Activation of JAK/STAT pathway transduces cytoprotective signal in rat acute myocardial infarction (original) (raw)

Abstract

Objectives: We reported that the activation of gp130 transduced hypertrophic and cytoprotective signals via Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in cardiac myocytes. Recent in vivo experiments have demonstrated that the JAK/STAT pathway is activated in acute pressure overload hearts. The present study was designed to examine whether the JAK/STAT pathway is also activated in acute myocardial infarction (AMI) and to determine its pathophysiological roles in ischemic heart disease. Methods and results: AMI model was generated by the ligation of proximal left anterior descending coronary artery of male Wistar rat. They were sacrificed at various time points ranging from 1 to 24 h after coronary ligation and their hearts were examined. Tyrosine phosphorylation of STAT3 was observed in the myocardium obtained from both the ischemic area and the healthy border area adjacent to the infarcted area. The AMI rats were next randomly assigned to two groups, one with coronary ligation only (group M), and the other with coronary ligation with AG-490 treatment (1 mg/kg i.v., every 4 h), a specific JAK2 inhibitor (group A). In group A, phosphorylation of STAT3 was significantly suppressed and caspase-3 activity and Bax expression were significantly increased in the myocardium after AMI. In group M, few apoptotic myocytes were identified in the border area by means of TUNEL assay. However, a significant increase in apoptotic cells was observed in group A. Conclusions: Administration of JAK2 inhibitor resulted in deterioration of myocardial viability in AMI hearts. The JAK/STAT pathway is activated in AMI myocardium and plays a pivotal role in cytoprotective signaling.

Time for primary review 23 days.

1 Introduction

A large number of studies have shown that cytokines share signaling pathways including activation of protein tyrosine kinases which is required for subsequent cellular responses. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a newly discovered intracellular signal transducing pathway that is activated by various cytokines [1–3] and growth factors [4,5]. The JAK family includes JAK1, JAK2, JAK3 and Tyk2 and is activated through interaction with various growth factor and cytokine receptors. JAK-mediated tyrosine phosphorylation of STAT family members promotes the translocation of these transcription factors to the nucleus and which promote the gene transcription.

It has been reported that JAK2 kinase plays an essential role in cross-talk of the JAK/STAT pathway [6,7]. AG-490 is a potent and selective inhibitor of JAK2 kinase, having no effect on the kinase activity of other protein tyrosine kinases [8]. AG-490 belongs to the tyrphostin family of tyrosine kinase inhibitors which inhibits protein tyrosine kinases by binding to the substrate binding site. Recently, AG-490 was found to inhibit angiotensin II (Ang II)- and platelet derived growth factor (PDGF)-induced aortic vascular smooth muscle cell proliferation [9].

The role of the JAK/STAT pathway in cardiac pathophysiology has been explored using the interleukin (IL)-6 family of cytokines [10–12]. Recent in vivo studies have demonstrated that acute pressure overloading activates JAK1, JAK2 and Tyk2 kinases as well as STAT1, 2 and 3 in the rat heart [13]. The activation of gp130 is reported to transduce cytoprotective signal against stressful conditions, such as serum depletion, hypoxia, or doxorubicin, by activating the JAK/STAT pathway [14,15]. In addition, cardiac specific disruption of gp130 was reported to present cardiomyopathy in response to mechano-stress with an increase in apoptosis [16]. Thus the inactivation of JAK/STAT pathway resulting from the loss of gp130 may be a key event in the transition from cardiac hypertrophy to heart failure.

