defined serum-free medium can be reduced by treatment with CT-1 [11,12] and this effect was inhibited by inactivation of p42/p44 MAP kinase, which had no effect on the hypertrophic response to CT-1 [12]. This effect, together with the expression of CT-1 in the early mouse embryonic heart tube, suggests that CT-1 may have significant effects on cardiac survival which are important during heart development [11]. Moreover, CT-1 may also play a key role in protecting cardiac cells against stressful stimuli. Thus, treatment of cultured neonatal cardiocytes with CT-1 protects them against subsequent exposure to thermal or hypoxic stress both in terms of the total amount of cell death observed and the extent of apoptosis [13].

Hence, we demonstrate for the first time that CT-1 can have a protective effect when added at the time of reoxygenation following simulated ischaemia as well as when added prior to the ischaemic period and that the protective effects observed when CT-1 is added either prior to or after the simulated ischaemia are blocked by an inhibitor of the p42/p44 MAPK pathway.

	2 Materials and methods

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
2.1 Cell culture
Cardiomyocytes from the hearts of 2-day-old neonatal rats (Sprague–Dawley), were prepared by a modification of a previously published protocol [14]. This investigation conforms with UK Home Office Guidelines and UCL Animal Ethical Committee procedures for animal work. The cells were dispersed in a series of incubations at 37°C in a nominally calcium free, HEPES-buffered salt solution containing pancreatin (0.6 mg/ml, Gibco-BRL) and type II collagenase (0.5 mg/ml at approximately 266 units/mg, Worthington Biochemical Corporation). The dispersed cells were preplated for 1 h to allow contaminating fibroblasts to attach. The myocytes free within the culture media were plated on 24 well gelatin-coated plates at a density of 1x10⁵ cells/well. The cardiac myocytes were cultured at 37°C, 21% O₂–5% CO₂ in 4:1 Dulbecco's Modified Eagles medium/medium 199 (Gibco-BRL) supplemented with 10% (v/v) horse serum, 5% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin for 24 h. The media was then replaced with low serum (1% v/v FCS) Dulbecco's modified medium/medium 199 with penicillin/streptomycin (minimal media) for 48 h. At this point the cells were ready for experimentation, as a spontaneous culture of beating myocytes had formed.

2.2 Simulated ischaemia
To simulate ischaemia in vitro, cultured cardiocytyes were incubated in 1 ml of simulated ischaemia buffer (137 mM NaCl, 12 mM KCl, 0.49 mM MgCl₂, 0.9 mM CaCl₂.2 H₂O, 4 mM HEPES, 20 mM sodium lactate, pH6.2: [15]) for 6 h in an atmosphere of 5% CO₂ and 95% argon at 37°C producing a PO₂ level of 4 mmHg, 0.2% final oxygen concentration. This buffer is a modification of that devised by Esumi et al. [16] to simulate the extra cellular milieu that occurs during myocardial ischaemia in vivo resulting in the appropriate concentrations of potassium, hydrogen and lactate ions.

CT-1 at a concentration of 2 ng/ml was added to the cardiac myocytes 0, 30, 60 and 120 min prior to a lethal simulated ischaemic insult and then removed just prior to the insult. For assays of the protective effect of CT-1 added prior to ischaemia, the cells were analysed immediately after the ischaemic period. Subsequent to this injury cell survival was assessed by trypan blue exclusion and apoptotic nuclei were assayed by the end labelling of DNA 3' ends with dUTP-FITC using a modification of the TUNEL method [17].

