Cardiotrophin-1 Induces Heat Shock Protein Accumulation in Cultured Cardiac Cells and Protects them from Stressful Stimuli

J Mol Cell Cardiol 30, 849–855 (1998)

Cardiotrophin-1 Induces Heat Shock Protein Accumulation in Cultured Cardiac Cells and Protects them from Stressful Stimuli A. Stephanou1, B. Brar1, R. Heads2, R. D. Knight3, M. S. Marber2, D. Pennica4 and D. S. Latchman1 1

Department of Molecular Pathology. Windeyer Institute of Medical Sciences, University College London Medical School, The Windeyer Building, 46 Cleveland Street, London W1P 6DB; 2 Department of Cardiology, UMDS, St Thomas’ Hospital, London; 3National Heart and Lung Institute, Royal Brompton Hospital, London, UK; 4Department of Molecular Biology, Genentech Inc., South San Francisco, CA 94080, USA (Received 8 August 1997, accepted in revised form 23 January 1998) A. S, B. B, R. H, R. D. K, M. S. M, D. P  D. S. L. Cardiotrophin1 Induces Heat Shock Protein Accumulation in Cultured Cardiac Cells and Protects Them From Stressful Stimuli. Journal of Molecular and Cellular Cardiology (1998) 30, 849–855. Cardiotrophin-1 (CT-1) was originally identified as a molecule capable of inducing cardiac hypertrophy. We show here that treatment of cultured neonatal cardiocytes with CT-1 induces enhanced synthesis of the heat shock proteins hsp70 and hsp90, with hsp70 levels being enhanced three-fold and hsp90 levels being enhanced seven-fold. Such CT-1-treated cells are protected against subsequent exposure to severe thermal or ischaemic stress, as assayed both by measures of total cell death, such as trypan blue exclusion and LDH release, and by measures of apoptosis, such as propidium-iodidestaining and TUNEL-labelling. Hence, CT-1 can induce the protective hsps and protect cardiac cells from diverse stresses.  1998 Academic Press Limited K W: Heat shock proteins; Cardioprotection; CT-1; Apoptosis.

Introduction Cardiotrophin-1 (CT-1) was originally isolated as a factor capable of inducing cardiac myocyte hypertrophy (Pennica et al., 1995a). Subsequent studies demonstrated that CT-1 induces a hypertrophic response which is distinct from that seen with aadrenergic stimulation, and which may be more relevant to the volume rather than pressure overload-induced hypertrophy, which produces cardiac failure in humans (Wollert et al., 1996). Further

investigation established that CT-1 is a member of a family of cytokines including interleukin-6 (IL-6) and leukaemia inhibitory factor (LIF) (Pennica et al., 1995b, 1996). The receptor for each of these factors contains a common, ubiquitously expressed cell surface polypeptide known as gp130 (for review see Kishimoto et al., 1994, 1995). In addition, the IL-6 receptor contains a specific receptor component (IL-6R) which is unique to this receptor (Kishimoto et al., 1994; 1995). In contrast, LIF and CT-1 share a common second receptor component which is

Please address all correspondence to: D. S. Latchman, Dept of Molecular Pathology, Windeyer Institute of Medical Sciences, University College London Medical School, The Windeyer Building, 46 Cleveland Street, London W1P 6DB, UK.

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known as LIF receptor sub-unit b (Pennica et al., 1995b; Wollert et al., 1996). We have previously shown in non-cardiac cells that treatment with IL-6 can induce enhanced expression of the heat shock proteins hsp70 and hsp90 (Stephanou et al., 1997). It would be of particular interest to determine whether similar treatment of cardiac cells with this family of cytokines would induce enhanced hsp synthesis. Thus, it has previously been shown that over expression of hsps in cardiac cells, whether produced by mildly stressful stimuli (for review see Yellon and Latchman, 1992), or by artificial over expression in transfected cells (Heads et al., 1994, 1995; Cumming et al., 1996b), or in transgenic animals (Marber et al., 1995), can protect the cell against subsequent severe stress. Hence, the induction of hsps by a non-abusive procedure, such as a cytokine, might have significant therapeutic implications. Here, we show for the first time that treatment of cardiac cells with CT-1 results in the induction of hsp synthesis, and can therefore protect cardiac cells against subsequent exposure to severe thermal or ischaemic stress.

