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Free radical scavenging inhibits STAT phosphorylation following in vivo ischemia/reperfusion injury James McCormick,*,1 Sean P. Barry,*,1 Ahila Sivarajah,† Giorgio Stefanutti,§ Paul A. Townsend,‡ Kevin M. Lawrence,† Simon Eaton,§ Richard A. Knight,* Christoph Thiemermann,† David S. Latchman,* and Anastasis Stephanou*,2 *Department of Medical Molecular Biology, §Department of Surgery, The Institute of Child Health, University College London, London, UK; †Centre for Experimental Medicine, Nephrology and Critical Care, The William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, Queen Mary, University of London, Charterhouse Square, London, UK; and ‡Human Genetics Division, Southampton General Hospital, University of Southampton, Southampton, UK The signal transducer and activator of transcription (STAT) family are latent transcription factors involved in a variety of signal transduction pathways, including cell death cascades. STAT1 has been shown to have a crucial role in regulating cardiac cell apoptosis in the myocardium exposed to ischemia/ reperfusion (I/R) injury. The free radical scavenger, tempol, is known to have cardioprotective properties, although little is known about the molecular mechanism(s) by which it acts. In the present study, we assessed the levels of phosphorylated STAT1 and STAT3 and examined whether tempol was able to affect STAT activation after in vivo cardiac I/R injury. We observed a reperfusion time-dependent increase in the tyrosine phosphorylation of STAT1 and STAT3 at residues 701 and 705, respectively. Here we show for the first time that tempol dramatically reduced STAT1 and 3 phosphorylation. The reduction in STAT1 and 3 phosphorylation was accompanied by a concomitant decrease in cellular malondialdehyde (MDA) levels. To verify the role of STAT1 in modulating the cardioprotective effect of tempol, rats were injected with the STAT1 activator, IFN-␥, and tempol during I/R injury. We found that the presence of IFN-␥ abrogated the protective effects of tempol, suggesting that the protective effects of tempol may partly operate by decreasing the phosphorylation of STAT1. This study demonstrates that careful dissection of the molecular mechanisms that underpin I/R injury may reveal cardioprotective targets for future therapy.—McCormick, J,. Barry, S. P., Sivarajah, A., Stefanutti, G., Townsend, P. A., Lawrence, K. M., Eaton, S., Knight, R. A., Thiemermann, C., Latchman, D. S., Stephanou, A. Free radical scavenging inhibits stat phosphorylation following in vivo ischemia/reperfusion injury. FASEB J. 20, E1404 –E1410 (2006)

ABSTRACT

blood flow to the left ventricle via the coronary artery, leading to cardiac stunning and cell death, which can lead to loss of cardiac output (1). Although both necrotic and apoptotic cell death occur during ischemia, apoptotic cell death appears to be enhanced after the re-establishment of coronary blood flow (reperfusion) (2, 3). Inhibition of the apoptotic process in cardiac myocytes exposed to simulated ischemia/reperfusion injury (I/R) or in the ischemic myocardium using specific caspase inhibitors results in reduced infarct size (4, 5). Reactive oxygen species (ROS) are generated during ischemia and reperfusion injury (6), and these free radicals promote the onset of cell death, resulting in characteristic cellular changes including lipid peroxidation, protein denaturation, and double-strand breaks in DNA (6). The generation of ROS during I/R has been demonstrated to induce apoptosis in the cardiac myocyte (7). Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a low molecular weight agent capable of scavenging superoxide anions, and offers an advantage over many other ROS scavengers as it is able permeate biological membranes in vitro (8). Moreover, tempol was shown to reduce infarct size after IR injury (8). The janus-kinase (JAK) family of proteins are activated in response to various physiological insults and cytokines, leading to the phosphorylation of signal transducer and activator of transcription (STAT) proteins (9, 10). Phosphorylation of STAT1 and STAT3 at tyrosine residues 701 and 705, respectively, results in their dimerization, leading to nuclear translocation. STAT1 is thought to promote the onset of apoptosis while STAT3 is believed to abro1

Key Words: tempol 䡠 heart 䡠 rat

Myocardial ischemia results from the occlusion of E1404

These authors contributed equally to this work. Correspondence: Department of Medical Molecular Biology, The Institute of Child Health, University College London, 30 Guilford St., London, WC1N 1EH, UK. E-mail: [email protected] doi: 10.1096/fj.06-6188fje 2

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gate the effects of STAT1 (11). Once in the nucleus, STAT proteins regulate the expression of pro- and antiapoptotic genes, including caspase 1 (9, 10). Both STATs can also be phosphorylated on serine residue 727, which is thought to be necessary for maximum activation, although the precise mechanism is poorly understood. Previously we demonstrated that STAT1 plays an important role in promoting apoptotic cell death in cardiac myocytes and also in the intact heart after I/R injury (11–14). These studies therefore suggest STAT1 as a therapeutic target in the ischemic myocardium. It has been reported that the polyphenolic agent epigallocatechin-3-gallate (EGCG), a major constituent of green tea, is a potent inhibitor of STAT-1 phosphorylation and activation (15). We recently showed that EGCG reduced STAT-1 phosphorylation and protected cardiac myocytes against I/R–induced apoptotic cell death. More interesting is the fact that EGCG reduced the infarct size and improved hemodynamic recovery and ventricular function in the ischemic/reperfused rat heart (16). Here we demonstrate, using an in vivo model, that STAT1 and STAT3 phosphorylation and myocardial infarct size can be reduced by injection of the free radical scavenger, tempol. Moreover, IFN-␥, a potent activator of STAT1, not only enhanced infarct size but overcame the protective effects of tempol. The results presented here demonstrate a potential mechanism of action for tempol and indicate the therapeutic use of free radical scavengers in postischemic insults.

