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Cardiovascular Research 2002 53(4):902-910; doi:10.1016/S0008-6363(01)00531-4
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Cardiotrophin-1 (CT-1) can protect the adult heart from injury when added both prior to ischaemia and at reperfusion

Zhihong Liaoa,1, Bhawanjit K. Brara,1, Qing Caib, Anastasis Stephanoua, Rhona M. O'Learyc, Diane Pennicad, Derek M. Yellonb and David S. Latchmana,*

aInstitute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK
bHatter Institute of Cardiology, University College Hospitals, London, UK
cDepartment of Protein Sciences, Genentech Inc., South San Francisco, CA, USA
dDepartment of Molecular Oncology, Genentech Inc., South San Francisco, CA, USA

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

Received 11 July 2001; accepted 5 November 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: To determine whether the cytokine cardiotrophin-1 (CT-1) can protect the adult heart against ischaemia/reperfusion when added either prior to ischaemia or at reperfusion. Background: CT-1 has previously been shown to protect cultured embryonic or neonatal cardiocytes from cell death. To assess the therapeutic potential of CT-1, it is necessary to determine whether this effect can be observed in adult cardiac cells both in culture and most importantly in the intact heart. Methods: We examined the protective effect of CT-1 both in cultured adult rat cardiocytes and in the rat intact heart. In both cases, the cardiac cells were exposed to hypoxia/ischaemia followed by reoxygenation/reperfusion and CT-1 was administered either prior to hypoxia/ischaemia or at reoxygenation/reperfusion. Results: CT-1 has a protective effect in reducing ischaemic damage in the intact heart ex vivo as assayed by infarct size to area at risk ratio (20% compared to 35%). Similar protective effects against cell death were noted in adult cells in vitro. Both in vitro and ex vivo CT-1 can exert a protective effect when added at the time of reoxygenation/reperfusion as well as prior to the hypoxic/ischaemic stimulus (cell death reduced from 50 to 20% in TUNEL assay, infarct size to zone at risk ratio reduced from 35 to 20%). These protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway. Conclusion: CT-1 can protect adult cardiac cells both in vitro and in vivo when added both prior to or after the hypoxic/ischaemic stimulus. The potential therapeutic benefit of CT-1 when added at the time of reperfusion following ischaemic damage is discussed.

Condensed abstract

The cytokine cardiotrophin-1 (CT-1) has previously been shown to have protective effects in embryonic or neonatal cardiac cells in culture. Here we show for the first time that CT-1 can protect adult cardiac cells both in culture and in the intact heart exposed to ischaemia/reperfusion. Moreover, this protective effect can be observed when CT-1 is added at reperfusion after ischaemia as well as prior to ischaemia. The ability of CT-1 to protect the intact adult heart when given at reperfusion suggests it may have therapeutic potential in the clinical situation.

KEYWORDS Apoptosis; Cytokines; Hypoxia/anoxia; Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. 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), CNTF, oncostatin M and IL-11 [3,4]. The receptor for each of these factors contains a common component known as gp130 (for review see Refs. [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 embryonic or neonatal 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].

These findings clearly indicate a protective effect for CT-1 in cultured embryonic or neonatal cardiocytes. However, in contrast to the hypertrophic effects of CT-1, such protective effects have not yet been demonstrated in the intact heart, although a protective effect of CT-1 has been demonstrated using atrial appendages derived from adult human cardiac patients [14]. Indeed, CT-1 has never been tested for its protective effect in cultured adult as opposed to embryonic or neonatal cardiac cells. This is of particular importance since CT-1 has been shown to induce the synthesis of protective heat shock proteins such as hsp70 or hsp90 and other protective stimuli which induce these proteins have been shown to be less effective in cells from aged compared to younger animals both in terms of hsp induction and protective effect [15,16]. Moreover, a wide variety of differences exist between cultured neonatal and adult cardiac cells [17,18]. Thus, neonatal cardiac cells are not fully differentiated and retain the ability to undergo one cell division following plating. In contrast, cultured adult cardiac cells are non-dividing and are terminally differentiated. In addition, since mitochondrial structure/arrangement in the cell and energy metabolism differ in adult compared to neonatal myocytes, the responses of adult myocytes to ischaemia differ from those of neonatal cells and therefore need to be studied directly. Similarly, cardiac cells from animals of different ages show differences in contractility and its response to calcium channel antagonists [19].