Materials and Methods Cell culture Ventricular myocytes from the hearts of 2-day-old neonatal rats (Sprague–Dawley), were prepared by a modification of a previously published protocol (Simpson and Savion, 1982). Cells were preplated for 1 h to remove fibroblasts (Simpson and Savion, 1982) and myocytes were cultured as described previously (Simpson and Savion, 1982; Chien et al., 1985). 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 at least 30 min to allow contaminating fibroblasts to attach. The myocytes free within the culture media were plated on sixwell gelatin-coated plates at a density of 1.5–2 million cells/well. The cardiac myocytes were cultured at 37°C, 21% 02–5% 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 serum-free Dulbecco’s modified medium/

Medium 199 with penicillin/streptomycin (minimal media) for 48 h, and preconditioned as follows. For the cell ischaemic preconditioning, the minimal media of six wells of cardiomyocyte cell cultures was replaced with 1 ml of a buffer designed to simulate ischaemia. This ‘‘simulated’’ ischaemic buffer consisted of 137 m NaCl, 3.58 m KCl, 0.49 m MgCl2, 0.9 m CaCl2.2 H2O, 4 m HEPES supplemented with 0.75 sodium dithionite, 10 m deoxyglucose, 20 m lactate and 12 m hydrogen (pH 6.5) ion concentration to inhibit glycolysis (Wollert et al., 1996). The cells were then exposed for 2 h to simulated ischaemia in an atmosphere of 5% CO2 and 95% argon at 37°C within a tissue culture chamber that we had available. The ‘‘simulated’’ ischaemia buffer was removed from the cells and replaced with minimal medium and the cells returned to 37°C, 21% 02–5% CO2 for 48 h. To precondition the cells with CT-1, 1 ng/ml and 0.1 ng/ml of this cytokine (Genentech Ltd) was incubated with six wells of cardiac myocytes within 1 ml of minimal media for 24 h. For a control, minimal media that did not contain CT-1 was incubated with six wells of cardiac myocytes. Subsequent to this incubation period, all the cells were exposed to ‘‘lethal’’ simulated ischaemia and ‘‘lethal’’ heat shock treatment. For lethal ischaemia, the cells were incubated in 1 ml of the ‘‘simulated’’ ischaemia buffer, as described above, for 6 h in an atmosphere of 5% CO2 and 95% argon at 37°C. This buffer was devised by Esumi et al. (1991), to simulate the extracellular milieu occurring during myocardial ischaemia with the approximate concentrations of potassium, hydrogen and lactate ions occurring in vivo. For lethal heat shock treatment, the cells were incubated in a water bath, heated to 43°C for 2 h with 1 ml of ischaemic buffer that was pre-heated to 43°C.

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 (12 000×g for 10 min), the supernatant was aspirated and the cardiocytes were resuspended in a suitable volume of PBS (100 ll). 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

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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.

Lactate dehydrogenase activity (LDH) LDH activity released from cardiac cells, subjected to ‘‘lethal’’ simulated ischaemia and heat shock, was determined using a LDH assay kit (Sigma). LDH catalyses the reduction of pyruvate to lactate, resulting in an equimolar amount of NADH being oxidised to NAD. The rate of increase in absorbance at 495 nm is directly proportional to lactate dehydrogenase activity in the sample. Aliquots of the minimal ischaemic buffer used for ‘‘lethal’’ simulated ischaemia and heat shock were tested for LDH activity as a marker of cell damage at the end of each lethal stress. LDH activity was expressed as LDH activity/cell protein content (Pierce, BCA protein assay, Rockford, IL, USA).

