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Cardiovascular Research 2003 57(2):333-346; doi:10.1016/S0008-6363(02)00664-8
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts

Dominic C.H Nga, Naomi W Courta, Cristobal G dos Remediosb and Marie A Bogoyevitcha,c,*

aCell Signalling Laboratory, Biochemistry and Molecular Biology in the School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
bMuscle Research Unit, Institute for Biomedical Research, Department of Anatomy and Histology, University of Sydney, Sydney, NSW, Australia
cWestern Australian Institute for Medical Research, Perth, WA, Australia

* Corresponding author. Tel.: +61-8-9380-1348; fax: +61-8-9380-1148. marieb{at}cyllene.uwa.edu.au

Received 21 May 2002; accepted 5 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: The signal transduction pathways mediating the progression to failure have been intensively studied in a variety of in vitro and in vivo animal models. Recently, acute activation of the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) has been observed in the heart, but whether this is sustained in ischemic heart disease (IHD) or dilated cardiomyopathy (DCM) has not been previously addressed. Methods: We assessed the tyrosine phosphorylation of STAT1, 3, 5 and 6 in ventricular samples of explanted human hearts with IHD (n=11) and DCM (n=9) as an indication of STAT activation. Samples from normal donor hearts (n=9) acted as controls. In parallel, we also assessed protein expression and phosphorylation of three major families of mitogen-activated protein kinases (MAPKs); ERK, p38 MAPK and c-Jun NH2-terminal kinase (JNK). Results: All STAT isoforms were significantly phosphorylated in DCM. In contrast, only the phosphorylation of STATs 1 and 5 were significantly enhanced in IHD. Expression of total STAT protein remained unchanged. For the MAPKs, significant phosphorylation of p38MAPK was only observed in IHD. In contrast, there was no change in ERK or JNK activation despite abundant protein expression. Conclusions: We have shown that different members of the STAT transcription factor family are chronically phosphorylated in the failing heart as a result of IHD (STAT1 and 5) or DCM (STAT1, 3, 5 and 6). In contrast, IHD but not DCM showed significant p38MAPK phosphorylation. Whilst the differences noted between IHD and DCM may reflect different initiating events, the common activation of STATs 1 and 5 suggests that these transcription factors may play a common role regulating the progression of heart failure.

KEYWORDS Cardiomyopathy; Heart failure; Ischemia; Protein phosphorylation; Signal transduction

This article is referred to in the Editorial by V.P.M. van Empel and L.J. de Windt (pages 294–297) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The signal transduction pathways that trigger and/or mediate the transition of cardiac hypertrophy to failure have come under increasing scrutiny as potential targets for therapeutic intervention [1,2]. Substantial work with in vivo and in vitro animal models has implicated the mitogen-activated protein kinase (MAPK) family of serine/threonine protein kinases as important regulators in the hypertrophic processes [1,3]. However, when the MAPKs have been examined in human patients with hypertrophy or heart failure, no consistent trend has been observed [4–7]. Some of the differences may reflect the different disease etiologies of the groups studied. Alternatively, other signalling pathways may make an equal or greater contribution.

The JAK/STAT pathway, in which the JAK family of tyrosine kinases directly phosphorylate the STAT transcription factors, was originally identified following cytokine stimulation [8], and has since been shown to be activated following the exposure of isolated cardiac myocytes to cytokines such as leukemic inhibitory factor [9]. Subsequently, STAT3 has been implicated as a mediator of cardiac hypertrophy and an inducer of vascular endothelial growth factor [10,11]. More recently, a cardioprotective role for STAT3 has been suggested [12,13]. Other STAT isoforms, including STAT5 and STAT6, were also activated following angiotensin II stimulation of the heart [14].