Here we demonstrate that CT-1 has a protective effect in both cultured adult cardiac cells and in the intact adult heart ex vivo subjected to ischaemia/reperfusion. Moreover, we demonstrate that both in vitro and ex vivo, CT-1 can have a protective effect when added at the time of reperfusion/reoxygenation as well as when added prior to the ischaemic/hypoxic period and that these protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Culture of adult cardiac cells
Adult cardiac myocytes were isolated using a combination of enzymatic and mechanical dispersion after Langendorff perfusion of the whole heart [20–22]. The hearts from adult female Sprague–Dawley rats, ranging in age from 2 to 7 months, were excised and then rapidly mounted on to the aortic cannula of a Langendorff perfusion system. The perfusion flow rate was kept constant at 9 ml/min per g of heart tissue. All buffers were heated to 37 °C and oxygenated with medically supplied oxygen (BOC gases). Initially, the heart was perfused for 2 min with modified Krebs–Hensliet (KH) buffer containing low concentration of physiological Ca2+. This contained 130 mM NaCl, 5.4 mM KCl, 5 mM HEPES, 0.4 mM NaH2PO4, 3.5 mM MgCl2, 750 µM CaCl2·2H2O, 10 mM glucose, pH 7.4, O2–CO2 (95:5). The hearts were then perfused with the above buffer containing 100 µM EGTA for 4 min and finally with a Collagenase type 2 enzyme solution consisting of KH buffer supplemented with 0.8 mg/ml Collagenase type 2 (Worthington Biochem) and 200 µM Ca2+ for 15 min.

The left ventricle was removed from the heart and cut into small pieces to increase the surface area for enzymatic digestion in oxygenated "shake solution" [KH buffer supplemented with 0.8 mg/ml collagenase type 2, 10% bovine serum albumin (BSA) (Sigma), 200 µM Ca2+] for 5 min in a 37 °C shaking water bath. The digested heart tissue was passed through nylon gauze for filtration with restore solution consisting of KH buffer supplemented with 10% BSA, 200 µM CaCl2. The cells were sedimented under gravity for 5–10 min and isolated cells were washed with restore solution twice. The cells were then washed with 50% (v/v) restore solution and 50% (v/v) DMEM (Gibco) supplemented with 80 µM EGTA, 1% (v/v) penicillin/streptomycin (PS) (Gibco) (wash solution) twice and finally with the wash solution once. The cells were then plated at a concentration of 2·105 cells/ml in DMEM containing 80 µM EGTA, 1% (v/v) PS and 1% (v/v) fetal calf serum (FCS) and plated on 3 cm dishes that were coated with Laminin (Sigma) at a concentration of 15 µg Laminin/ml in phosphate-buffered saline (PBS) for 2 h at room temperature in a laminar flow hood, prior to plating. For the annexin V labelling and TUNEL studies, cardiac myocytes were plated onto tissue culture slides (NUNC). The cells were pre-plated for 2 h and media was replaced with fresh media for 24 h prior to experimentation. Cells were maintained in a 37 °C Heraeus incubator in a humidified atmosphere under 5% CO2 in air.

2.2. Hypoxia
To produce hypoxia in vitro, cultured cardiocytes were incubated in 1 ml of hypoxic buffer (137 mM NaCl, 12 mM KCl, 0.49 mM MgCl2, 0.9 mM CaCl2·2H2O, 4 mM HEPES, 20 mM sodium lactate, pH 6.2) for 6 h in an atmosphere of CO2–argon (5:95) at 37 °C producing a PO2 level of 4 mmHg [23]. CT-1 (Genentech) at a concentration of 20 ng/ml was added to the cardiac myocytes for 24 h and removed prior to a lethal hypoxic insult (Fig. 1). For assays of the protective effect of CT-1 added prior to hypoxia the cells were analysed immediately after the hypoxic period. The MEK1 inhibitor (PD98059) (New England Biolabs, Beverly, MA, USA) [24,25] was stored at –20 °C at a concentration of 100 mM and was added at a concentration of 50 µM, 10 min prior to the addition of CT-1.