Apoptosis is a form of programmed cell death that has been found to occur in different types of cells at various stages of development [17]. Recently, several studies of apoptosis in the heart have showed that apoptosis contributes to cardiac myocyte loss in pressure overload-induced hypertrophy [18], ischemia–reperfusion injury [19], myocardial infarction [20,21], and long-standing heart failure [22,23]. In acute myocardial infarction (AMI), cardiac myocytes die as a result of apoptosis as well as necrosis. Apoptosis is detected mainly in the hypo-perfused myocardium near the infarcted area in a rat AMI model [24]. However, a few myocytes underwent apoptosis even in non-ischemic healthy area, including the border area adjacent to the infarcted area [25,26]. AMI heart is characterized by a complex of various stressful states featuring reduction in oxygen supply followed by inflammatory responses and mechanical stretching. This implies that two main cellular events induce apoptosis, hypoxia and mechanical loading.

In the present study, we first investigated whether the JAK/STAT pathway (Fig. 1) is activated in rat AMI heart, and demonstrated substantial activation of STAT3 especially in non-ischemic healthy border area. We next examined the pathophysiological role of STAT3 activation in AMI heart by inhibiting the JAK/STAT pathway using AG-490. Pretreatment with AG-490 resulted in an enhancement of apoptosis in non-ischemic healthy border area of the infarcted myocardium.

Scheme of JAK/STAT pathway mediated anti-apoptotic signaling.

Fig. 1

Scheme of JAK/STAT pathway mediated anti-apoptotic signaling.

2 Methods

2.1 Reagents

AG-490 was purchased from Calbiochem-Novabiochem (La Jolla, CA, USA), recombinant mouse leukemia inhibitory factor (LIF) and anti-phosphotyrosine (4G10) from Upstate Biotechnology (Lake Placid, NY, USA) and proteinase K from Merck (Darmstadt, Germany). Medium-199 (M-199; Flow Laboratories) and newborn calf serum (NCS; Gibco) were used for cell culture. Rabbit anti-JAK1, anti-JAK2, anti-STAT1, anti-STAT3, anti-Bax, anti-Bad, anti-Bcl-2, and anti-Bcl-xL polyclonal antibodies were purchased from Santa Cruz Biotechnology, and phospho-STAT1, STAT3, Akt, P38 and extracellular signal-regulated kinase (ERK) from New England Biolabs (Beverly, MA, USA). All other chemicals were reagents of molecular biology grade obtained from standard commercial sources.

2.2 Rat myocardial infarction model

Male Wistar rats (Nippon Dobutsu, Osaka, Japan), 4-week-old and weighing about 100 g were anesthetized with ether and intubated under direct visualization. Controlled ventilation with room air was provided by using a rodent ventilator (Respirator, Shinano, Tokyo, Japan). The heart was explored through a left intercostal thoracostomy, and the proxymal portion of the left anterior descending (LAD) coronary artery was surgically ligated with a 7-0 silk suture (Nescosuture, Nippon Shoji, Osaka, Japan) as previously described [27]. This ligation created a large antero-lateral wall infarction, and the appearance of cyanosis and bulging of the myocardium was evidence of successful coronary ligation. After the ligation, chest was closed, the intratracheal tube was removed and the rats were allowed to recover. Animals were killed at various time points ranging from 1 to 24 h after coronary ligation. Sham-operated animals (with thoracostomy and intubation without coronary ligation) served as controls.

The heart was excised and divided transversely at the level of the papillary muscle. Delineation of the ischemic–infarcted area of the heart was obtained by means of injection of 1% Evans blue dye into the inferior vena cava before sacrifice. The viable area stained blue, whereas the ischemic–infarcted area remained pale. Three different myocardial samples were obtained from an ischemic area, the region adjacent to the ischemic myocardium (a border area), and a remote area.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and Osaka University Animal Care guidelines.

2.3 Cell culture

Primary cultures of neonatal rat cardiac myocytes were prepared from the ventricles of 1-day-old Sprague–Dawley rats (Nippon Dobutsu, Osaka, Japan) as previously described [11]. In brief, cardiac myocytes were plated onto 35- or 60-mm culture dishes at a density of 1.0×105 cells/cm2 and cultured in M-199 supplemented with 10% NCS and 0.1 mM bromodeoxyuridine. The medium was changed to M-199 and 10% NCS 24 h after seeding. We obtained >95% myocytes in cultures prepared with this procedure. The medium was changed to M199, 6 h before the experiments.