To investigate the downstream signalling pathways, which mediate CT-1 cardioprotection against ischaemic injury, the cardiac myocytes were incubated for 10 min in 1 ml of minimal medium containing signal transduction inhibitors, prior to a 30-min incubation with 2 ng/ml of CT-1. The MEK1 inhibitor (PD98059) (New England Biolabs Inc, Beverly MA 01915) has been shown to act in vivo as a highly selective inhibitor of MEK 1 activation and the p44/p42 MAP kinase cascade [18]. The inhibitor was stored at –20°C at a concentration of 100 mM and was used at a concentration of 50 µM. A highly specific inhibitor of p38 MAP kinase (SB 203580 (Calbiochem)) was stored in the dark at a concentration of 100 mM in 50 mg/ml DMSO and was used at a concentration of 10 µM [19]. Following the lethal ischaemic insult the cardiac myocytes were assessed for cell survival by trypan blue exclusion and apoptotic nuclei were assessed by the end labelling of DNA 3' ends with dUTP-FITC using a modification of the TUNEL method [17].

2.3 Reoxygenation
For analysis of the protective effect of CT-1 added at the time of reoxygenation, cardiac cell cultures were exposed to 6 h of lethal ischaemia as described above and then incubated in a normoxic environment for 2 h (reoxygenation). At the point of reoxygenation the ischaemic buffer was removed and replaced with 1 ml minimal media with and without the addition of 2 ng/ml of CT-1 for 2 h. To investigate the downstream signalling pathway which mediates CT-1 cardioprotection in reperfusion injury the cardiac myocytes were incubated for 10 min in 1 ml of minimal medium containing signal transduction inhibitors, prior to the 2-h incubation with 2 ng/ml of CT-1. Following reoxygenation, the cardiac myocytes were assessed for cell survival by trypan blue exclusion and apoptotic nuclei were assessed by the end labelling of DNA 3' ends with dUTP-FITC using a modification of the TUNEL method.

2.4 Determination of cardiocyte viability
After the cardiocyte cultures were subjected to lethal ischaemia, they were washed with phosphate buffered saline (PBS), trypsinised for 2 min in 0.25 mg/ml trypsin in versene (GIBCO BRL) and then neutralised by the addition of new born calf serum. Cells were centrifuged (12 000xg for 10 min). The supernatant was aspirated and the cardiocytes were resuspended in a suitable volume of PBS (100 µl). Following addition of an equal volume of 0.8% trypan blue in PBS, the cells were counted in a haemocytometer. The number of dead cells with disrupted membranes (blue cells) in a total of 250 cells was counted in triplicate, for each well of plated cells. Cell death is represented as the mean percentage of blue cells/total cells.

2.5 Assessment of apoptosis
2.5.1 Tunel labelling
Apoptotic nuclei were assessed by the end labelling of DNA 3' ends with dUTP-FITC, using a modification of the TUNEL method [17]. For TUNEL, cardiac myocytes were plated onto 1% gelatin (Sigma)-coated 24-well tissue culture dishes and TUNEL assays were performed after the cells were fixed in 4% paraformaldehyde for 30 min at 25°C and washed with PBS three times. Terminal deoxynucleotidyl transferase reaction solution, containing 2 mM fluorescein-conjugated dUTP and 10 units of terminal deoxynucleotidyl transferase (Boehringer Mannheim) was added to the cells for 2 h in a 37°C humidified incubator. After washing with PBS the cells were then imaged with fluorescent microscopy.

2.5.2 Annexin V staining
Annexin V, an anticoagulant is a member of a family of proteins, which binds to negatively charged phospholipids, although it binds with highest affinity to phosphatidyl serine (PS). PS translocation occurs early in apoptosis when the cell membrane is still intact. To investigate whether CT-1 inhibited apoptosis in reperfusion injury by annexin V labelling, the cardiac myocytes were exposed to 2 h of ischaemia. The ischaemic buffer was removed and the cells were washed with ice cold PBS, pH7.4. One hundred µl of diluted annexin V-FITC solution (Sigma) [containing 10 µg/ml FITC-labelled annexin V diluted in Hepes binding buffer (10 mM Hepes, pH7.4, 140 mM NaCl, 5 mM KC1, 5 mM CaC1₂)] was added to each well of cells. The cells were incubated at room temperature, in the dark for 2 h on a swirling base. Following the incubation with the label, the cells were fixed with 1% paraformaldehyde for 30 min at 25°C and washed with PBS three times. The cells were then imaged with fluorescent microscopy and the percentage of annexin V positive cells expressed as a percentage of total cells. A minimum of 250 cells were scored, three times per well of cells.