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Figure 1 Left-hand panel: Western blot of extracts from culture of untreated cardiomyocytes (c) or parallel cultures treated with 1 ng/ml CT-1 (CT-1) with antibodies to hsp70 or hsp90. Right-hand panel: Coomassie blue stained gel of total protein from the two samples to show equal loading.

Statistics Assessment of apoptosis Apoptotic nuclei were assessed by the end-labelling of DNA 3′ ends with dUTP-FITC, using a modification of the TUNEL method (Gavrielli et al., 1992), and by flow cytometry using propidium iodide (Melino et al., 1994). For TUNEL, cardiac myocytes were plated onto 1% gelatin (Sigma)-coated 24well tissue culture dishes, and FACS analysis was performed on cells plated on 9 cm3 tissue culture plates. Cardiac myocytes were then exposed to 6 h of ischaemia in the presence and absence of CT-1 (1 ng/ml). 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 m 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. For FACS analysis, 1×106 cells were pelleted by centrifugation at 1200 rpm for 5 min and then resuspended in 500 ll of cold PBS and 500 ll of methanol:acetone (4:1) o/n at 4°C. The cells were then pelleted and resuspended in 50 ll of RNase (20 lg/ml) for 15 min at 37°C. Subsequent to this incubation period, 100 ll of propidium iodide (40 lg/ml) was added to the cells, which were then incubated in the dark for 20 min at 37°C. DNA fluorescence was measured using a FACScan (Becton-Dickinson).

The unpaired Student’s t-test was used to identify significant differences between control and experimental groups. Statistical significance was assumed at P<0.05 level.

Western blotting Cardiomyocytes which had either been left untreated or exposed to CT-1 (1 mg/ml) for 24 h were harvested, and Western blot analysis was performed as described previously (Stephanou et al., 1997), with the AC88 antibody to hsp90 or commercially available antibody to the inducible form of hsp70 (Stressgen Biotechnologies).

Results To test the effect of CT-1 on hsp expression in cardiac cells, a dose of 1 ng/ml was added to cultures of cardiac myocytes, and the cells were subsequently harvested for Western blotting with specific antibodies to hsp70 and hsp90. The cells treated with 1 ng/ml of CT-1 showed a clear enhancement of both hsp70 and hsp90 levels compared to control untreated cells (Fig. 1). Similar induction of hsp70 and hsp90 by CT-1 was observed in six independent experiments. By scanning densitometric analysis, CT-1 was shown to produce an approximately threefold increase in hsp70 levels and an approximately

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Figure 2 Cell death as assayed by the percentage of cells unable to exclude trypan blue (a) or units of LDH activity released per mg of protein (b) when untreated cells, CT-1 treated cells or cells pre-conditioned with mild ischaemia are exposed to subsequent severe ischaemia. In each case, values are the mean of three independent experiments, each of which contained nine replicates of each experimental treatment (i.e. n=27 for each treatment). Bars indicate the standard deviation of the mean.

seven-fold increase in hsp90 levels. The degree of hsp accumulation induced by 1 ng/ml of CT-1 was similar to that observed in these cells following exposure to mild ischaemia in parallel experiments in accordance with our previous results (Cumming et al., 1996a), whilst 0.1 ng/ml of CT-1 induced less hsp accumulation (data not shown). Hence, CT1 at a dose of 1 ng/ml can indeed induce enhanced accumulation of both hsp70 and hsp90. In our previous experiments, overexpression of hsp70 in primary cardiocytes (Cumming et al., 1996b), or in the H9 cardiac cell line (Heads et al., 1994) by transfection, protects the cells against subsequent exposure to severe thermal or ischaemic stress. Similarly, overexpression of hsp90 protects both primary cardiocytes (Cumming et al., 1996b) and H9 cells (Heads et al., 1995) from thermal but not ischaemic stress. We therefore investigated whether pre-treatment with CT-1 would similarly protect cardiac myocytes from subsequent exposure to severe thermal or ischaemic stress. In these experiments, pre-treatment with 0.1 ng/ ml of CT-1 had no effect on the ability of the cells to survive a subsequent severe lethal ischaemic stress, as assayed both by the number of dead cells unable to exclude trypan blue [Fig. 2(a)], or by the level of lactate dehydrogenase (LDH) released into the medium by dying cells [Fig. 2(b)]. In contrast, pre-treatment with 1 ng per ml CT-1 produced a