The importance of the JAK/STAT pathways has been further underscored by their activation in myocardial ischemia [15] and that STATs 1 and 3 can mediate preconditioning protection [16,17]. Despite this increasing interest, no studies have addressed STAT isoform phosphorylation in the human heart. In the present study, we have evaluated the phosphorylation of STAT1, STAT3, STAT5 and STAT6 in ventricular samples taken from transplant patients with heart failure due to ischemic heart disease (IHD) or patients with idiopathic dilated cardiomyopathy (DCM) but little or no IHD. We also evaluated the phosphorylation of the MAPK family members, ERK, JNK and p38. We show a marked enhancement in the phosphorylation of STAT 1, 3β, 5 and 6 in DCM. STAT phosphorylation in IHD was lower, more variable, and only significant for STATs 1 and 5. However, in IHD, p38 MAPK was also significantly phosphorylated. Thus, DCM is accompanied by robust JAK/STAT signalling in the absence of significant MAPK signalling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Patient characteristics
Human failing heart samples (n=20) were collected from patients undergoing heart transplantation at St. Vincent's Hospital, Sydney, Australia, and categorized according to their disease status as ischemic heart disease (IHD, n=11) or dilated cardiomyopathy without ischemia (DCM, n=9). Collection of this material complied with approvals from the Human Ethics Committee of the University of Sydney (00/02/11) and from the Human Ethics Research Committee of St Vincent's Hospital (H91/048/1), and conformed with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3). Patients were predominantly male reflecting the prevalence of DCM and myocardial infarction in the Australian population. It was therefore not possible to test the relationship between the sex of the individual and any observed changes in phosphorylation.

The samples were snap frozen in liquid nitrogen within 5–40 min of the loss of coronary circulation. All samples were transmural sections of the anterior free left ventricle wall. IHD samples were taken from an ~1 cm region surrounding the infarcted areas with care taken to avoid including the scar tissue. Non-failing samples (n=9) were from unused donors that were either incompatible or where there were unacceptable risks associated with the donor hearts (e.g. by the discovery of a renal tumor). Table 1 summarises the clinical data together with the patient codes used in subsequent data presentation.


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  		Table 1 Patient characteristics

 
2.2 Protein lysate preparation
All tissues were snap-frozen, ground to a fine powder under liquid nitrogen then resuspended in lysis buffer (20 mM HEPES pH 7.7, 2.5 mM MgCl2, 0.1 mM EDTA, 20 mM β-glycerophosphate, 100 mM NaCl; supplemented immediately prior to use with 0.05% (v/v) Triton X-100, 0.5 mM dithiothreitol, 0.1 mM Na3VO4, 20 mg/ml leupeptin, 100 mg/ml phenylmethanesulfonyl fluoride and 20 mg/ml aprotinin) at a 1:5 (w/v) ratio. Following centrifugation (20 000xg, 10 min, 4 °C), the supernatant was collected as the tissue lysate and protein concentrations determined using the Bio-Rad protein assay.

2.3 Immunoblot analysis of MAPK and STAT phosphorylation
Lysates (40 µg protein per lane) were separated by SDS–PAGE, transferred to nitrocellulose membranes, then probed with the antibodies described below. Antibody binding was detected with chemiluminescence using either Supersignal reagents (Pierce) or Lumilight PLUS reagents (Boehringer) for enhanced sensitivity.

The antibodies employed were: anti-phosphotyrosine 4G10 from Upstate Biotech.; anti-total STAT1, STAT3 (N-terminal) and STAT6 from Transduction Labs; STAT3 (C-terminal), STAT5 (G-2) and anti-total MAPKs (p38, JNK and ERK) from Santa Cruz; phosphospecific antibodies recognising STATs and MAPKs were from Cell Signalling Technology with the exception of phopho-STAT1 Ser727 specific antibody, which was from Pharmingen.

All immunoblot signals were quantitated using the Luminescent Image Analyzer LAS-1000 and ImageGauge v3.0 software. The numbers of samples analysed (n=29 human heart samples) prevented analysis for each protein on a single gel. Therefore, we loaded protein lysates from control and LIF-treated neonatal rat cardiac myocytes [18] as controls. We used the LIF-treated sample as a reference point in all quantitation of the phosphorylated STATs, thereby allowing samples on separate gels to be compared.