2.6 Activation of ERK1/2, p42/44 kinase and STAT-3 phosphorylation by CT-1
Cardiac myocytes were cultured in serum free media for 24 h at 37°C in a humidified atmosphere of 5% CO₂, 21% O₂ (normoxic environment) and treated with CT-1 for 10, 20, 40, 60, 120, 360 min in six well culture dishes. Cells were harvested immediately in 200 µl of ice cold lysis buffer containing 0.1% (v/v) 2-mercaptoethanol, 0.01% (v/v) Triton-X-100, 1 mM EDTA, 1 mM EGTA, 10 mM Tris pH 7.4, 0.2 mM Sodium vanadate, 0.2 mM phenyl methyl sulfonylfluoride. An aliquot of cell lysate was retained for protein content determination (Bio-Rad). Cellular proteins were resuspended in an equal volume of 2x sample treatment buffer (STB; 100 mM Tris (pH 6.8), 200 mM DTT, 4% (w/v) SDS, 0.2 (w/v)% bromophenol blue, 20% (w/v) glycerol). The samples were boiled for 3 min and proteins were electrophoresed on a 12% SDS–PAGE gel and were subsequently transferred onto hybond C nitrocellulose membranes and then probed for 2 h at room temperature using antibodies specific for phosphorylated/active ERK1/2, p42/44 and STAT3. The membranes were washed in PBS/0.05% (v/v) Tween and incubated with a peroxidase-conjugated antibody. Immunoreactive bands were visualised by enhanced chemiluminescence (ECL). The relative protein levels were determined using densitometry (Bio Rad) normalising to the actin band on a duplicate Coomassie Brilliant Blue R250 (BDH) stained gel and probing the membranes with anti-actin antibody.

2.7 Antibodies for western blotting
Anti-active MAPK pAb, rabbit ERK1/2, p42/44 antibody was obtained from Promega, Madison, W1 53711-5399 USA (Cat. No. V8031). Antibody to phosphorylated-STAT3, P-STAT3 (B7), (Cat. No. Sc-8059) mouse monoclonal IgG_I was obtained from Santa Cruz Biotechnology, supplied by Insight Biotechnology Ltd., Wembley, Middlesex, UK. Anti-actin goat polyclonal antibody (Cat. No. (C-11): SC-1615) was supplied by Insight Biotechnology. Secondary antibodies used were HRP-conjugated anti-mouse (Cat. No. P0260) and anti-rabbit IgG (Cat. No. P0448) and anti-rabbit IgG (Cat. No. P0160) were obtained from DAKO A/s, Denmark. For Western blot analysis all primary antibodies were used at a dilution of 1/1000. Secondary antibodies were used at a dilution of 1/2000. Blots were scanned using an Epson perfection photoscanner and gene tools from Syngene.

2.8 Statistical analysis
Data are expressed as means±S.D. Single factor ANOVA was performed for each group of treatments and significance was assumed when the P-value was less than 0.05. Differences among means were compared within the treatment groups using Bonferroni's post-hoc test. The experiments were repeated as indicated in the figure legends, each n number corresponds to the mean of three random fields per well of cells with a minimum of 250 cells scored per view. In all cases, comparisons were performed between untreated samples and those treated with CT-1 or between a sample treated with an inhibitor and the same treatment without the inhibitor.