Figure 1
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  		Fig. 1 Protocol used to test the protective effect of CT-1 added either prior to hypoxia or at reoxygenation.

 
2.3. Reoxygenation
For analysis of the protective effect of CT-1 added at the time of reoxygenation, cardiac cell cultures were exposed to 1 h of lethal hypoxia as described above and then incubated in a normoxic environment for 2 h (reoxygenation) (Fig. 1). At the point of reoxygenation the hypoxic buffer was removed and replaced with 1 ml minimal media with and without the addition of 20 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 20 ng/ml of CT-1.

2.4. TUNEL labelling
DNA degradation was assessed by the end labelling of DNA 3' ends with dUTP-FITC, using a modification of the TUNEL method [26] as previously described [27–29].

2.5. Annexin V staining
Surface staining with annexin V to test for the translocation of phosphatidyl serine to the outer surface of the plasma membrane which occurs early in apoptosis [30] was carried out as previously described [27–29]. Only cells staining positive for annexin V and negative for propidium iodide were scored as apoptotic to eliminate membrane permeable necrotic cells which take up both stains.

2.6. Animals
Male Sprague–Dawley rats (280–400 g body weight) were used. All animals were obtained from the same supplier, fed a standard pelleted diet with free access to water, housed under the same conditions and received humane care in accordance with the Guidelines on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office (London, UK).

2.7. Heart perfusion and treatment protocol
The animals were anaesthetised by i.p. administration of sodium pentobarbital (55 mg/kg) and were given heparin (300 IU). The hearts were excised, placed in chilled buffer solution and within 1 min mounted on a constant pressure (80 mmHg) Langendorff perfusion apparatus. They were perfused retrogradely through the ascending aorta with a modified KH hydrogencarbonate buffer containing (in mM): NaCl 118.5, NaHCO3 25, KCl 4.8, MgSO4 1.2 KH2PO4 1.2, CaCl2 1.7, glucose 12. All solutions were filtered through a Whatman microfiber filter (2.0 µm pore), gassed with O2–CO2 (95:5) and maintained at 37 °C with the help of water heated double jacket chambers. The temperature was permanently monitored by a thermocouple inserted in the right ventricle. The pH of the perfusate was ascertained with a blood gas analyser (AVL type 993; AVL Instruments, Stone, UK) and adjusted as necessary to maintain pH close to 7.4 (±0.05) by modifying the gas output. A latex isovolumic balloon was introduced into the left ventricle through an insertion in the left atrial appendage and inflated to give a preload of 2–15 mmHg. The balloon catheter was attached by a pressure transducer to a chart recorder (Lectromed, Welwyn Garden City, UK). Left ventricular developed pressure, heart rate and coronary flow were registered at regular intervals.

A 4/0 silk suture on a round bodied surgical needle (Mersilk type W546, Ethicon, Edinburgh, UK) was passed under the left main coronary artery and the ends of the suture were passed through a small plastic tube to form a snare. Regional ischaemia was induced by tightening the snare and clamping in place with haemostat forceps. Reperfusion was instituted by releasing the ends of the suture.