2.4 Statistical analysis
Quantitated results were expressed as mean±S.E. Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by Fisher's least-square difference test (P<0.05).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Phosphorylation of STAT proteins in failing human heart
Although previous studies have investigated the differences in signalling proteins between the normal and diseased states of the heart, the activation of the STAT family of proteins has not been extensively studied in the human heart [4,6]. We first examined the tyrosine phosphorylation of the STAT proteins in a series of failing human hearts (IHD, DCM) and donor human hearts. As these donors acted as control reference points for comparison with failing hearts, we refer to these as ‘controls’ (CON) throughout this study.

STAT1 Y701 phosphorylation, which is required for its transcriptional activation, was significantly elevated in IHD (2.5-fold, P<0.05) and DCM (3.3-fold, P<0.01) when compared to the control donor hearts (Fig. 1A). The phosphorylation of STAT1 in the individual samples was generally consistent within each group (Fig. 1B–D, top panels). Immunoblotting for total STAT1 protein showed evidence for the expression of STAT1{alpha} (91 kDa) and STAT1β (84 kDa) spliceforms in these hearts [19] and indicated that STAT1 phosphorylation was not related to differences in total STAT1 expression (Fig. 1B–D, bottom panels). Quantitation of total STAT1 protein bands confirmed that there was no significant change in the amount of STAT1 protein between groups (data not shown). The lanes loaded with lysates from control and LIF-stimulated neonatal rat cardiac myocytes, labelled as ‘Con’ and ‘LIF’ in Figs. 1–4GoGoGo, were used as a reference point in the quantitation of phosphorylated STATs and allowed comparison between all immunoblots using a single antibody.


Figure 1
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  		Fig. 1 STAT1 phosphorylation is enhanced in IHD and DCM. Ventricular samples were obtained from patients: (A) without cardiovascular disease (control; C1–C9), (B) with IHD (ischemic heart disease; I1–I11) and (C) DCM (dilated cardiomyopathy; D1–D9) and immunoblotted for STAT1 Tyr701 phosphorylation. (A) Phospho-STAT1 bands were quantified by lumi-imaging and expressed as arbitrary units. Values are represented as mean±S.E. * Indicates a statistical difference (ANOVA) of phospho-STAT1 levels in IHD (P<0.05) and DCM (P<0.01) when compared to control hearts. Representative phospho-STAT1 blots of (B) control, (C) IHD and (D) DCM hearts show the data for all individual patients (top panels). Total STAT1 protein was confirmed by stripping each membrane and reblotting with an antibody for both phosphorylated and unphosphorylated STAT1 (bottom panels). Identical control or LIF-treated rat neonatal cardiac myocytes served as a reference points for quantification.

 

Figure 2
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  		Fig. 2 STAT3β phosphorylation is enhanced in DCM. Ventricular samples from control (C1–C9), IHD (I1–I11) and DCM (D1–D9) patient groups were immunoblotted for STAT3 {alpha}/β Tyr705 phosphorylation. (A) Phospho-STAT3β bands were quantified by lumi-imaging and expressed as arbitrary units. Values are represented as mean±S.E. * Indicates a statistical difference (ANOVA) of phospho-STAT3β levels in DCM (P<0.01) when compared to control hearts. Representative phospho-STAT3 blots of (B) control, (C) IHD and (D) DCM hearts show the data for all individual patients (top panels). Total STAT3 {alpha}/β protein was then determined by stripping the respective membranes and reblotting with an antibody raised against the N-terminal portion of STAT3 (middle panels). Blotting with a STAT3{alpha} C-terminal antibody identified the larger band as STAT3{alpha} (bottom panels). ‘LIF’ and ‘Con’ indicate rat cardiac myocyte reference lanes for quantification.

 

Figure 3
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  		Fig. 3 STAT5 phosphorylation is enhanced in IHD and DCM. Ventricular samples from control (C1–C9), IHD (I1–I11) and DCM (D1–D9) patient groups were immunoblotted for STAT5 Tyr694 phosphorylation. (A) Phospho-STAT5 bands were quantified by lumi-imaging and expressed as arbitrary units. Values are represented as mean±S.E. * Indicates a statistical difference (ANOVA) of phospho-STAT5 levels in IHD (P<0.05) and DCM (P<0.01) when compared to control hearts. Representative phospho-STAT5 blots of (B) control, (C) IHD and (D) DCM hearts show the data for all individual patients (top panels). Total STAT5 protein was determined by stripping the respective membranes and reblotting with an antibody for both phosphorylated and unphosphorylated STAT5 (bottom panels). ‘LIF’ and ‘Con’ indicate rat cardiac myocyte reference lanes for quantification.

