Copyright © 2004, European Society of Cardiology
STAT3 mediates cardioprotection against ischemia/reperfusion injury through metallothionein induction in the heart*
aDepartment of Molecular Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita City, Osaka, 565-0871, Japan
bDepartment of Clinical Evaluation of Medicines and Therapeutics, Osaka University, 1-6 Yamadaoka, Suita City, Osaka, 565-0871, Japan
cDepartment of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita City, Osaka, 565-0871, Japan
dLaboratory of Immune Regulation, Graduate School of Frontier Bioscience, Osaka University, 1-3 Yamadaoka, Suita City, Osaka, 565-0871, Japan
* Corresponding author. Tel.: +81 6 6879 8252; fax: +81 6 6879 8253. Email address: fujio{at}phs.osaka-u.ac.jp
Received 23 June 2004; revised 9 October 2004; accepted 13 October 2004
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentReferences |
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Objective: Activation of signal transducer and activator of transcription 3 (STAT3) was reported to be correlated with myocardial protection against ischemia/reperfusion (I/R) injury in ischemic preconditioning. Here, we tested the causality between STAT3 activity and cardioprotection. We also addressed the molecular mechanism for its cardioprotection.
Methods and results: Cardiac-specific transgenic mice expressing constitutively active STAT3 (TG) were generated and exposed to I/R injury. TG hearts exhibited infarcts that reduced by 60.3% in size, compared with nontransgenic littermates (NTG). By measuring dichlorofluorescein (DCF) and 8-isoprostane, reactive-oxygen-species (ROS)-induced metabolites, it was revealed that ROS were generated to lesser extent in TG hearts than in NTG in response to I/R stress. In parallel, ROS scavengers, metallothionein1 (MT1), and metallothionein2 (MT2) were markedly up-regulated in TG hearts. Finally, homozygous deletion of the MT1 and MT2 genes abrogated cardioprotective effect of STAT3 against I/R injury with the cancellation of its ROS-scavenging effects.
Conclusions: Activation of STAT3 protects myocardium from I/R injury in vivo. STAT3 mediates cardioprotection at least partially through MT1 and 2. STAT3 is a potential therapeutic target for I/R injury.
KEYWORDS Ischemia/reperfusion; Metallothionein; STAT3
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentReferences |
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Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor that was originally identified as a mediator of cytokine signaling [1]. Interleukin-6 family cytokines activate glycoprotein 130 (gp130) and phosphorylate STAT3, leading to its translocation from the cytoplasm to the nucleus, where it activates the transcription of the target genes [2]. STAT3 contributes to a diverse array of cell functions such as proliferation [3], differentiation [4,5], and survival [6,7]. In cardiac myocytes, STAT proteins are activated by extracellular stimuli, including catecholamines [8] and mechanical stretch [9], through autocrine/paracrine system. Activated STAT proteins up-regulate cardioprotective genes, including bcl-xL [10] and VEGF [11,12]. Thus, it could be proposed that STAT proteins play an important role in the maintenance of cardiac function, however, the molecular mechanisms remain to be fully elucidated especially under pathophysiological conditions in vivo.
Acute coronary syndrome is a major cause of death [13]. Early recanalization is recommended to rescue the myocardial damage by ischemia [14], but this strategy is accompanied by tissue damage, known as reperfusion injury. Large quantities of reactive oxygen species (ROS) are generated in the reperfused myocardium, resulting in cell damage [15,16]. Clinical and experimental studies demonstrated that ischemic preconditioning reduces reperfusion injury [17,18]. Interestingly, STAT3 is activated by ischemic preconditioning, and STAT3 activity correlates with the protective effects of preconditioning [19]. However, the causality between STAT3 activation and its protective effects against the reperfusion injury has not been established under these conditions.
In the present study, we examined the cardioprotective effects of STAT3 against ischemia/reperfusion (I/R) injury in the heart by using transgenic mice expressing active form of STAT3. We also investigated the mechanisms for STAT3-mediated resistance to I/R injury.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentReferences |
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2.1. Transgenic mice
Generation of cardiac-specific transgenic mice expressing constitutively active STAT3 (TG) has been described previously [12]. In the present study, TG mice were backcrossed with C57BL/6 by more than four generations to generate TG mice on C57BL/6 background. The experiments were performed by using two independent lines of TG with the same results.
The constitutively active mutant of STAT3 shows the conformational changes by intramolecular S–S bonding and exhibits biological activities [7]. Therefore, its activities are regulated not by the expression level but by intramolecular S–S bond formation. DNA-binding activity of STAT3 from TG hearts is about twofold compared with that from nontransgenic littermates (NTG), suggesting that STAT3 in TG hearts is activated at physiological level, not at nonphysiologically high level [12]. The care of all animals was in compliance with the Osaka University Animal Care Guidelines. The present study conforms to 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).