Six groups are included in the study (Fig. 6). Group (i): control, (n=8) 20 min perfusion without intervention followed by 35 min coronary occlusion and 120 min reperfusion; Group (ii): CT-1 pre-ischaemia, (n=8) 30 min perfusion of 10 µg CT-1/1 of the buffer was followed by a 35 min period of ischaemia and 120 min reperfusion. Group (iii): (n=8) following 35 min ischaemia CT-1 (10 µg/l) was perfused for 30 min at the beginning of 120 min reperfusion. Group (iv): (n=8) following 35 min ischaemia PD (5 µM) and CT-1 (10 µg/l) was perfused together for 30 min at the beginning of 120 min reperfusion. Group (v): (n=6) following ischaemia PD (5 µM) alone was perfused for 30 min at the beginning of 120 min reperfusion. Group (vi): (n=8) CT-1 (10 µg/l) was perfused during the 35 min ischaemia. Infarct size was measured as previously described [27].


Figure 6
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  		Fig. 6 Protocol used to test the protective effect of CT-1 in the isolated heart on a Langendorff perfusion apparatus and to investigate the effect of PD98059 on the protection.

 
2.8. Statistical analysis
Data for in vitro experiments are expressed as means±S.D. Single-factor analysis of variance (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 the Student's t-test. The experiments were repeated at least three times for each experiment, each n number corresponds to the mean of three random fields per well of cells with a minimum of 250 cells scored per view. Infarct size data were tested for group differences by one-way ANOVA combined with Tukey's posthoc test. ANOVA, P values and the Student's t-test values that were less than *<0.05, were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Previous animal work showing protective effects of CT-1 has been conducted in embryonic or neonatal cardiac cultures which are of limited relevance to any potential therapeutic situation [11–13]. We therefore wished to evaluate the protective effect of CT-1 in cultures from adult rats and compare it to that observed in neonatal cultures. Accordingly, CT-1 was added at different doses to cardiocyte cultures derived from rats of different ages and 24 h later the cells were exposed to hypoxia (see Fig. 1 for details of the experimental protocol). Cell survival was assayed by the trypan blue exclusion assay which measures total cell death, both necrotic and apoptotic on the basis of the ability of live cells to exclude trypan blue.

As illustrated in Fig. 2, a dose of 20 ng/ml was able to produce a similar statistically significant protection against hypoxia in cells from neonatal rats aged 2 days or adult rats aged 2, 4 and 7 months. Interestingly, however, whilst a significant protective effect could be observed in the neonatal cultures at a dose of CT-1 of 2 ng/ml in accordance with our earlier results [13], this was not seen in the adult cultures. In other experiments, doses of 5 ng/ml or 10 ng/ml of CT-1 were also ineffective in adult cultures whereas 30 ng/ml CT-1 had the same effect as 20 ng/ml (data not shown). Hence, adult cultures can be protected against subsequent exposure to hypoxia by CT-1 but a higher dose is required compared to that needed in cultures from younger neonatal animals. 20 ng/ml CT-1 was used in all subsequent experiments.


Figure 2
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  		Fig. 2 Protective effect of different doses of CT-1 added prior to hypoxia in cardiac cultures prepared from neonatal or adult rats of the indicated ages. Cell death was assayed by trypan blue exclusion immediately after exposure to simulated ischaemia. In each case, cell death in the CT-1 treated cells was expressed as a percentage of the amount of death observed in untreated cultures from each age animal exposed to hypoxia (set at 100%). Values are the mean of three experiments whose standard deviation is shown by the bars. *=P<0.05.

 
As well as investigating the effect of CT-1 using the trypan blue exclusion assay we also utilised the TUNEL labelling and annexin V assays to measure the effect on cell death using two additional methods in adult cultures. In these experiments (Fig. 3), CT-1 treatment produced a clear protective effect in cultures of adult cells as assayed by the two different methods confirming the protective effect of CT-1 in cultured adult cardiac cells exposed to hypoxia.


Figure 3
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  		Fig. 3 Protective effect of 20 ng/ml CT-1 added prior to hypoxia in cultures from 6-month-old rats as assayed by TUNEL or annexin V staining immediately after the ischaemic period. Values are the mean of three experiments whose standard deviation is shown by the bars. *=P<0.05.