 

Figure 4
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  		Fig. 4 STAT6 phosphorylation is enhanced in IHD. Ventricular samples from control (C1–C9), IHD (I1–I11) and DCM (D1–D9) patient groups were immunoblotted for STAT6 Tyr641 phosphorylation (top panels). (A) Phospho-STAT6 bands were quantified by lumi-imaging and expressed as arbitrary units. * Indicates a statistical difference (ANOVA) of phospho-STAT6 levels in DCM (P<0.01) when compared to control hearts. Values are represented as mean±S.E. Representative phospho-STAT6 blots of (B) control, (C) IHD and (D) DCM hearts show the data for all individual patients (top panels). STAT6 protein was determined by stripping the respective membranes and reblotting with an antibody for both phosphorylated and unphosphorylated STAT6 (bottom panels). ‘LIF’ and ‘Con’ indicate rat cardiac myocyte reference lanes for quantification.

 
Many cytokines that lead to the activation and phosphorylation of STAT1 also promote STAT3 phosphorylation and activation. When we investigated the phosphorylation of STAT3 Y705, we found significant phosphorylation in DCM (7.4-fold, P<0.01) but not IHD (Fig. 2A). PhosphoSTAT3 was strongly detected in all DCM samples (Fig. 2D, upper panel), whereas there was greater variation in the IHD samples (Fig. 2C, upper panel). More strikingly, when we compared these blots with total STAT3 immunoblots (Fig. 2B–D, middle and bottom panels), this phosphorylation corresponded to the lower STAT3 band (80 kDa) detected with an N-terminal antibody. A C-terminal truncation isoform of STAT3 has been denoted STAT3β and has been shown to be generated by differential splicing [20]. Therefore, in DCM samples the phosphorylation of the STAT3β isoform was detected (Fig. 2B–D, upper panels). Quantitation of total STAT3 bands again indicated that the observed enhanced phosphorylation was not due to a significant difference in protein expression of the STAT3β isoform (data not shown).

Given that STAT1 and STAT3 act downstream of gp130 signalling by the interleukin-6 cytokine family [10], and that these cytokines (e.g. LIF or cardiotrophin-1) are elevated in heart failure [21–23], we evaluated tyrosine phosphorylation of gp130. Following gp130 immunoprecipitation and phosphotyrosine immunoblotting, we were unable to detect changes in gp130 receptor tyrosine phosphorylation in IHD or DCM despite positive results with LIF-treated cultured cardiac myocytes (results not shown). This suggests that other cytokines may be involved in stimulating or maintaining STAT1 and STAT3β phosphorylation in the failing heart. Alternatively, the negative feedback pathways which limit STAT activation under normal conditions, may be compromised in IHD or DCM.

We broadened the scope of this study and examined the phosphorylation of both STAT5 and STAT6. The phosphorylation of STAT2 and STAT4 were not investigated because phospho-specific antibodies were not commercially-available. As shown in Fig. 3A, STAT5 Y694 phosphorylation was enhanced in IHD (3.5-fold, P<0.05) and DCM (5.8-fold, P<0.01). Although demonstrating a significant positive trend, STAT5 phosphorylation in IHD was variable such that three individuals (I6, I9 and I10) showed no phosphorylation (Fig. 3C, top panel). With the DCM samples, only one individual (D9), did not show strong STAT5 phosphorylation (Fig. 3D). Again, elevated STAT5 phosphorylation was not due to a significant change in protein expression of total STAT5 (Fig. 3B–D, bottom panels) because no differences in total STAT5 could be observed (data not shown).