Metallothionein1 (MT1) and metallothionein2 (MT2) knocked-out mice (MT–/–), obtained from Jackson Labs, were backcrossed with C57BL/6 by six generations to obtain MT-null mice on C57BL/6 background.
2.2. Murine ischemia/reperfusion model in vivo
Murine ischemia/reperfusion model was generated as described previously [20], with minor modification. Male mice weighing 25 to 30 g were anesthetized with pentobarbital sodium (40 mg/kg) and ventilated with 80% oxygen. Left-side thoracotomy was performed to reach the heart. The left coronary artery was ligated with a suture under the edge of auricle. After 60-min occlusion, the ligature was loosened, and reperfusion was confirmed visually by the rapid restoration of blood flow accompanied by a change in the appearance of the ischemic myocardium from pale to bright red. After 120-min reperfusion, the ligature was briefly retied and 0.5 ml of 4% Evans Blue (Sigma) in PBS was immediately injected into the left carotid artery to identify the area at risk (AAR). The body temperature was maintained at 37.0 °C during the operation.
In some experiments, we examined the infarct area (IA) after 30-min ischemia followed by 24-h reperfusion.
Sham operation group underwent thoracotomy and penetration by the suture around the left coronary artery without ligation.
Mice (n=159) underwent surgical procedure, including sham operation, in total. Thirteen mice died of ventricular arrhythmia or heart failure. Four mice died of bleeding by penetration of ventricle. Twelve mice were excluded, with mainly technical troubles such as inadequate staining (n=5), inadequate ventilation (n=2), and unconfirmed reperfusion (n=5). The operative mortality was 11% in this study.
Infarct area sizes and risk area sizes were evaluated as described previously [20]. Briefly, the isolated heart was sectioned with a vibrating blade microtome (Leica) into five slices perpendicular to the long axis. To delineate the infarct area (IA), the slices were incubated in 2% triphenyltetrazolium chloride (TTC; Sigma) in PBS at 37 °C for 20 min. Left ventricular area (LVA), AAR, and IA on both sides of each slice were measured by using the Macscope program. The LVA, AAR, and IA for each slice were determined by taking the average of the areas of the basal and apical side of each slice. Infarct weight was determined as follows: {(I1 x WT1)+(I2 x WT2)+(I3 x WT3)+(I4 x WT4)+(I5 x WT5)}, where I indicates the ratio of IA over LVA for each slice, and WT indicates the weight of each slice. Risk area weight was calculated in the same way. Percent myocardial infarction (%MI) was calculated as the ratio of the infarct weight to risk zone weight.
2.3. Measurement of tissue concentration of 8-isoprostane
To evaluate ROS production, we measured tissue concentration of 8-isoprostane, a nonenzymatical metabolite from ROS [21] by enzyme immunoassay (EIA) as described previously [22] with modifications. At the end of 90-min reperfusion, the hearts were homogenized in a polytron homogenizer. The homogenates were hydrolyzed by incubation in 0.5 ml of 15% KOH at 40 °C for 90 min, and proteins were removed by adding 2 ml of ethanol containing 0.01% butylhydroxytoluene followed by centrifugation at 1500 x g for 10 min. The supernatants were evaporated by means of vacuum centrifugation and resolved in 1.5 ml of the neutralizing buffer (pH 7.4). After purified with the 8-isoprostane Affinity Column (Cayman Chemical), 8-isoprostane was concentrated in 150 µl of EIA buffer and measured by using EIA Kit (Cayman) according to manufacturers' protocol. The tissue 8-isoprostane level was expressed as nanogram per gram tissue.
2.4. Visualization and estimation of reactive oxygen species with 2',7'-dichloro-dihydrofluorescin diacetate
The 2',7'-dichlorodihydrofluorescin diacetate (H2DCFDA, Molecular Probes) was dissolved in DMSO at a concentration of 25 mg/ml. After being diluted with 50% ethanol to a final concentration of 2.5 mg/ml, 6 µg/g body weight was administered intravenously. After being loaded with H2DCFDA, the heart was exposed to I/R injury as described above. Ten minutes after reperfusion, the heart was isolated and sectioned with a vibrating blade microtome. The fluorescence images were obtained by LAS-3000 system (Fuji Photo Film) with a cooled digital CCD camera. Excitation of 2',7'-dichlorofluorescein (DCF), an oxidized form of H2DCFDA, was produced by using a blue-single-quantum-well (SQW)-light emitting diode (LED), and emitted fluorescence was collected through a Y515 filter. Fluorescent images were processed by using Multi Gauge image analyzing software (Fuji Photo Film). Background fluorescence intensity (arbitrary unit/mm2) was defined as that in the myocardium loaded without H2DCFDA. The fluorescence intensities at 10 randomly selected points in the risk area and five points in the nonrisk area were measured. By subtracting background intensity, the average intensity at the risk area (FRA) and that at the nonrisk area (FNA) were calculated. The ratio of FRA to the FNA was determined as the relative fluorescence intensity.