highly significant increase in cell survival, compared to untreated cells. This was observed both by measuring the percentage of cells unable to exclude trypan blue, where the effect observed was almost as strong as that produced by conditioning with mild ischaemia [Fig. 2(a)] and was highly statistically significant (P<0.0001), as well as by measuring LDH release from dying cells [Fig. 2(b), P<0.03]. A similar protective effect of pre-treatment with 1 ng/ml of CT-1 was also observed when the treated cells were exposed to a subsequent lethal heat shock. In this case, however, the protective effect was somewhat less than that observed against lethal ischaemia, and only the effect on the percentage of cells able to exclude trypan blue was statistically significant compared to control cells (P<0.0001) [Figs. 3(a), (b)]. The better protective effect observed with trypan blue exclusion may suggest that these cells may exclude trypan blue under conditions where some leakage of enzymes still occurs. Hence, a mild protective effect may only be observed by this means and not by measuring LDH release. We next wished to determine whether the protective effect observed with CT-1 was due to a reduced rate of apoptosis (programmed cell death) following exposure to ischaemia. We therefore quantitated the extent of such death using both FACS analysis of propidium iodide stained cells to

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Figure 3 Cell death as assayed by the percentage of cells unable to exclude trypan blue (a) or units of LDH activity released per mg of protein (b) when untreated cells or CT-1 treated cells are exposed to subsequent lethal heat shock. The results in untreated cells not exposed to lethal heat shock are shown for comparison. In each case, values are the mean of three independent experiments, each of which contained nine replicates of each experimental treatment (i.e. n=27). Bars indicate the standard deviation of the mean.

measure the percentage of apoptotic cells with less than 2 N DNA content [Fig. 4(a)] and TUNEL labelling to measure the proportion of cells containing DNA breaks [Fig. 4(b)]. It is clear from these experiments that CT-1 treatment at a dose of 1 ng/ ml, sufficient to induce hsp expression, is able to significantly reduce the extent of apoptosis as measured by two independent assays.

Discussion The findings presented here demonstrate for the first time that CT-1 is able to induce enhanced accumulation of hsp90 and hsp70 in cultured cardiac cells, and can protect these cells from subsequent exposure to severe thermal or ischaemic stress. Numerous previous studies have demonstrated that similar induction of the hsps, and protection against subsequent severe stress, can be achieved by exposure of cardiac cells to mildly stressful stimuli, such as mild elevations in temperature or short exposure to ischaemia (for review see Yellon and Latchman, 1992). Similarly, transfection of cultured cardiac cells with genes encoding hsps can protect them against subsequent thermal or ischaemic stress (Heads et al., 1994, 1995; Cumming et al., 1996b), and the same effect is observed in the hearts of transgenic animals overexpressing hsp70 (Marber et al., 1995).

However, relatively few studies have demonstrated elevation of hsp synthesis and a protective effect using pharmacological agents which do not induce cellular damage thereby resulting in a stress response. It has been shown that the tyrosine kinase inhibitor herbimycin-A can induce hsp70 synthesis and protect cardiomyocytes from subsequent severe stress (Morris et al., 1996). However, herbimycinA is likely to have a wide variety of other effects on cellular physiology, due to its ability to inhibit tyrosine kinase activity, and the concentrations required to protect cultured cells cannot be readily achieved in vivo. In contrast, our data indicate that similar induction of hsps and protective effects can be achieved with CT-1, a factor which is naturally expressed by myocardial cells (Sheng et al., 1996a). Indeed, whilst our data represents the first demonstration that CT-1 can protect against severe thermal or ischaemic stress, previous studies have demonstrated that the apoptotic death of unstressed myocytes in defined serum-free medium can be reduced by treatment with CT-1 (Sheng et al., 1996b). It is clear, therefore, that CT-1 can protect cardiac cells both against the cell death which occurs when cells are plated out in culture, and against the more extensive death which is observed in cells exposed to severe stress. These findings suggest, therefore, that CT-1 may have therapeutic potential in the protection of the