Further analysis of STAT6 Y641 phosphorylation demonstrated significant elevation in DCM (11-fold, P<0.01), but not IHD (Fig. 4A). When the individual samples were considered, only two IHD samples (I3 and I8) showed strong phosphorylation (Fig. 4C, top panel). For DCM, only one individual (D9) failed to show strong STAT6 phosphorylation (Fig. 4D, top panel). Elevated STAT6 phosphorylation was not due to a significant change in protein expression of total STAT6 (Fig. 4B–D, bottom panel) and no significant differences in total STAT6 could be observed (data not shown). Given that D9 appeared as an outlier for both STAT5 and STAT6 phosphorylation, we re-examined the patient characteristics which showed that D9 had the highest left ventricular ejection fraction (LVEF), highest left ventricular end diastolic diameter (LVEDD), highest precapillary wedge pressure (PCWP), lowest cardiac output (CO) and lowest cardiac index (CI). Thus, we hypothesise that the difference in STAT5 and STAT6 phosphorylation reflects the severity of their haemodynamic parameters. Testing this hypothesis clearly needs additional collection of ventricular tissues from patients with similar parameters.

Taken together, our analysis suggests selective phosphorylation of STAT1 and STAT5 is involved in heart failure following ischemia whereas a broader range of STAT phosphorylation typifies DCM.

3.2 Phosphotyrosine profiles of human hearts
Tyrosine phosphorylation regulates the activities of many upstream signalling proteins [24]. Given the enhanced STAT tyrosine phosphorylation, we evaluated whether the tyrosine phosphorylation profiles of other signalling proteins differed in the failing and control donor heart groups. Western blotting with an anti-phosphotyrosine antibody (4G10) is a standard procedure to reveal changes in phosphorylation on tyrosine residues of intracellular proteins [25]. This procedure showed that many phosphotyrosine-containing proteins were present in the control, IHD and DCM hearts (Fig. 5). The tyrosine phosphorylation profiles of individuals within the control group were variable, particularly among the high molecular weight proteins (between 92 and 220 kDa markers as denoted in Fig. 5 with arrows). Specifically, major differences can be observed when C2 and C4 are compared with C3 and C5 (Fig. 5A). In contrast, the tyrosine phosphorylation profiles of the failing hearts showed greater similarity (Fig. 5B and C), especially in DCM where the high molecular weight proteins appear uniformly high for the individuals we tested (Fig. 5C).


Figure 5
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  		Fig. 5 Phosphotyrosine profile of IHD and DCM human heart samples. Ventricular samples from (A) control (C1–C9), (B) IHD (I1–I11) and (C) DCM (D1–D9) patient groups were immunoblotted with anti-phosphotyrosine antibodies. Arrows to the left of panel A indicate the positions of two bands that were variable in intensity in control hearts. These positions are also marked by arrows in B and C, thus showing the greater consistency in their intensities in the individual IHD and DCM samples.

 
The molecular weights of many growth factor receptors and cytokine receptors are consistent with the sizes of these larger bands. This suggests that these proteins may be activated in the diseased heart and raises the possibility that upstream signalling mechanisms contribute to the increased STAT phosphorylation. The consistent profiles in DCM and IHD further suggest that similar signalling mechanisms may underlie the progression to failure.

3.3 Activation/phosphorylation of MAPKs in human heart
Given the interest in the role of MAPKs in cardiac hypertrophy and failure [2–7], we investigated the phosphorylation/activation of the MAPK family in IHD and DCM samples. Immunoblotting for T180/Y182 phosphorylated p38MAPK revealed a significant increase in IHD (3.3-fold, P<0.05) (Fig. 6A). Despite a positive trend, phospho-p38MAPK was not significantly different in DCM (Fig. 6A). Blotting for total p38MAPK protein confirmed constant protein expression in the sample groups (Fig. 6B–D, bottom panels).


Figure 6
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  		Fig. 6 MAPK phosphorylation in failing and ischemic human hearts. Ventricular samples from control (C1–C9), IHD (I1–I11) and DCM (D1–D9) patient groups were immunoblotted for p38MAPK Thr180 and Tyr182 phosphorylation. (A) Phospho-p38MAPK bands were quantified by lumi-imaging and expressed as arbitrary units. Values are represented as mean±S.E. * Indicates a statistical difference (ANOVA) of phospho-p38MAPK levels in IHD (P<0.01) when compared to control hearts. Representative phospho-p38MAPK blots of (B) control, (C) IHD and (D) DCM hearts show the data for all individual patients (top panels). Total p38MAPK protein expression was determined by immunoblot analysis of protein lysates with an antibody for both phosphorylated and unphosphorylated p38MAPK (bottom panels). (E) lysates from control, IHD and DCM patient groups were immunoblotted with an antibody for both phosphorylated and unphosphorylated ERK (top panel) and JNK (bottom panel). Representative blots show data of four individuals from each sample group and are typical of the total protein expression levels within their respective group.