2.5. Western blot analyses
Western blot analyses were performed as described previously [12]. The antibodies against phospho-extracellular-signal-regulated kinase (ERK), phospho-Akt, and Akt were purchased from Cell Signaling, antibodies against STAT3, ERK1, ERK2, and cdk4 were from SantaCruz Biotechnology, and anti-Flag antibody was from Upstate Biotechnology. Antimetallothionein antibody was from Trans Genic. ECL system (Amersham) was used for detection.
2.6. Northern blot analyses
Northernblot analyses were performed as described previously [11]. In brief, 10 µg of total RNA was size-fractionated and blotted onto a nylon membrane (Hybond N+, Amersham). After 20 min of prehybridization at 60 °C, the membranes were hybridized with probes labeled with 32P at 60 °C for 1 h. The oligonucleotide probes for MT1, MT2, manganese super oxide dismutase (MnSOD), and inducible NO synthetase (iNOS) mRNA were as follows: 5'-CCGAGATCTGGTGAAGCTGGAGCTACGG-3' for MT1, 5'-ATGGCGAGTGGAGGCGGCGGTTGAAGAT-3' for MT2, 5'-ATTGAGGTTTACACGACCGCTGCTCTCC-3' for MnSOD, 5'-TTCTGTGCTGTCCCAGTGAGGAGCTGCG-3' for iNOS. These probes were labeled with 32P by using a 5'-end labeling kit (Amersham). The probe for GAPDH mRNA, kindly donated by Dr. Chien, KR (University of California San Diego.), was labeled with 32P by Megaprime labeling kit (Amersham).
2.7. Statistical analysis
Data are expressed as mean ± S.E.M. Statistical analysis was performed by t test or one-way ANOVA and Scheffe's test for multiple comparison.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentReferences |
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3.1. Transgenic hearts expressing active STAT3 showed the resistance to I/R injury
To examine the cardioprotective effects of STAT3 activation in vivo, TG hearts expressing constitutively active STAT3 were exposed to I/R injury. As shown in Fig. 1A and B, the infarct size in transgenic myocardium (TG) was reduced by 60.3% (n=8) compared with that of nontransgenic littermates (NTG; n=9). The infarct size of NTG hearts was 57% in average, which is almost consistent with the previous study [20], indicating that the surgical procedure was reproduced according to previous works. The ratios of risk zone weight to the left ventricular weight (%RZ) were similar in TG and NTG hearts (63.1 ± 10.0% and 64.9 ± 7.9%, respectively).
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As STAT3 is reported to induce cardiotrophin-1 [23], a member of IL-6 related cytokines, other signal transducing molecules downstream of gp130, such as Akt and ERK, were analyzed by Western blotting by using phospho-specific antibodies. As shown in Fig 1C, neither Akt nor ERK were activated in TG hearts, indicating that STAT3 is exclusively activated in TG hearts.
Following the myocardial damage by I/R, infarct area continues to expand due to the inflammatory reaction by infiltrating neutrophils until about 24 h after reperfusion [24]. Thus, we examined the effect of STAT3 activation on ischemia/reperfusion injury 24 h after reperfusion. In some preliminary studies, nontransgenic hearts were exposed to 60-min ischemia followed by 24-h reperfusion. Under this condition, the mortality was about 40%, which could bias the data concerning infarct area. Therefore, to reduce the mortality, we established a mild injury model with less than 20% of mortality by shortening ischemia time from 60 to 30 min. Thus, TG and NTG hearts were subjected to 30-min ischemia followed by 24-h reperfusion (Fig. 2). The ratios of infarct size to risk area size were estimated as 11.2 ± 5.8% and 40.9 ± 4.1% in TG and NTG hearts, respectively (p<0.005), suggesting that the STAT3-mediated myocardial protection is sufficient to prevent the injury expansion induced by the inflammatory reaction.