2.4 Immunoprecipitation

Frozen tissue samples (about 30 mg) were homogenized by using a homogenizer from Polytron (Kinematica, Switzerland) with RIPA buffer containing 30 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 10 mM Na4P2O7, 10 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF and 1 μg/ml aprotinin. After centrifugation, the lysates containing equal amounts of protein (2 mg) were incubated with 1 μg of anti-STAT3 antibody for 12 h at 4°C before the addition of protein A–sepharose for 2 h. The immunoprecipitates were washed three times with TBS buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5 mM, 1 mM sodium orthovanadate), eluted with 30 μl of sample buffer (62.5 mM Tris, pH 7.4, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol), and boiled for 10 min. The samples were then centrifuged at 5000 rpm for 1 min, and the supernatants were collected and stored at −80°C until assay. The lysis buffer was changed to the sample buffer in the case of incubation with phospho-STAT1 and phospho-STAT3 antibodies, while the other procedures were the same as described above. The cultured cardiac myocytes were rapidly rinsed with ice-cold PBS and solubilized with RIPA buffer for JAK1 and JAK2, or with sample buffer for STAT1, STAT3, mitogen-activated protein kinase (MAPK), P38, c-Jun N-terminal kinase (JNK) or Akt. The lysates were treated as described above.

2.5 Western blot analysis

The samples were separated in a 10% or 20% SDS–PAGE, and they were electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilon, Millipore) with a transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol). Membranes were incubated with a blocking buffer (TBS, 0.05% Tween 20, 5% nonfat milk) for 1 h at room temperature, and probed with anti-phosphotyrosine antibody to detect phosphorylated JAK1, JAK2, STAT1 or STAT3. After washing with TBST buffer (TBS–0.05% Tween 20), membranes were incubated with peroxidase-conjugated anti-mouse IgG antibody for 1 h. An enhanced chemiluminescence detection system (Amersham) was employed to visualize the peroxidase reaction products. The membranes were then stripped and reprobed with anti-JAK1, JAK2, STAT1 or STAT3 antibody.

2.6 Measurement of caspase-3 activity

The activity of caspase-3 was determined with the caspase-3 cellular activity assay kit (Biomol Research, PA, USA) according to the manufacturer's instructions. The activity was measured by means of detection of chromophore _p_-nitroanilide after cleavage from the labeled substrate Asp–Glu–Val–Asp (DEVD)–_p_-nitroanilide as previously described [28]. In brief, tissue samples (10 mg) were solubilized by using a Polytron homogenizer with a cell lysis buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA). Equal amounts of protein lysates were then reacted with 50 μM DEVD–_p_-nitroanilide for 1 h at 37°C. The activity was determined in a microtiter plate-reader at 405 nm and the results were calibrated with known concentrations of _p_-nitroanilide.

2.7 Apoptosis assay

To visualize apoptotic nuclei in cardiac myocytes, in situ labeling of fragmented DNA was performed as previously described using Apoptag kit (Oncor) [16]. The heart was fixed in 4% paraformaldehyde and embedded in parafin. A terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay was performed on 5-μm sections according to the manufacturer's instructions and with 30 min of labeling reaction. Sections were mounted on gridded coverslips and visualized by light microscope. For each section, the number of TUNEL-positive myocyte nuclei in a 0.0625 mm2 area was scored in healthy border area of AMI heart by using a gridded reticule ×400. The total number of myocyte nuclei in the same area was also counted in serial sections stained with hematoxyline and eosin as described previously [24]. Data is reported as the mean±S.D. of three sections from three different hearts.

2.8 Statistical analysis

Statistical analysis was performed by means of Student's t test and one-way ANOVA followed by the Bonferroni procedure for multiple group comparisons. A value of P<0.05 was considered significant.