 
In contrast, we found no significant phosphorylation of ERK and JNK in IHD, DCM or donor hearts and confirmed this negative result with activity assays specific for these kinases (data not shown). Immunoblotting confirmed ERK and JNK expression and, as shown in the representative samples in Fig. 6E, total ERK and JNK protein levels remained unchanged in all samples. Thus, not all MAPKs are phosphorylated in the failing heart. The selective activation of MAPK families may be involved in the different categories of failure.

3.4 Serine phosphorylation of STATs 1 and 3 in human heart
Given the significant p38MAPK phosphorylation in IHD and the previous evidence of serine/threonine kinases involved in regulating the activity of STAT protein through phosphorylation on C-terminal serine residues, we next looked for a correlation between p38MAPK phosphorylation and STAT serine phosphorylation in the failing human hearts. p38MAPK has been previously shown to regulate the activity of STAT1 through phosphorylation on serine 727, in a cardiomyocyte model of ischemic disease [26]. Likewise, various serine kinases have been implicated in regulating STAT3 activity in vitro [27,28]. However, by immunoblotting for STAT1 Ser727 phosphorylation, we found no significant differences in IHD, DCM and control hearts (Fig. 7A–D) but all hearts showed high STAT1 Ser727 phosphorylation. Similarly, phosphorylation of Serine 725 on STAT3 remained unaltered in the failing hearts, however, in this case, there was little to no serine phosphorylation detected in all sample groups (data not shown).


Figure 7
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  		Fig. 7 STAT1 Serine 727 phosphorylation is unchanged in IHD and DCM. Ventricular samples from control (C1–C9), IHD (I1–I11) and DCM (D1–D9) patient groups were immunoblotted for STAT1 Ser727 phosphorylation. (A) Phospho-STAT1 S727 bands were quantified by lumi-imaging and expressed as arbitrary units. Values are represented as mean±S.E. Representative phospho-STAT1 S727 blots of (B) control, (C) IHD and (D) DCM hearts show the data for all individual patients (top panels). Total STAT1 protein was determined by stripping the respective membranes and reblotting with an antibody for both phosphorylated and unphosphorylated STAT1 (bottom panels). (E). Protein lysates from neonatal rat cardiomyocytes, treated with sorbitol (0.5 M) or left untreated as a control, were immunoblotted for STAT1 Ser727 phosphorylation (top panel) and p38MAPK Thr180 and Tyr182 dual-phosphorylation (bottom panel).

 
To confirm our ability to detect changes in STAT1 Ser727 phosphorylation, we exposed cultured myocytes to agents such as hyperosmolarity (0.5 M sorbitol), anisomycin (50 ng/ml), arsenite (50 µM), okadaic acid (100 µM) or hydrogen peroxide (1 mM) which are known p38MAPK activators. As shown in Fig. 7E for hyperosmolarity, enhanced p38MAPK phosphorylation and enhanced STAT1 Ser727 phosphorylation could be observed. Similar results were obtained with the other agents tested (results not shown). Thus, in this in vitro setting and in agreement with previous literature, a correlation between p38 phosphorylation and STAT1 Ser727 phosphorylation could be observed.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Considerable evidence from clinical studies shows that enhanced cytokine levels accompany cardiovascular disease [29,30]. The possibility that these cytokines then activate numerous intracellular signalling pathways to contribute to disease progression has been increasingly explored as a basis for developing new therapeutic strategies to treat the failing heart. Rather than focusing on specific cytokines, several recent studies have profiled signal transduction proteins that might be subsequently activated in the hypertrophic or failing human heart. These studies have predominantly investigated the serine/threonine protein phosphorylation events with a major focus on the MAPK families, p90 ribosomal S6 kinase, PI3-K/Akt and calcineurin [4,6,7]. However, the tyrosine phosphorylation of the STAT family of transcription factors accompanies a number of experimental models of cardiac hypertrophy, ischemia/reperfusion and dilated cardiomyopathy [12,14,18,31]. In light of these animal-based studies, it is surprising that the tyrosine phosphorylation of STATs in human heart failure had not been addressed.