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3.2. ROS generation was remarkably inhibited in TG hearts
As ROS play important roles in I/R injury [25], we evaluate ROS production by measuring tissue concentration of 8-isoprostane [21] (Fig. 3). In sham-operated hearts, there were no differences in the concentrations of 8-isoprostane between TG (n=3) and nontransgenic NTG (n=3). NTG hearts (n=4) showed threefold induction of 8-isoprostane following I/R injury that is significantly higher than sham-operated NTG (p<0.005). Importantly, 8-isoprostane generation followed by I/R was significantly inhibited in TG hearts compared with NTG (p<0.005 versus NTG after I/R). Since accumulation of 8-isoprostane reflects total production of ROS, these data suggest that activation of STAT3 inhibited ROS generation induced by the exposure to I/R, accompanying the myocardial protection.
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To address ROS-scavenging effects of TG myocardium, we directly measured ROS by using DCF immediately after reperfusion. We administered H2DCFDA prior to I/R injury and measured the fluorescence intensity of DCF 10 min after reperfusion. As shown in Fig. 4A and B, ROS production in the risk area was inhibited in TG hearts by 84.0% compared with NTG. By histological analyses, infiltrating neutrophils were neither detected in TG nor in NTG myocardium 10 min after reperfusion (data not shown), suggesting that STAT3 activation transduces antioxidative signals in myocardium, independent of inflammatory cells.
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3.3. STAT3 activation up-regulates MT1 and MT2 in the hearts
To clarify the molecular mechanism of the ROS-scavenging effects of STAT3 activity, we examined the expression of antioxidative genes (Fig. 5). Northern blot analyses demonstrated that MT mRNAs were up-regulated in TG hearts but not MnSOD or iNOS (Fig. 5A). Furthermore, Western blot analyses showed that the expression of MT proteins was increased in TG hearts (Fig. 5B). These findings suggest that MT1 and 2 are candidate genes responsible for STAT3-mediated cardioprotection against I/R.
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3.4. Ablation of MT1 and MT2 genes abolishes cardioprotective effects of STAT3 activities in TG hearts
To examine a role of MT induction in STAT3-mediated cardioprotection, we generated the transgenic mice with the overexpression of active STAT3 on MT–/– or MT–/+ background (designated as TG-MT–/– or TG-MT–/+, respectively). First, it was confirmed by Western blotting that MT expression was abrogated in TG-MT–/–, although the expression level of active STAT3 was equivalent to that in TG-MT–/+ (Fig. 6A).
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To address whether ischemia affects the expression of STAT3 and MT and modifies the cardioprotective effects as a result, we quantified the cardiac expression of these proteins before and after ischemia. As shown in Fig. 6B, the expression of STAT3 and MT was not altered by exposure to 1-h ischemia. To examine the time course of STAT3 activation and MT up-regulation, LIF was intravenously injected, and the expression of MT mRNA and protein was analyzed. It was revealed that MT mRNA started to increase 3 h after LIF injection and that the up-regulation of MT protein was detected 8 to 24 h after injection (data not shown).
Next, we subjected double mutant mice (MT–/–, MT–/+, TG-MT–/–, and TG-MT–/+) to I/R injury. As shown in Fig. 7A, on MT–/+ background, active STAT3 transgenic hearts exhibited remarkable reduction in infarct size by 63.6% compared with nontransgenic MT–/+ littermate hearts. Homozygous depletion of MT genes in TG mice (TG-MT–/–) remarkably abolished STAT3-mediated reduction in infarct size compared with TG-MT–/+ hearts. There was no significant difference in infarct size among MT–/+, TG-MT–/–, and MT–/– littermates. The ratio of risk area to left ventricle was similar in MT–/+, MT–/–, TG-MT–/+, and TG-MT–/– hearts (60.3 ± 3.2%, 58.9 ± 2.7%, 57.8 ± 5.7%, and 60.6 ± 3.4%, respectively).
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Finally, we analyzed ROS production in MT-null mice by measuring DCF (Fig. 7B). In nontransgenic mice, MT-null mice did not show increase in ROS production in response to I/ R compared with MT–/+. Importantly, the homozygous ablation of MT genes in TG mice abrogated STAT3-mediated reduction in ROS generation in infarct areas compared with TG-MT–/+ hearts.
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentReferences |
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In the present study, we provided the evidence that cardiac-myocyte-specific activation of STAT3 mediates a protective signal against I/R injury in vivo. Transgenic hearts expressing constitutively active STAT3 showed resistance against I/R injury. Furthermore, ROS generation was remarkably inhibited in transgenic hearts in accordance with up-regulation of ROS scavengers MT1 and MT2. Finally, STAT3 activities could not protect myocardium from I/R injury in MT-null mice, indicating that MT proteins play an important role in STAT3-mediated cardioprotection.