	3 Results

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
In our previous experiments [13] we observed a protective effect of CT-1 when it was added 24 h before exposure of cultured cardiocytes to simulated ischaemia. To investigate the time required for this protective effect, we added CT-1 at progressively shorter intervals before exposure to the simulated ischaemia. As shown in Fig. 1, a clear protective effect against the cell death induced by this stress was observed even when the CT-1 was added only 30 min before the stressful stimulus and then removed prior to exposure to the stress. Thus 64.8% of the cells failed to exclude trypan blue in control cultures exposed to simulated ischaemia and CT-1 pre-treatment for different periods reduced this to 51.2% (30 min), 42.8% (60 min) or 38.0% (120 min). In contrast, no effect was observed when the CT-1 was added immediately before the stressful stimulus and then removed.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 CT-1 (2 ng/ml) was added to the cardiac myocytes and either immediately removed prior to a 6 h simulated lethal ischaemic stress or the cells were incubated with CT-1 under normoxic conditions for 30, 60 or 120 min prior to the stress. The cells were then harvested and cell death was assessed by trypan blue exclusion as described in Materials and methods. The immediate addition and then removal of CT-1 failed to protect the cardiac myocytes from simulated lethal ischaemic stress (P=0.53 n=9) compared to the untreated cells exposed to the same stress. An incubation period of 30 min with CT-1 resulted in protection from simulated lethal ischaemic stress (P=0.005, n=10) which was also observed when the CT-1 was incubated with the cells for 60 (P=0.0009, n=7) and 120 min (P=0.00007 n=8). P Value for ANOVA group differences=1x10–12, *P<0.05.

 
In these and all other experiments, the total number of trypan blue positive and negative cells counted at the end of the experiment after ischaemia was the same as the number of cells counted prior to treatment indicating that dead cells were not being missed by this procedure (data not shown). Although these experiments involved analysing the cells immediately after the period of simulated ischaemia, the protective effect of a 30 min pre-treatment with CT-1 was also observed when cells were exposed to simulated ischaemia followed by reoxygenation for 24 h prior to harvesting confirming that this effect could be observed after a long period of reoxygenation (Fig. 2). As expected, enhanced cell death was observed in the control cells following exposure to reoxygenation (P<0.05, compare Figs. 1 and 2) since such enhanced cell death during reperfusion following ischaemia has previously been observed in the heart in vivo [20].

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Cardiac myocytes were cultured with or without CT-1 for 30 min under normoxic conditions. The CT-1 was removed from the cells, which were then exposed to a 6 h simulated lethal ischaemic stress. The cells were then incubated in growth media for 24 h under normal conditions. Subsequently cell death was assessed by trypan blue exclusion. CT-1 protected the cardiac myocytes from cell death for 24 h of reoxygenation following ischaemic injury as cell death decreased from 82.8% in the untreated cells compared to 48.2% in the CT-1 treated cells (*P<0.05). n=10 for each treatment.

 
Although we previously observed that the protective effect of CT-1 was correlated with its ability to induce enhanced synthesis of the heat shock proteins hsp70 and hsp90 [13] the rapid protective effect of CT-1 suggested that this might be independent of protein synthesis. To investigate whether a protective effect of CT-1 could be obtained in the absence of de novo protein synthesis, CT-1 was added to cardiac myocytes either alone or in the presence of the protein synthesis inhibitor cycloheximide, 1 h prior to exposure to the stress. In all experiments on the protective effect of CT-1 added prior to simulated ischaemia, cells were analysed immediately after the period of simulated ischaemia. In these experiments (Fig. 3) the addition of cycloheximide had no effect on the protective effect of CT-1 indicating that this effect does not appear to depend upon de novo protein synthesis in the treated cells.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 CT-1 (2 ng/ml) was added to cardiac myocytes for 60 min in the presence and absence of cycloheximide (1 µg/ml). The CT-1/cycloheximide was removed from the cells, which were then exposed to a 6 h simulated lethal ischaemic stress. The cells were then harvested and cell death was assessed by trypan blue exclusion as described in the Materials and methods. CT-1 treated cells exhibited protection compared to the control (P<0.05) and this was also observed for the cells treated with cycloheximide and CT-1 compared to cycloheximide treated controls (*P<0.05). n=8 for each treatment.