 
The protective effect of CT-1 against cell death in neonatal cardiocytes grown in serum-free medium or exposed to hypoxia 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 [12,29]. The protective effect of CT-1 against hypoxia was therefore investigated in our adult cultures using the trypan blue exclusion assay in the presence or absence of this inhibitor. In these experiments (Fig. 4) the PD98059 p42/p44 MAPK inhibitor completely blocked the protective effect of CT-1 in cultures from 6-month-old animals. This suggests that, as with the protective effect in neonatal cultures, the protective effect of CT-1 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. Similar inhibition of the protective effect of CT-1 by PD98059 was also observed in TUNEL and annexin V assays (data not shown).


Figure 4
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  		Fig. 4 Effect of the p42/p44 MAPK inhibitor PD98059 on the protective effect of CT-1 as assayed by trypan blue exclusion immediately after the hypoxic period. Values are the mean of three experiments whose standard deviation is shown by the bars. *P<0.05.

 
Having established the pattern of protection by CT-1 when added before hypoxia 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 hypoxic period and cells analysed after two h reoxygenation (see Fig. 1 for experimental protocol). In these experiments we were able to observe a protective effect using trypan blue, TUNEL labelling or staining with labelled annexin V (Fig. 5) when CT-1 was added at the time of reoxygenation.


Figure 5
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  		Fig. 5 Protective effect of CT-1 against cell death induced by hypoxia followed by reoxygenation. CT-1 was added immediately prior to reoxygenation and total cell death was assayed by trypan blue exclusion, TUNEL or annexin V staining after 2 h of reoxygenation. Values are the mean of three experiments whose standard deviation is shown by the bars. *P<0.05.

 
The protective effect of CT-1 when added prior to hypoxia and more particularly its protective effect when added immediately prior to reoxygenation in cultures of cardiac cells from adult rats suggested a potential therapeutic use for this factor in individuals suffering cardiac ischaemic episodes. Despite this protective effect in vitro however, no protective effect of CT-1 against cell death in the intact adult heart has, to our knowledge, been demonstrated either prior to or after the ischaemic period. We therefore investigated the protective effect of CT-1 in the intact heart perfused ex vivo in a Langendorff perfusion apparatus (see Fig. 6 for the experimental protocol used in these experiments). In these experiments, no alteration in haemodynamic parameters such as left ventricular developed pressure, heart rate and coronary flow rate was observed in any of the groups either at baseline or following ischaemia (Tables 1 and 2Go). Similarly, the sizes of the zone at risk were similar in all groups (Table 3). However, CT-1 had a clear protective effect when added prior to the period of ischaemia, significantly reducing the degree of damage observed in the heart as measured by the ratio of infarct size to zone at risk (Fig. 7 and Table 3).


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  		Table 1 Baseline haemodynamics of all groups

 

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  		Table 2 Percentage decrease in haemodynamics from baseline values in all groups

 

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  		Table 3 Body weight and risk to left ventricular volume ratio and Infarct to risk volume ratio of all groups

 

Figure 7
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  		Fig. 7 Infarct size compared to the zone at risk in hearts subjected to ischaemia/reperfusion on a Langendorff perfusion apparatus. Results are shown for control hearts, or hearts given CT-1 either pre-ischaemia, during ischaemia or at reperfusion and for hearts given CT-1 with PD98059 or PD98059 alone at reperfusion. The results for each group are shown together with the mean value and the number of animals (n). The bars show the standard error of the mean. Both groups of CT-1 treated hearts showed significant protection compared to controls (P<0.05 in each case).