3 Results

3.1 Tyrosine phosphorylation of STAT3 in AMI hearts

To examine whether the JAK/STAT pathway is activated in AMI, we initially examined the tyrosine phosphorylation of STAT3 in rat AMI hearts. As shown in Fig. 2, STAT3 was not phosphorylated in sham-operated rats but significant tyrosine phosphorylation of STAT3 was observed in ischemic, remote and border areas 6 h after coronary ligation. More intense phosphorylation of STAT3 in the border area than in ischemic and remote areas was observed at 1 h and continuing to 24 h after coronary ligation. These results indicate that STAT3 was transiently activated, especially in the healthy border area adjacent to the infarcted myocardium, after coronary ligation.

Fig. 2

Time course of STAT3 phosphorylation after AMI in rat hearts. Tissue samples obtained from three different areas (ischemic, border and remote areas) were examined at various times after coronary ligation. (A) Equal amount of proteins from border area of rat AMI heart obtained 0, 1, 4, 6, 12 and 24 h after coronary ligation were lysed with RIPA buffer, immunoprecipitated with anti-STAT3 antibody for 4 h at 4°C, separated by 10% SDS–PAGE, and transferred onto an Immobilon-P membrane. The blot was probed with anti-phosphotyrosine antibody (top). The blot was reprobed with anti-STAT3 antibody (bottom). (B) Equal amount of proteins from sham and AMI hearts (ischemic area and remote area) 1 and 6 h after coronary ligation were treated as described above. Representative bands corresponding to the molecular weight of STAT3 (92 kDa) are presented in (A) and (B). Five separate experiments from five different hearts showed the similar results.

3.2 JAK2 inhibitor suppressed STAT3 phosphorylation in AMl hearts

To evaluate the physiological significance of STAT3 phosphorylation in the border area, we used AG-490 to inhibit the JAK/STAT pathway in rat AMI hearts. Tyrosine phosphorylation of STAT3 and STAT1 was evaluated in border area 4 h after coronary ligation with or without AG-490 treatment. As shown in Fig. 3, pretreatment with 1 mg/kg AG-490, i.v., 30 min before the coronary ligation significantly inhibited the phosphorylation of STAT3. STAT1 was weakly phosphorylated in AMI heart, however it was not significantly affected with AG-490 treatment. In addition, AG-490 neither affected ERK nor P38 MAPK activation.

Fig. 3

Effects of AG-490 on STAT3 and STAT1 activation after AMI. Myocardial tissue samples were obtained from the healthy border area at 4 h after AMI. AG-490 (1 mg/kg) was administered intravenously 30 min before coronary ligation. Equal amount of proteins (20 μg) were separated by 10% SDS–PAGE and transferred onto an Immobilon-P membrane. The blot was probed with phospho-specific STAT3, STAT1 ERK or P38 antibody, and then reprobed with anti-STAT3 or -STAT1 antibody. (B) Phosphorylation of STAT3 and STAT1 was quantified by densitometric analysis of the autoradiogram and results were expressed as ratio of relative intensity obtained from p-STAT3 and STAT3 or p-STAT1 and STAT1. Data are expressed as mean±S.D. from four separate experiments. *, P<0.05 vs. AG (−).

In subsequent experiments, AMI rats were randomly assigned to coronary occlusion only (group M) or coronary occlusion with AG-490 treatment (1 mg/kg, i.v., 30 min before ligation and every 4 h thereafter) (group A).