 
In view of the rapid protective effect of CT-1 and its lack of dependence upon protein synthesis, we wished to investigate the signalling pathways which were involved in the protective effect against simulated ischaemia and which had not previously been investigated. Previous studies with members of the IL-6 family have indicated that they can activate both the p42/p44 MAP kinase pathway resulting in the phosphorylation of the transcription factor NF-IL6 and the Jak/STAT pathway resulting in the phosphorylation of the STAT-3 transcription factor [5,8–10].

To confirm that these effects were also observed in our system, we treated cardiocytes with CT-1 for various periods and assayed cell extracts by western blotting with antibodies specific for the active phosphorylated form of the p42/p44 MAPK enzymes or the phosphorylated form of STAT-3. As illustrated in Fig. 4 the phosphorylated form of the p42/p44 MAPK (ERK 1/2) enzymes was only observed in the CT-1 treated cells (panel a) whilst enhanced levels of phosphorylated STAT-3 were observed in CT-1 treated cells compared to untreated cells (panel b). In contrast no phosphorylation of p38 MAPK or JNK was observed in the CT-1 treated cells using a specific antibody indicating that CT-1 does not significantly activate these pathways (data not shown). Hence both the p42/p44 MAPK and Jak/STAT pathways are activated by CT-1 in our system. Enhanced phosphorylation of p42/p44 MAPK was also observed in cardiac cells exposed to ischaemia/reoxygenation and treated with CT-1 compared to the level observed in cells exposed to ischaemia/reoxygenation alone (panels c and d).

View larger version (64K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Western blots of cardiac cell extracts. Each experiment was repeated three times and a typical blot is shown. Panel a shows the result of blotting extracts from cells treated with CT-1 or left untreated for various periods using antibodies to the active phosphorylated form of the p42/p44 MAPK (ERK 1/2) enzymes or to actin. Panel b shows the result using an antibody specific for the phosphorylated form of STAT-3 in CT-1 treated or untreated (NT) cells. Panel c shows the level of phosphorylated p42/p44 and of total p42/p44 in cells exposed to ischaemia/reoxygenation and either left untreated or treated with CT-1 in the presence or absence of PD98059 (50 µM). Panel d shows the results of densitometric scanning of three replicate blots of the type shown in panel c. Values show the mean and the bars indicate the standard deviation.

 
The protective effect of CT-1 against apoptosis in cardiocytes grown in serum-free medium has previously been shown to be inhibited by PD98059, a specific inhibitor of the p42/p44 MAP kinase pathway which acts by inhibiting the activity of the upstream activator of these kinases MEK1. As expected, the enhanced phosphorylation of p42/p44 induced by CT-1 was blocked by addition of PD98059 (Fig. 4c and d).The protective effect of CT-1 against simulated ischaemia was therefore investigated using the trypan blue exclusion assay in the presence or absence of this inhibitor as well as in the presence or absence of the p38 MAP kinase inhibitor SB203580 [19]. In these experiments (Fig. 5) the PD98059 p42/p44 MAPK inhibitor completely blocked the protective effect of CT-1 and a dose response experiment showed that this inhibitory effect could be achieved at very low doses of the inhibitor down to 0.5 µM (data not shown). In contrast, no inhibition of the protective effect was observed with the p38 MAPK inhibitor even though the concentration of this inhibitor used was sufficient to block the p38 MAPK phosphorylation which occurs during reoxygenation (data not shown) in agreement with the work of others using this inhibitor concentration [21,22].