 
Most importantly however, a significant protective effect was also observed when CT-1 was added immediately prior to reperfusion and subsequent to the period of ischaemia. Hence, CT-1 can protect against injury when added after the ischaemic period and unlike the great majority of proposed protective agents (such as β-blockers, calcium antagonists and nitrates) can have an effect when added after, as well as before, the period of ischaemia. Moreover, as observed in our in vitro experiments, the protective effect of CT-1 added at reperfusion was abolished by addition of PD98059 indicating it could be prevented by an inhibitor of the p42/p44 MAPK pathway. Interestingly CT-1 also produced a protective effect when added during ischaemia. This effect is likely to be partly dependent on the small amount (approximately 6%) of collateral flow, which occurs in the rat [31]. However, it is also likely to be due to this CT-1 entering the heart at reperfusion on release of the occlusion and producing a protective effect in this manner.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. 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. Many studies in this area have been carried out with cardiac cultures from neonatal animals since these are easier to culture than those from adult animals. However, these neonatal cells are different from adult cells in their ability to divide in culture and are not fully differentiated in terms of morphology, biochemistry, etc. Most importantly, they differ in terms of energy metabolism from adult cells and hence are likely to respond to ischaemia differently. It is necessary therefore to confirm any work carried out in neonatal cardiocyte cultures by carrying out similar experiments using adult cardiac cells.

Here we show that the cytokine CT-1 has a protective effect in cultured cardiac cells from adult animals as well as its previously demonstrated protective effect in cardiac cells from neonatal or embryonic animals [11–13]. These studies in rat ventricular myocytes are in agreement with a recent study which showed a protective effect of CT-1 in human atrial appendages taken from adult human cardiac patients [14]. The protective effect of CT-1 in adult cells, is of particular importance in view of the differences between adult and neonatal cardiac cells and indeed we observed that higher doses of CT-1 were required for the protective effect in adult cultures. This requirement for a higher concentration of CT-1 may reflect a lower concentration of either the gp130 or LIF receptor β components of the CT-1 receptor in adult cells or a lower sensitivity of intra-cellular signalling pathways to activation by CT-1. Further studies will be necessary to investigate these possibilities.

Most importantly, we observed a protective effect of CT-1 in the intact perfused heart exposed to ischaemia, the first time such an effect has been demonstrated in this situation. Moreover, both in the cultured adult cells and in the intact adult heart CT-1 can be effective at minimising the damage caused by a period of ischaemia/hypoxia followed by reperfusion/re-oxygenation when given at the time of reperfusion/re-oxygenation as well as prior to the ischaemic/hypoxic period. Hence, CT-1 is able to exert a protective effect against the damage, which occurs during reperfusion/re-oxygenation following a period of ischaemia/hypoxia.

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 [32]. In addition studies by Foreman and co-workers [33,34] have suggested that adenosine given at reperfusion in animal models can protect against lethal reperfusion injury although this was not seen by others [35,36].

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 [27,28] 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 in adult cells and previously in neonatal cells [29].

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 [8–10,37]. In neonatal cardiocytes 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 [12,29]. In contrast, the hypertrophic effect of CT-1 was not inhibited by inhibition of the p42/p44 MAPK pathway [12]. In the work presented here, we have similarly demonstrated that the protective effect of CT-1 in adult cardiac cells both in vitro and in the intact heart ex vivo 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 [8,37]. 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 in neonatal cardiocytes 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 [38]. We also observed such induction in adult cells in our study (data not shown), although it is still unclear whether the induction of these proteins is essential for the protective effect of CT-1.

Further studies will be required in order to demonstrate the protective effect of CT-1 in the intact adult animal as well as 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 in adult cardiac cells. 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 in the adult heart when given at the time of reperfusion following an ischaemic episode.

Time for primary review 29 days.


   Acknowledgements
 
We are extremely grateful to Professor Chris Proud, Dr. Lijun Wang and Dr. Xuemin Wang (University of Dundee, Dundee, UK) for help in setting up the adult cardiac cultures. This work was supported by the British Heart Foundation and the Wellcome Trust. A.S. is a BHF Intermediate Fellow. Z.L. was supported by a Royal Society Britain–China Fellowship.