In this study, we present evidence of elevated tyrosine phosphorylation of STATs in the failing human heart. It was not possible to define correlations between STAT isoform phosphorylation and specific individual parameters such as sex, age or haemodynamic measurements. However, by grouping data from hearts depending on disease etiology (i.e. IHD (n=11) versus DCM (n=9)), we showed the significant tyrosine phosphorylation of STAT1, STAT3β, STAT5 and STAT6 in DCM when compared to control donor hearts. For IHD, the greater variability between individuals resulted in our analysis only showing significant phosphorylation of STAT1 and STAT5.

Previous work in animal model systems has shown the tyrosine phosphorylation of STAT5 and STAT6 following ischemia/reperfusion of the rat heart [14] and the phosphorylation of STAT1 and STAT3 in the preconditioned rat heart [16]. The differences in STAT isoform activation noted in these studies when compared to our current results might be explained by differences between a chronic human condition that is associated with prolonged STAT activity and the acute ischemic episode in the experimental models. However, the general consensus to be derived from the animal model studies suggests that JAK/STAT signalling pathways when activated in the heart facilitate cardioprotection.

It is not possible to directly address a role for STAT-mediated protection in the human heart as we currently do not have access to interventions to accelerate or inhibit failure in the different patient groups. However, there is at least one additional complication in extrapolating the animal model data to predict the role of STATs in facilitating failure or protecting the human heart during failure. Specifically, we emphasise that we have shown selective STAT3β phosphorylation in the absence of STAT3{alpha} phosphorylation. To our knowledge, this is the first study demonstrating STAT3β phosphorylation in the heart although its phosphorylation has been recently shown in other chronic, non-cardiac diseases [32,33]. These {alpha} and β isoforms of STAT3 are spliceforms from a single gene with STAT3β being a C-terminal truncation of STAT3{alpha} [20]. Thus, we could detect STAT3β with a commercial STAT3 N-terminal-specific antibody, but not the antibody generated to a STAT3{alpha} C-terminal peptide. No STAT3β C-terminal antibody is currently available and a survey of previous literature shows that STAT3{alpha} antibodies have been extensively used for immunoprecipitation studies detailing STAT3 activation in the heart [10,34]. Furthermore, studies on the effects of STAT3 overexpression have evaluated the STAT3{alpha} isoform and inadvertently disregarded the contribution of STAT3β [12].

We can only speculate on the role of STAT3β activation in the failing heart. Evidence that STAT3β binds consensus DNA sequences in a sustained fashion but fails to activate transcription to a significant degree compared to wild-type STAT3{alpha}, has led to the suggestion that STAT3β functions as a dominant-negative regulator of transcription [20]. Therefore, although STAT3{alpha} may transduce a cardioprotective signal in response to ischemia and reperfusion [12], it may be that a STAT3β pro-death pathway predominates during the progression into failure.

The consistent phosphorylation of STAT1, STAT3β, STAT5 and STAT6 in DCM is consistent with previous reports of elevated serum cytokine levels in chronic heart failure [29,30]. It is not clear how a broad activation of STAT isoforms might contribute to the disease progression in DCM, but the consistency between samples likely reflects the nature of DCM as a global failure of the ventricular tissue. This contrasts the more variable levels of STAT activation in IHD, which would be expected to reflect the greater disease heterogeneity.

Our phosphotyrosine immunoblots have also provided evidence that tyrosine kinase activities may differ in the failing and normal human heart. We noted a change in tyrosine phosphorylation of several high molecular weight proteins with more consistent phosphorylation observed in failing heart samples. This suggests a common signalling mechanism may control the progression into cardiac failure. As mentioned, these high molecular weight bands are consistent with the sizes of many growth factor and cytokine receptors. STAT proteins are generally phosphorylated by such receptors with intrinsic or associated kinase activity [35].