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 CT-1 was cultured with cardiac myocytes for 30 min under normoxic conditions in the presence and absence of PD98059 (50 µM) or SB203580 (10 µM). The inhibitors were added to the cells 30 min before the addition of CT-1. The CT-1 and inhibitors were removed from the cells, which were then exposed to a 6 h simulated lethal ischaemic stress (I). The cells were harvested and cell death was assessed by trypan blue exclusion as described in Materials and methods. CT-1 significantly protected the cardiac myocytes from ischaemic injury (P=0.028) compared to untreated cells (NT). PD98059 inhibited the protective effect of CT-1 (P=0.0016). SB203580 had no effect on the protective effect of CT-1 (P=0.97). PD98059 alone had no significant effect on survival after hypoxic/ischaemic stress whilst SB203580 alone was cardioprotective (P=0.0007). P Value for ANOVA group differences 2x10–6. n=6 for all treatments.

 
This suggests that, as with the protective effect against serum starvation, the protective effect of CT-1 against ischaemic stress involves the MEK1 kinase, which specifically activates the p42/p44 MAP kinase pathway. No significant effect of PD98059 on cell survival was observed in the absence of CT-1 confirming that the effect of PD98059 specifically involved blocking the protective effect of CT-1. Abolition of p38 MAPK activity with SB 203580 did itself produce a protective effect (Fig. 5) in agreement with previous reports on the detrimental effects of activating this kinase [21,23]. SB203580 had no effect on the protective effect of CT-1, suggesting that p38 MAPK was not involved in this effect, although the protective effect of SB203580 alone means that this cannot be definitively excluded.

As well as investigating the effect of the different inhibitors using the trypan blue exclusion assay, which measures total cell death, both necrotic and apoptotic, we also utilised the TUNEL labelling assay to measure the effect on apoptotic cell death. In these experiments (Fig. 6), CT-1 treatment produced a clear protective effect. This protective effect was abolished by PB98059 but not by SB203580. This further supports the role of the p42/p44 MAPK pathway in mediating the protective effect against cell death when CT-1 is added prior to the insult.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Percentage of TUNEL positive cells in the cardiac myocytes treated with CT-1 and the various signal transduction inhibitors 30 min prior to exposure to simulated lethal ischaemia (I). The cells were fixed using 4% paraformaldehyde and the % of TUNEL positive nuclei were analysed using fluorescein-12 dUTP and terminal transferase (as described in Materials and methods). The CT-1 had a protective effect (P<0.05) which was abolished by PD98059 (P<0.05) but not by SB203580. The inhibitors alone had no statistically significant effect on the number of TUNEL positive cells. n=8 for each treatment.

 
Having established the pattern of protection by CT-1 when added before simulated ischaemia we wished to establish whether CT-1 could have a protective effect when added solely during the period of reoxygenation. In these experiments, CT-1 was added at the end of the period of simulated ischaemia and cells analysed after 2 h reoxygenation. Most interestingly, we found that CT-1 had a protective effect against both the total amount of cell death observed (Fig. 7) and the amount of apoptosis as measured by TUNEL labelling (Fig. 8) or staining with labelled annexin V (Fig. 9) when added at the time of reoxygenation. This is the first time that a protective effect of CT-1 has been demonstrated when it is added after rather than before the ischaemic stimulus. Moreover, this effect can be demonstrated both by assaying total cell death and by assaying apoptotic cell death using two assays which measure two different aspects of apoptosis, namely DNA degradation (TUNEL) and translocation of phosphatidyl serine to the plasma membrane (annexin V).

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Cardiac myocytes were exposed to simulated lethal ischaemic injury for 6 h. Immediately after this period, the stimulated ischaemic buffer was removed and replaced with normal growth medium supplemented with CT-1 (2 ng/ml) in the presence and absence of PD98059 (50 µM), or SB203580 (10 µM). The inhibitors were added to the cells 10 min before the addition of CT-1. The cells were incubated under normoxic, oxygenated conditions for 2 h. Subsequent to this period the cells were harvested and cell death following ischaemia/reoxygenation (IR) was assessed by trypan blue exclusion as described in Materials and methods, CT-1 significantly protected the cardiac myocytes from injury when added at the point of reoxygenation (P=0.016). PD98059 partially inhibited the protective effect of CT-1 (P=0.0165) SB203580 had no effect on the protective effect of CT-1 (P=0.938). PD98059 alone had no statistically significant effect on cell survival whilst SB203580 alone had a protective effect (P=0.015). P Value for ANOVA group differences 3.44x10–7. n=6 for each treatment.