   Notes
 
1 These authors contributed equally to the work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Pennica D., King K.L., Shaw K.J., et al. Expression cloning of cardiotrophin 1 a cytokine that induces cardiac myocyte hypertrophy. Proc. Natl. Acad. Sci. USA (1995) 92:1142–1146.[Abstract/Free Full Text]
  2. Jin H., Yang R., Keller G.A., et al. In vivo effects of cardiotrophin-1. Cytokine (1996) 8:920–926.[CrossRef][Web of Science][Medline]
  3. Pennica D., Shaw K.J., Swanson T.A., et al. Cardiotrophin-1: biological activities and binding to the leukemia inhibitory factor receptor/gp130 signalling complex. J. Biol. Chem. (1995) 270:10915–10922.[Abstract/Free Full Text]
  4. Pennica D., Wood W.I., Chien K.R. Cardiotrophin-1: a multifunctional cytokine that signals via L.I.F. receptor gp130 Dependent pathways. Cytokine Growth Factor Rev. (1996) 7:81–91.[CrossRef][Medline]
  5. Kishimoto T., Taga T., Akira S. Cytokine signal transduction. Cell (1994) 76:253–262.[CrossRef][Web of Science][Medline]
  6. Kishimoto T., Akira S., Narazaki M., Taga T. Interleukin-6 family of cytokines and gp130. Blood (1995) 86:1243–1254.[Free Full Text]
  7. Wollert K.C., Taga T., Saito M., et al. Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. J. Biol. Chem. (1996) 271:9535–9545.[Abstract/Free Full Text]
  8. Nakajima T., Kinoshita S., Sasagawa T., et al. Phosphorylation at threonine-235 by a ras-dependent mitogen activated protein kinase cascade is essential for transcription factor NF-IL6. Proc. Natl. Acad. Sci. (1993) 90:2207–2211.[Abstract/Free Full Text]
  9. Akira S., Nishio Y., Inoue M., et al. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91 related transcription factor involved in the gp130 mediated signalling pathway. Cell (1994) 77:71.
  10. Wegenka U.M., Buschmann J., Lutticken C., Heinrich P.C., Horn F. Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level. Mol. Cell. Biol. (1993) 13:276–288.[Abstract/Free Full Text]
  11. Sheng Z., Pennica D., Wood W.I., Chien K.R. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development (1996) 122:419–428.[Abstract]
  12. Sheng Z., Knowlton K., Chen J., et al. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen activated protein kinase dependent pathway. J. Biol. Chem. (1997) 272:5783–5791.[Abstract/Free Full Text]
  13. Stephanou A., Brar B., Heads R., et al. Cardiotrophin-1 induces heat shock protein accumulation in cultured cardiac cells and protects them from stressful stimuli. J. Mol. Cell. Cardiol. (1998) 30:849–855.[CrossRef][Web of Science][Medline]
  14. Ghosh S., Ng L.L., Talwar S., Squire I.B., Galiñanes M. Cardiotrophin-1 protects the human myocardium from ischemic injury: Comparison with the first and second window of protection by ischemic preconditioning. Cardiovasc. Res. (2000) 48:440–447.[Abstract/Free Full Text]
  15. Bongrazio M., Comini L., Gaia G., Bachetti T., Ferrari R. Hypertension, ageing and myocardial synthesis of heat-shock protein 72. Hypertension (1994) 24:620–624.[Abstract/Free Full Text]
  16. Locke M., Tanguay R.M. Diminished heat shock response in the aged myocardium. Cell Stress Chaperones (1996) 1:251–260.[CrossRef][Web of Science][Medline]
  17. Simpson P., Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating non-myocardial cells. Circ. Res. (1982) 50:101–116.[Free Full Text]
  18. Navaratnam V. Heart muscle: ultrastructural studies. (1988) Cambridge University Press.
  19. Nair R.R., Nair P. Age-dependent variation in contractility of adult cardiac myocytes. Int. J. Biochem. Cell Biol. (2001) 33:119–125.[CrossRef][Web of Science][Medline]
  20. Wang X., Levi A.J., Halestrap A.P. Kinetics of the sarcolemmal lactate carrier in single heart cells using BCECF to measure pHi. Am. J. Physiol. (1994) 267:H1759–H1769.[Web of Science][Medline]