3.3 Treatment with JAK2 inhibitor increased caspase-3 activity in AMI hearts

Previous studies have demonstrated that activation of caspase-3 leads to nucleosomal fragmentation of DNA, a hallmark of apoptosis, and is substantially elevated in AMI [29]. As shown in Fig. 4, caspase-3 activity in group M increased to 1.2 times the control level at 4 h and to 1.3 times control level at 18 h after coronary ligation. Group A had significantly high caspase-3 activity at 18 h after coronary ligation (2.0 times control level, P<0.05 vs. group M). The addition of a specific caspase-3 inhibitor, DEVD-CHO (20 μM), to the sample reaction mixtures suppressed caspase-3 activity to control level, indicating that the measured activities of caspase-3 were quite specific.

Fig. 4

Caspase-3 activities in AMI hearts. Caspase-3 activities were measured by the caspase-3 cellular activity assay kit as described in Methods. Closed bar and open bar indicate with or without AG-490 treatment. AG-490 was administered intravenously 30 min before coronary ligation and every 4 h after ligation at a dose of 1 mg/kg. DEVD-CHO (20 μM) was used as a chemical inhibitor for caspase-3 and added to the reaction mixture when indicated. Data are expressed as mean±S.D. from four samples. #, P<0.05 vs. control; *, P<0.05.

3.4 Treatment with JAK2 inhibitor increased Bax expression in AMI hearts

To determine the underlying molecular mechanism of the JAK2 inhibitor-induced increase in caspase-3 activity, the expressions of apoptosis-related proteins, such as Bax, Bad, Bcl-xL and Bcl-2, were examined by Western blot analysis. The amount of Bax protein was significantly increased in group A 12 h after coronary ligation (Fig. 5A), while the levels of Bcl-xL, Bcl-2 and Bad did not differ significantly between group A and group M (Fig. 5B). Treatment with AG-490 did not change the level of Bax in the control myocardium through 12 h.

Fig. 5

Expression of apoptosis related proteins in AMI hearts. Myocardial tissue samples were obtained from the border area of AMI hearts with or without AG-490 treatment, 0, 4 and 12 h after coronary ligation. Equal amount of protein (30 μg) was separated by 20% SDS–PAGE gel and immunoblotted with anti-Bax (A), anti-Bcl-xL (B), anti-Bcl-2 (B), or anti-Bad (B) antibody. Representative bands corresponding to the molecular weight of each protein are presented. AG; intravenous administration of AG-490 every 4 h (1 mg/kg) to the control, M; AMI and A; AMI with AG-490 treatment. Lower panel in (A) the level of Bax protein was quantified by densitometric analysis of the autoradiogram and results were expressed as ratio of relative intensity compared with control heart. Data are mean±S.D. from four samples. *, P<0.05 vs. M.

3.5 Apoptotic myocytes increase in JAK2 inhibitor-treated AMI hearts

To assess the occurrence of apoptosis in situ in healthy border area of AMI heart, the TUNEL assay was performed. Although few TUNEL-positive cells were observed in border areas of the myocardium at 4 and 8 h after coronary ligation in group M and group A, there was a significant increase in TUNEL-positive cells in the border area of group A 24 h after ligation (Fig. 6). On the other hand, control hearts with AG-490 treatment exhibited few TUNEL-positive cells throughout the period of observation (data not shown). These results suggest that the JAK2-dependent pathway protects against myocyte apoptosis induced by AMI.

Fig. 6

Effects of AG-490 on TUNEL-positive nuclei in AMI. Apoptotic cardiac myocytes were measured by TUNEL technique at 4, 8 and 24 h after coronary ligation. Data are expressed as TUNEL-positive nuclei/mm2 in border area of AMI heart. Six fields in border area were examined for each heart. AG; intravenous administration of AG-490 every 4 h (1 mg/kg) to the control, M; AMI and A; AMI with AG-490 treatment. Data are expressed as mean±S.D. from three hearts. *, P<0.05.