 

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Cardiac myocytes were exposed to simulated lethal ischaemic injury for 6 h. Immediately after this period, the simulated ischaemic buffer was removed and replaced with normal growth medium supplemented with CT-1 (2 ng/ml) in the presence and absence of PD98059 (50 µM), or SB203580 (10 µM). The inhibitors were added to the cells 10 min before the addition of CT-1. The cells were incubated under normoxic, oxygenated conditions for 2 h. Subsequent to this period the cells were harvested and apoptotic cell death was assessed by TUNEL labelling as described in Materials and methods. CT-1 significantly protected the cardiac myocytes from injury when added at the point of reoxygenation (P=0.0001). PD98059 inhibited the protective effect of CT-1 (P=0.001) and SB203580 (P=0.0047) also had an inhibitory effect. Neither of the inhibitors alone had any significant effect on the number of TUNEL labelled cells observed in the absence of CT-1. P Value for ANOVA group differences 1.57x10–5. n=8 for each treatment.

 

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 Cardiac myocytes were exposed to simulated ischaemic injury for 2 h. Immediately after this period, the simulated ischaemic buffer was removed and replaced with normal growth medium supplemented with CT-1 (2 ng/ml) in the presence and absence of PD98059 (50 µM), or SB203580 (10 µM). The inhibitors were added to the cells 10 min before the addition of CT-1. The cells were incubated under normoxic, oxygenated conditions for 2 h. Subsequent to this period the cells were harvested and apoptotic cell death was assessed by Annexin-V labelling as described in Materials and methods. CT-1 significantly protected the cardiac myocytes from ischaemic injury when added at the point of reoxygenation (P=0.0007). PD98059 inhibited the protective effect of CT-1 (P=0.00078) as did SB (P=0.017) although to a much smaller extent. PD98059 alone had no statistically significant effect on the number of annexin V labelled cells observed in the absence of CT-1 whilst SB203580 alone had some protective effect (P=0.014). P Value for ANOVA group differences 1.45x10–7. n=6 for each treatment.

 
As in the case of the protective effect observed when CT-1 was added before the period of simulated ischaemia, the protective effect observed during re-oxygenation was inhibited by PD98059, but not to the same extent by SB203580. This indicates that this effect is similarly likely to be dependent primarily upon activation of the p42/p44 MAP kinase pathway rather than upon the activation of the p38 MAP kinase pathway. It is possible however, that the p38 MAPK pathway may play some role since an effect of SB203580 on the protective effect of CT-1 was observed in the TUNEL assay. No statistically significant effect of PD98059 alone on cell survival was observed with any of the assays confirming that the effect of PD98059 was specific for CT-1, as in the experiments involving pre-treatment with CT-1. A similar protective effect of CT-1 added at reoxygenation was also observed in experiments where the cells were incubated for up to 96 h in normal medium prior to assay confirming that it could be observed following prolonged periods of reoxygenation (data not shown). Interestingly, addition of CT-1 both prior to ischaemia and at the time of reoxygenation did not provide enhanced protection compared to either treatment alone (data not shown).

	4 Discussion

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
The development of methods for reducing the damage suffered by individuals undergoing episodes of cardiac ischaemia is evidently of central importance in cardiology. As a result of intensive studies, it has been shown that treatments such as pre-conditioning with a series of short ischaemic episodes or treatment with a variety of agents can be effective at minimising such damage in the intact animal. However, such treatments are effective at minimising damage only if given prior to the severe ischaemic episode and are therefore limited in their clinical applicability since the majority of individuals present with an ongoing ischaemic episode.