  21. Lundgren E., Borg T., Mardh S. Isolation and characterisation and adhesion of calcium-tolerant myocytes from the adult rat heart. J. Mol. Cell. Cardiol. (1984) 16:355–362.[CrossRef][Web of Science][Medline]
  22. Piper H.M., Probst I., Schwartz P., Hutter F.J., Spiekermann P.G. Culturing of calcium stable adult cardiac myocytes. J. Mol. Cell. Cardiol. (1982) 14:397–412.[CrossRef][Web of Science][Medline]
  23. Esumi K., Nishida M., Shaw D., Smith T.W., Marsh J.D. NADH measurements in adult rat myocytes during stimulated ischaemia. Am. J. Physiol. (1991) 260:H1743–H1752.[Web of Science][Medline]
  24. Dudley D.T., Pang L., Decker S.J., Bridges A.J., Saltiel A.R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA (1995) 92:7686–7689.[Abstract/Free Full Text]
  25. Alessi D.R., Cuenda A., Cohen P., Dudley D.T., Saltiel A.R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J. Biol. Chem. (1995) 46:27489–27494.
  26. Gavrieli Y., Sherman Y., Ben-Sasson S.A. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J. Cell Biol. (1992) 119:493–501.[Abstract/Free Full Text]
  27. Brar B.K., Jonassen A.K., Stephanou A.S., et al. Urocortin protects against ischemic and reperfusion injury via a MAP-kinase dependent pathway. J. Biol. Chem. (1999) 275:8508–8514.[CrossRef][Web of Science]
  28. Jonassen A.K., Brar B.K., Mjos O.D., et al. Insulin administered at reoxygenation exerts a cardioprotective effect in myocytes by a possible anti-apoptotic mechanism. J. Mol. Cell. Cardiol. (2000) 32:757–764.[CrossRef][Web of Science][Medline]
  29. Brar B.K., Stephanou A.S., Liao Z., et al. Cardiotrophin-1 can protect cardiac myocytes from injury when added both prior to simulated ischaemia and at reoxygenation. Cardiovasc. Res. (2001) 51:265–274.[Abstract/Free Full Text]
  30. Andree H.A., Reutelingsperger C.P., Hauptmann R., et al. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J. Biol. Chem. (1990) 265:4923–4928.[Abstract/Free Full Text]
  31. Maxwell M.P., Hearse D.J., Yellon D.M. Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc. Res. (1987) 21:737–746.[Abstract/Free Full Text]
  32. Mullane K.M., Young M. Pathophysiology of reperfusion and reperfusion injury. Yellon D.M., Jennings R.B., eds. (1991) New York: Raven Press. 59–83. chapter 4.
  33. Norton E.D., Jackson E.K., Virmani R., Forman M.B. Effect of intravenous adenosine on myocardial reperfusion injury in a model with low myocardial collateral blood flow. Am. Heart J. (1991) 122:1283–1291.[CrossRef][Web of Science][Medline]
  34. Forman M.B., Velasco C.E., Jackson E.K. Adenosine attenuates reperfusion injury following regional myocardial ischaemia. Cardiovasc. Res. (1993) 27:9–17.[Free Full Text]
  35. Vander Heide R.S., Reimer K.A. Effect of adenosine therapy at reperfusion on myocardial infarct size in dogs. Cardiovasc. Res. (1996) 31:711–718.[Abstract/Free Full Text]
  36. Goto M., Miura T., Iliodoromitis E.K., et al. Adenosine infusion during early reperfusion failed to limit myocardial infarct size in a collateral deficient species. Cardiovasc. Res. (1992) 25:943–949.[CrossRef][Web of Science]
  37. Akira S., Isshiki H., Sugita T., et al. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. (1990) 9:1897–1906.[Web of Science][Medline]
  38. Stephanou A., Amin V., Isenberg D.A., et al. Interleukin-6 activates heat shock protein 90β gene expression. Biochem. J. (1997) 321:103–106.[Web of Science][Medline]

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D. J. Hausenloy and D. M. Yellon
Cardioprotective growth factors
Cardiovasc Res, July 15, 2009; 83(2): 179 - 194.
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