3.6 Effects of JAK2 inhibitor on JAK/STAT, the mitogen-activated protein kinase (MAPK), and phosphatidylinositol (PI)-3 kinase signaling in cardiac myocytes

To determine the effects of AG-490 on signaling pathway in cardiac myocyte, particularly on signaling via gp130, the activation of JAK1, JAK2, STAT1, STAT3, P38, ERK, JNK, and Akt was examined by Western blot analysis. As shown in Fig. 7, 5 min after stimulation with 102 U/ml LIF, phosphorylation of JAK1, JAK2, STAT1 and STAT3 was observed in cardiac myocytes. Pretreatment with 50 μM AG-490 significantly suppressed all phosphorylation except for that of JAK1. Furthermore, AG-490 had no effect on the phosphorylation of P38, ERK, JNK or Akt. This in vitro study thus demonstrated that AG-490, a specific inhibitor of JAK2 kinase, reduced the phosphorylation of STAT1 and STAT3 only through the activation of JAK2, and had no effect on either the MAPK family or phosphatidylinositol 3-kinase (PI 3-K) signaling.

Fig. 7

Effects of AG-490 on gp130-dependent signaling pathway in cardiac myocytes. (A) Phosphorylation of JAK/STAT family (JAK1, JAK2, STAT1 and STAT3) (B) Phosphorylation of MAPK family (JNK, ERK and P38) and Akt. Cardiac myocytes were cultured with M-199 and 10% NCS for 2 days and starved for 6 h. The cells were stimulated with 102 U/ml LIF for 5 min with or without 1 h pretreatment with 50 μM AG-490. The cells were lysed with RIPA buffer, immunoprecipitated with anti-JAK1 or anti-JAK2 antibody for 4 h at 4°C, separated by 10% SDS–PAGE and transferred onto an Immobilon-P membrane. The blot was probed with anti-phosphotyrosine antibody (4G10) and then reprobed with anti-JAK1 or anti-JAK2 antibody. When anti-phospho-STAT1, STAT3, JNK, ERK, P38, or Akt antibody was used, the cells were lysed with SDS buffer, separated by 10 or 20% SDS–PAGE and probed. The blot was reprobed with anti-STAT1 or STAT3 antibody. Experiments were repeated three times with similar results. Lower panel in (B); Coomassie blue staining. (C) Phosphorylated level is normalized by the results with reprobing with each antibody as described in Fig. 3B. Data are mean±S.D. from four samples. *, P<0.05 vs. LIF (+).

4 Discussion

The major new findings of this study, in which we systematically examined the spatial activation of STAT3 in the myocardium after acute ligation of the LAD coronary artery in rats, is that STAT3 is activated after the infarction. Interestingly, activation of STAT3 was not confined to the ischemic infarcted area but was in fact more prominently activated in the healthy border area, where myocardial remodeling was taking place. The border area of AMI is commonly considered to represent the tissue adjacent to the central area of maximal ischemia in which myocytes have suffered reversible injury [25]. This border area is affected by various factors, such as hypoxia, inflammatory responses and mechanical stretching [24,26]. Although STAT3 was also phosphorylated in the ischemic area and this phosphorylation might be the result of many cellular events as mentioned above, the phosphorylation level was very low. The healthy viable area can be expected to be the most affected by the abnormal passive stretching produced by the bulging of the necrotic area during ventricular contraction. Moreover, diastolic overloading is known to affect the myocardium distant from the ischemic area, so that these mechanical forces may induce cell death in this area as well. However, not only cardiac myocytes but also cardiac non-myocytes such as cardiac fibroblasts would be a possible cellular source for STAT3 activation in AMI heart.

Several observations made in this study have important implications for the understanding of the contribution of cytokines to AMI. Previously it was thought that cytokines mainly contribute to immunological responses, and STAT3 was identified originally as an acute-phase response factor activated by IL-6 [3]. STAT3 is activated by a variety of cytokines, including the IL-6 family of cytokines, granulocyte colony stimulating factor, epidermal growth factor, interferon-γ, leptin, IL-2, and IL-10 [30]. The phosphorylation of STAT3 observed in AMI heart might be caused by ligands which activate gp130, such as cardiotrophin-1, IL-6 and LIF, or which activate JAK2, such as Ang II or PDGF. Stretching of cardiac myocytes is known to release Ang II [31], and this autocrine/paracrine-secreted Ang II might also in combination with other growth factors activate the JAK/STAT pathway.