Here we show for the first time that the cardiac cytokine CT-1 can be effective at minimising the damage caused by a period of simulated ischaemia followed by re-oxygenation when given at the time of re-oxygenation as well as prior to the ischaemic period. Hence, CT-1 is able to exert a protective effect against the damage, which occurs during re-oxygenation following a period of simulated ischaemia.

To date no studies have demonstrated conclusively that pharmacological agents given at the time of reperfusion can protect the myocardium from reperfusion induced lethal injury. Although controversial, some studies have demonstrated however that an inhibition of leukocyte activation is useful at the time of reperfusion [24]. In addition studies by Forman's group [25,26] have suggested that adenosine given at reperfusion in animal models can protect against lethal reperfusion injury although this was not seen by others [27,28].

However recently we have investigated the ability of specific factors to protect against injury when given at reperfusion. In this respect we have recently demonstrated that factors such as urocortin and insulin both appear able to protect the myocardium directly if given at the onset of reperfusion in a similar manner to that observed using CT-1 in the present study [29,30].

It has been shown that reperfusion/re-oxygenation following ischaemia results in apoptotic cell death both in vitro [31] and in the intact heart in vivo [32] and it appears that CT-1 exerts its protective effect by minimising such apoptotic cell death. Thus, previous studies by others demonstrated that CT-1 could minimise the apoptotic death of cardiocytes cultured in serum free medium [11,12] and we have shown that CT-1 also reduces the extent of apoptotic cell death in cardiocytes exposed to stressful stimuli, whether it is added prior to the period of hypoxia [13] or at the time of re-oxygenation (this study).

Cytokines of the IL-6 family such as CT-1 have previously been shown to activate both the p42/p44 MAP kinase pathway and the Jak/STAT pathway [8–10,33] and this was also observed in our study (Fig. 4). In cardiocytes exposed to serum free medium, the protective effect of CT-1 against apoptosis is dependent upon its ability to activate p42/p44 MAP kinase and appears to be independent of its activation of the Jak/STAT pathway [12]. In contrast, the hypertrophic effect of CT-1 was not inhibited by inhibition of the p42/p44 MAPK pathway [11]. In the work presented here, we have similarly demonstrated that the protective effect of CT-1 against either simulated ischaemia or re-oxygenation can be abolished by treatment with PD98059, a specific inhibitor of the p42/p44 MAP kinase pathway. Hence these novel protective effects are also likely to be dependent on this pathway.

IL-6 family cytokines are able to activate the NF-IL6 transcription factor by MAP kinase-dependent phosphorylation [8,33]. Hence, newly activated NF-IL6 could activate the transcription of genes encoding protective proteins resulting in the protective effect of CT-1. Indeed, we have previously demonstrated that CT-1 can enhance the expression of the protective heat shock proteins hsp70 and hsp90 whose transcription is known to be stimulated by activated NF-IL6 [13,34]. However, our observation that the protective effect of CT-1 does not require de novo protein synthesis suggests that this protective effect can be achieved by the phosphorylation of proteins other than transcription factors which do not need to stimulate new gene transcription/protein synthesis to have a protective effect.

Further studies will be required in order to demonstrate the protective effect of CT-1 in the intact animal and in the ex vivo heart preparation. Similarly, it will be important to identify the mechanisms by which CT-1 activation of the p42/p44 MAP kinase pathway results in enhanced protection. This is of particular importance since it may well lead to a method of activating this protective pathway either with CT-1 or related agents without activating the potentially damaging hypertrophy pathway which is also activated by CT-1, hence allowing therapeutic advantage to be taken of the ability of CT-1 to have a protective effect when given at the time of reperfusion following an ischaemic episode.

Time for primary review 38 days.

	Acknowledgements

 
This work was supported by the British Heart Foundation. Z.L. was supported by a Royal Society Britain–China Fellowship.

	References

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 

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