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Figure 4 Percentage of apoptotic nuclei as measured by FACS analysis (a) or TUNEL labelling (b) in cells either left untreated or exposed to ischaemia with or without prior treatment with CT-1. Values are the means of three independent experiments, each of which contained nine replicates of each experimental treatment (i.e. n=27). Bars indicate the standard deviation of the mean.

heart from stress, particularly if its protective effects can be dissected away from the potentially damaging induction of cardiac hypertrophy. In this regard, it is of interest that stimulation of gp-130 receptor activity results in the activation of two distinct sets of cellular transcription factors, NFIL6/NF-IL6b which are phosphorylated by MAP kinase, and STAT-3 which is phosphorylated by Jak kinases (Nakajama et al., 1993; Akira et al., 1994). In the case of the acute phase genes, whose transcription is stimulated by IL-6 in the liver, some genes are stimulated solely via the NF-IL6/NF-IL6b pathway, whereas others are stimulated only by the STAT-3 pathway (Akira et al., 1990; Wegenka et al., 1993). Interestingly, it has recently been shown that the protective effect of CT-1 against apoptosis in unstressed myocytes appears to involve the MAP kinase pathway, whilst the stimulation of hypertrophy does not (Sheng et al., 1996b). Further studies of the pathways by which CT-1 stimulates hsp gene expression, cardiac cell protection against severe stress and cardiac cell hypertrophy, will evidently be necessary in order to determine whether different gp-130 activated pathways are involved in these effects. If this is indeed the case, it may be possible to devise novel CT-1 like agents having the protective effect of CT-1 but which do not stimulate the hypertrophic response. Whatever the case, the findings reported here,

when taken together with our previous observations that IL-6 induces hsp synthesis (Stephanou et al., 1997), indicate that the activation of the gp-130 pathway can result in elevated hsp synthesis and, hence, in cardiac protection. In view of the known protective effect of the hsps, it is likely that these two effects are correlated and that the protective effect of CT-1 involves hsp induction, although other mechanisms may also be involved. Indeed, previous findings showing that rabbit hearts can be protected from reperfusion injury via prior treatment with leukaemia inhibitory factor, were associated with enhanced synthesis of manganese superoxide dismutase, which has a protective effect by scavenging free radicals (Nelson et al., 1995). However, the ability of LIF to activate the gp-130 receptor suggests that enhanced hsp synthesis may also be involved in its protective effect in this system. Thus, the family of gp-130-activating cytokines clearly constitutes a new class of potential cardioprotective agents.

Acknowledgements This work was supported by the British Heart Foundation.