In addition, previous studies have reported increases in circulating cytokine levels in the heart failure [22,29,30]. A change in the profile of receptor tyrosine phosphorylation in heart failure is therefore consistent with the reported increase in STAT phosphorylation. This also suggests that the observed elevated STAT phosphorylation in DCM and, to a lesser extent, IHD samples, may be due to controlling upstream signalling pathways rather than an absence of negative regulation by STAT inhibitors, such as SOCS and PIAS [36]. Identification of the exact receptors leading to these phosphorylation changes in human heart failure is obviously a challenge for further investigation. Previous studies have reported elevated levels of several members of the IL-6 family of cytokines in heart tissue and circulating plasma in advanced heart failure patients [21–23]. However, our initial studies have been unsuccessful in detecting significant changes in gp130 phosphorylation. This does not eliminate the possible involvement of other receptor types. Clearly the contributions of cytokines and their activated signalling networks to failure of the heart is an important area for further investigation.

We have also demonstrated a significant increase in p38MAPK phosphorylation in IHD and this likely indicates its significant action [37]. A role of activated p38MAPK in the failure is likely given the recent in vivo animal work showing that the cardiac specific overexpression of this MAPK induces heart failure [38]. However, we have been unable to detect discernible changes in the activation or phosphorylation of the ERK or JNK MAPK families in either IHD or DCM (results not shown). Our inability to detect a change in ERK and JNK activity was not due to high basal activity in donor hearts. On the contrary, we were unable to detect significant levels of ERK and JNK phosphorylation or activity despite abundant levels of total protein. This is clearly inconsistent with previous studies, one reporting JNK and p38MAPK activity in IHD samples specifically and a second reporting elevated phosphorylation of all three MAPK families in a mixed heart failure group [4,6]. Although there is no clear explanation for these differences, this does highlight the limitations inherent in the correlative study of human patients.

Lastly, we have investigated the phosphorylation of specific serine residues in STAT1 and STAT3{alpha}. Numerous studies have demonstrated a role for MAPKs, including p38MAPK, in cross-talk regulation of the JAK/STAT pathway [26–28,39]. In particular p38MAPK has been shown to phosphorylate STAT1 on serine 727 in a rat cardiomyocyte model of ischemia [26]. In addition, p38MAPK has also been implicated in regulating the transcriptional activation of STAT3 by IL-6 in a hepatoma cell line [27]. Although we have demonstrated elevated p38MAPK phosphorylation in IHD, we failed to detect any significant change in the serine phosphorylation of STAT1 or STAT3{alpha}. Although there was considerable serine phosphorylation of STAT1 in IHD and DCM (Fig. 7B–D), a high level of serine phosphorylation in control samples suggested that it is unlikely that p38MAPK is involved at least in the final stages of failure. It could be argued that the constitutive serine phosphorylation of STAT1 might have been an artifact resulting from tissue collection and storage processes. However, this is unlikely, as we were unable to detect a similar extent of serine phosphorylation in STAT3{alpha} (data not shown).

Thus, we have presented the first evidence that JAK/STAT signalling can be significantly activated in human heart failure. We have demonstrated differential activation profiles of STAT and MAPK proteins in IHD and DCM. This reinforces the potential of anti-cytokine therapy in the treatment of heart failure and implicates intracellular cytokine-mediated mechanisms as therapeutic targets. The results presented in this study, in addition to others to date, support the idea that the process of heart failure is not associated with a global up-regulation of all signalling pathways. Rather selective activation of distinct pathways in the different etiologies of heart failure represents the more likely scenario. Of course there are many limitations in the study of human tissue made available only at the time of transplantation. Whilst we have addressed a number of these limitations throughout this paper, we would also emphasise that the phosphorylation we report reflects the sampling of the heterogeneous cell population making up the heart. We have been unable to use currently available phosphoSTAT antibodies in immunostaining protocols, but clearly the localisation of activated STATs to the cardiac myocyte or other cells in the failing heart is an important next step before the precise involvement of STAT proteins and their roles in regulating the various processes of heart failure can be clarified.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by funding from the National Heart Foundation of Australia.


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

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