First, we examined the antiapoptotic effects of STAT3 by TUNEL staining and found that TG hearts showed lower frequency of TUNEL-positive cardiomyocytes than NTG. However, antiapoptotic proteins, such as bcl-xL and bcl-2, were not up-regulated in TG hearts exposed to I/R (data not shown). Thus, we examined ROS generation in reperfused myocardium because high levels of ROS initiate irreversible chemical changes in lipids and proteins, resulting in cytotoxicity. As shown in the present study, large amounts of 8-isoprostane were produced by I/R injury in nontransgenic hearts but not in TG hearts. Moreover, using DCF, we also indicated that ROS production was inhibited in TG hearts immediately after reperfusion, when inflammatory reaction was not initiated. These data suggest that activation of STAT3 protects cardiac myocytes from reperfusion injury by scavenging ROS generated in myocardium. Since ROS mediate both necrotic and apoptotic cell death, STAT3 may inhibit both necrosis and apoptosis. These combined protective effects may explain the large reduction in the infarct size seen in TG hearts.
Here, we demonstrated that STAT3 TG showed the up-regulation of MT, an ROS scavenger, associated with reduction in ROS generation. Recently, it has been reported that the promoter region of the MT gene contains functional STAT3-regulatory sequence [26,27], providing the causality between STAT3 activation and MT induction. Consistently, we have also confirmed that activation of STAT3 by LIF up-regulates MT mRNA and protein both in vitro and in vivo (data not shown).
To reveal functional significances of MT in cardioprotection, we generated double mutant mice of active STAT3 transgenic mice on the MT-null background. TG-MT–/– mice exhibited normal growth, as is the case with MT–/– mice. Consistent with previous reports [12,23], TG-MT–/– hearts showed up-regulation of ANF and cardiotropin-1 and enhancement of capillary density compared with NTG; however, no significant differences were detected in the expression of these genes or in vasculature between TG-MT–/– and TG-MT–/+ hearts (data not shown) probably because there was no significant difference in active STAT3 expression level between TG-MT–/– and TG-MT–/+ hearts. In contrast, MT expression was completely ablated in TG-MT–/– hearts, which failed to show the resistance to I/R, indicating the importance of MT proteins in STAT3-mediated cardioprotection.
There was little difference in STAT3-mediated cardioprotection between TG-MT–/+ and TG-MT+/+ hearts. Thus, we quantified the expression of MT1 and 2 proteins in these mice. TG-MT+/+ hearts showed 1.3-fold increase in MT protein expression probably because MT protein expression was regulated by posttranscriptional mechanisms as well as by transcriptional mechanisms, as reported previously [28]. The small difference in MT expression between TG-MT–/+ and TG-MT+/+ hearts might account for the same level of protective effects in the transgenic mice on MT–/+ and MT+/+ backgrounds. It might also be possible that the expression level of MT proteins in TG-MT–/+ hearts is sufficient for full activities of STAT3/MT pathway-mediated cardioprotection against I/R.
In comparison with MT–/+, MT-null hearts showed no significant increase in ROS in response to I/R in nontransgenic mice. As the expression of MT protein was unchanged during ischemia, it could be proposed that basal level of MT expression in nontransgenic mice is not sufficient to scavenge ROS induced by I/R. Importantly, the expression of STAT3 was not affected by ischemia in TG-MT–/– and TG-MT–/+ hearts. Thus, it is unlikely that ROS production is enhanced in MT-null hearts, abrogating STAT3-mediated cardioprotection in TG-MT–/– hearts. Collectively, although we could not completely exclude the possibility of the involvement of other target genes of STAT3, activation of STAT3 prior to I/R could lead to remarkable cardioprotection against I/R through ROS scavengers. Supporting this hypothesis, we have confirmed that intravenous injection of LIF, an activator of STAT3, results in cardioprotection against I/R (data not shown). Importantly, the up-regulation of MT proteins was detected 6 h after LIF injection and was sustained for 24 h (data not shown).
In summary, we have demonstrated that cardiac-specific activation of STAT3 transduces antioxidative signals through MT and confers resistance against ROS stress in the heart. These findings suggest that activation of STAT3 could be a therapeutic strategy against myocardial injury.
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Acknowledgment |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAcknowledgmentReferences |
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We thank Jurei Hironaka, Kae Hoshide, and Yasuko Murao for their secretarial work.
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Notes |
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* This study was partially supported by Osaka Foundation for Promotion of Clinical Immunology (to Y.F. and to Y.O.). This work was also supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by Health and Labour Sciences Research Grant.
Time for primary review 21 days
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