Heat Shock Proteins in Cultured Cardiac Cells

References A S, I H, S T, T O, K S, N Y, N T, H T, K T, 1990. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J 9: 1897–1906. A S, N Y, I M, W X-J, W S, M T, Y K, S T, N M, K T, 1994. Molecular cloning of APRF, a novel IFN-stimulated Gene factor 3 p91 related transcription factor involved in the gp130 mediated signaling pathway. Cell 77: 63–71. C KR, S A, R R, C A, K Y, G MD, B LM, W JT, 1985. Release of arachidonate from membrane phospholipids in cultured neonatal rat myocardial cells during adenosine triphosphate depletion. J Clin Invest 75: 1770–1780. C DVE, H RJ, B NJ, Y DM, L DS, 1996a. The ability of heat stress and metabolic preconditioning to protect primary rat cardiac myocytes. Basic Res Cardiol 9: 79–85. C DVE, H RJ, W A, L DS, Y DM, 1996b. Differential protection of primary rat cardiocytes by transfection of specific heat stress proteins. J Mol Cell Cardiol 28: 2343–2349. E K, N M, S D, S TW, M JD, 1991. NADH measurements in adult rat myocytes during simulated ischaemia. Am J Physiol 29: H1743– H1752. G Y, S Y, B-S SA, 1992. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 119: 493–501. H RJ, L DS, Y DM, 1994. Stable high level expression of a transfected human hsp70 gene protects a heat-derived cell line against thermal stress. J Mol Cell Cardiol 26: 695–699. H RJ, Y DM, L DS, 1995. Differential cytoprotection against heat stress or hypoxia following expression of specific stress protein goes in myogenic cells. J Mol Cell Cardiol 27: 1669–1678. K T, T T, A S, 1994. Cytokine Signal Transduction. Cell 76: 253–262. K T, A S, N M, T T, 1995. Interleukin-6 family of cytokines and gp130. Blood 86: 1243–1254. M MS, M R, C S-H, S R, Y DM, D WH, 1995. Overexpression of the rat inducible 70 kiloDalton heat stress protein in transgenic mouse increases resistance of the heart to ischemic injury. J Clin Invest 95: 1446–1456. M G, A-P M, P L, C E, G V, D PJ, P M, 1994. Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells. Mol Cell Biol 14: 6584–6596. M SD, C DVE, L DS, Y DS,

855

1996. Specific induction of the 70 kDa heat stress proteins by the tyrosine kinase inhibitor herbimycinA protects rat neonatal cardiomyocytes: a new pharmacological route to stress protein expression. J Clin Invest 97: 706–712. N T, K S, S T, S K, N M, K T, A S, 1993. Phosphorylation at threonine-235 by a ras-dependent mitogen activated protein kinase cascade is essential for transcription factor NF-IL6. Proc Natl Acad Sci 90: 2207–2211. N SK, W GH, MC JM, 1995. Leukemia inhibitory factor and tumor necrosis factor induce manganese superoxide dismutase and protect rabbit hearts from reperfusion injury. J Mol Cell Cardiol 27: 223–229. P D, K KL, S KJ, L E, R J, L SM, D WC, K DS, Y R, C KR, B JB, W WI, 1995a. Expression cloning of cardiotrophin 1 a cytokine that induces cardiac myocyte hypertrophy. Proc Natl Acad Sci USA 92: 1142– 1146. P D, S KJ, S TA, M MW, S DL, Z KA, R A, T T, P NF, W WI, 1995b. Cardiotrophin-1: Biological activities and binding to the leukemia inhibitory factor receptor/ gp130 signaling complex. J Biol Chem 270: 10915– 10922. P D, W WI, C KR, 1996. Cardiotrophin-1: a multifunctional cytokine that signals via LIF receptor gp130 dependent pathways. Cytokine Growth Factor Rev 7: 81–91. S Z, K K, C J, H M, B JH, C KR, 1996a. Cardiotrophin-1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway. J Biol Chem 272: 5783–5791. S Z, P D, W WI, C KR, 1996b. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development 122: 419–428. S P, S S, 1982. Differentiation of rat myocytes in single cell cultures with and without proliferating non myocardial cells. Circ Res 50: 101–116. S A, A V, I DA, A S, K T, L DS, 1997. Interleukin-6 activates heat shock protein 90b gene expression. Biochem J 321: 103–106. W UM, B J, L C, H PC, H F, 1993. Acute-phase response factor, a nuclear factor binding is acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level. Mol Cell Biol 13: 276–288. W KC, T T, S M, N M, K T, G CC, V AB, H JK, P D, W WI, C KR, 1996. Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. J Biol Chem 271: 9535–9545. Y DM, L DS, 1992. Stress proteins and myocardial protection. J Mol Cell Cardiol 24: 113–124.