Several experiments have demonstrated that anti-apoptotic signals are transduced via JAK2. In hematopoietic cells, the kinase domain of JAK2 mediates the induction of Bcl-2 and inhibits cell death [32]. Additionally, in vitro studies have provided evidence that STAT3 plays important roles in the generation of anti-apoptotic signals such as JAK2 kinase, most likely through induction of the Bcl-2 or Bcl-xL genes [33]. To examine the physiological function of the JAK/STAT activation in AMI heart, AG-490 was used in the present study to inhibit the JAK2-dependent pathway. Treatment with JAK2 inhibitor reduced the phosphorylation of STAT3, and resulted in a significant increase in caspase-3 activity and Bax protein in AMI hearts. Therefore, our results suggested the possibility of an anti-apoptotic function of JAK2-dependent signaling in AMI hearts. We further examined the effects of AG-490 on other signaling cascades downstream of gp130 with the use of cultured cardiac myocytes. Although AG-490 inhibited STAT1 and STAT3 tyrosine phosphorylations, no effect was observed on the activation level of PI 3-K or MAPK. However, it is possible that other downstream signaling pathways via JAK2 may, with the exception of STATs, play pivotal roles in protection against myocardial apoptosis.

A recent in vivo study confirmed that staurosporine-induced apoptosis in cardiac myocytes was associated with increased caspase-3 expression [34]. In addition, caspase inhibitor was found to be effective in reducing myocardial reperfusion injury, and this effect was in part due to attenuation of cardiomyocyte apoptosis [35]. In the present study, caspase-3 activity slightly increased in the non-ischemic border area after AMI, and treatment with JAK2 inhibitor significantly increased this activity. This finding indicates that caspase families might play pivotal roles in myocardial apoptosis in ischemic heart disease.

Induction of apoptosis increased the level of Bax protein and enhanced formation of Bax–Bcl-2 complexes. These changes are important for the subsequent activation of caspase-3 and the progression of apoptosis [36,37]. The mechanical loading induced by AMI affected the expression of Bcl-2 and Bax in viable myocardium, which in turn triggered apoptosis and remodeling of the ventricular wall [38]. Number of apoptotic myocyte is reported to increase 7.6-fold after 24 h of stretching in association with an increase in the level of Bax [39]. In the present study, a few cardiac myocytes underwent apoptosis 24 h after coronary occlusion in the healthy border area of the AMI heart, although no significant change was detected in levels of apoptosis-related proteins. Inhibition of JAK2 by AG-490 significantly augmented myocyte apoptosis, as demonstrated by TUNEL assay, and increased level of Bax protein.

A recent study using ventricular restricted gp130 knockout mice revealed increased cardiac myocyte apoptosis after mechanical stress [16]. To date, few findings are available on the signaling mechanism of the JAK/STAT-induced anti-apoptotic function. However, the present study suggests that activation of the JAK/STAT pathway suppresses Bax expression in combination with or without as yet unidentified anti-apoptotic genes, and thus inhibits caspase-3 activation. In summary, STAT3 was activated especially in the healthy border area of rat AMI heart, mainly as a result of stretch-related stress. Inhibition of JAK/STAT activation in the healthy border myocardium increased Bax and caspase-3 activation, which resulted in augmentation of myocyte apoptosis in AMI hearts. The JAK/STAT pathway, especially the JAK2-dependent pathway, may have a cytoprotective effect on stretched myocardium in AMI hearts.

Acknowledgements

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and Grants from the Ministry of Health and Welfare of Japan and Takeda Science Foundation. We thank Ms. C. Kusunoki for her secretarial assistance.

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