described previously (24), using anti-STAT1, anti-STAT3, antiphospho ERK1/2 (Santa Cruz Biotechnology, CA, USA), antiphosphoSTAT1Y701 (Zymed, San Francisco, CA, USA), antiphosphoSTAT1S727 (Upstate, Cambridge, UK) antiphospho STAT3Y705, antiphospho STAT3727 (Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (Chemicon, Temecula, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were from Dako (Glostrup, Denmark). Densitometric analysis was performed using a GS-800 scanner and quantity one software (Bio-Rad, Herts, UK). All statistical analyses were performed using Graphpad Prism v4.0 (GraphPad Software, San Diego, CA, USA).

MATERIALS AND METHODS

Drug protocol

This study was performed in accordance with the United Kingdom, Home Office Animals (Scientific Procedures) Act 1986.

For MDA and STAT activation experiments, the following experimental groups were used: LAD coronary artery occlusion (25 min) and reperfusion (30 min) infused with 1) saline 5 min prior to the onset of reperfusion, 2) 100 mg/kg tempol 5 min prior to the onset of reperfusion, or 3) sham operation (n⫽4). For infarct measurements, rats were either sham operated or subjected to 25 min of ischemia and 2 h of reperfusion infused with 1) saline, 2) tempol, 3) IFN-␥, or 4) tempol ⫹ IFN-␥ (n⫽5).

In vivo ischemia/reperfusion injury in the rat Ischemia/reperfusion injury in the rat was performed as described previously (8). Briefly, male Wister rats (255–285 g) were anesthetized with sodium thiopentone (120 mg/kg; Intraval, Essex, UK) by intraperitoneal injection. Rats were subjected to 25 min of acute myocardial ischemia by occlusion of the left anterior descending coronary artery (LAD), followed by reperfusion, or were sham operated (no LAD occlusion). After reperfusion, the coronary artery was reoccluded; Evans blue dye (VWR, Dorset, UK) was injected into the left ventricle and the animals sacrificed by an overdose of anesthetic. The uncolored area (risk area) of the heart was removed from the blue area (nonrisk area).

Immunohistochemistry After the indicated treatments, the area at risk was separated and cut transversely into four slices. The tissue was then fixed in 10% formalin and embedded in paraffin. Immunohistochemical analysis was carried out on 4 ␮m sections with standard protocols using antibodies specific for phosphoSTAT3Y705 (Cell Signaling) and phospho-STAT1Y701 (Zymed, San Francisco, USA). Biotin-conjugated secondary antibodies were used (Vector Laboratories, Peterborough, UK), followed by streptavidin-biotin peroxidase complex solution (Dako). Color reactions were developed by incubating sections in 3⬘3-diaminobenzidine (Sigma-Aldrich, Dorset, UK) and images were captured on a Zeiss Axioscope 2 plus microscope. Determination of tissue malondialdehyde concentration Levels of malondialdehyde MDA in cardiac tissue were measured by HPLC as described previously (17). All reagents used were obtained from Sigma-Aldrich. Protein concentration in the homogenate was measured by the method of Peterson (25), and MDA was expressed as ␮mol/g protein.

Quantification of myocardial tissue injury At the end of the 2 h reperfusion period, the risk area was obtained (as above). The tissue from the risk area was cut into small pieces and incubated with p-nitroblue tetrazolium (NBT, 0.5 mg/ml) for 30 min at 37°C. The stained tissue was separated from the infarcted tissue, weighed, and the infarct size expressed as a percentage of the risk area.

Western blot analysis

Statistical analysis

Cardiac tissue was snap frozen in liquid nitrogen and lysed in radio-immuno-precipitation assay (RIPA) buffer (0.75 M NaCl, 5% (v/v) Nonidet P-40, 2.5% (w/v) deoxycholate, 0.5% (w/v) SDS, 0.25 M Tris-HCl pH 8.0, 10 mM DTT containing protease inhibitor cocktail). Western blots were carried out as

All data are expressed as a mean ⫾ se of n observations, where n represents the number of animals in the group. Hemodynamic parameters were analyzed via a 2-way ANOVA, followed by a Bonferroni post test. Risk area and infarct size were analyzed via a one-factorial ANOVA, followed by a Bonferroni

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(S727) was induced 15 min after reperfusion in the risk area and reached a maximum at 30 min. Levels of STAT1 Y701 phosphorylation decreased dramatically at 60 min of reperfusion and remained unchanged up to 120 min. Phosphorylation of STAT1 occurred predominantly in the risk area, the main area of ROS generation, highlighting the role of activated STAT1 in the damaged myocardium. In contrast to STAT1, tyrosine 705 (Y705) phosphorylation of STAT3 occurred during ischemia, but during reperfusion it followed kinetics similar to STAT1, with increasing levels of Y705 phosphorylation seen during reperfusion phase, reaching a maximum at 30 min (Fig. 1A). Phosphorylation of STAT3 at S727 occurred maximally during ischemia alone and remained relatively unchanged during reperfusion, suggesting that STAT3 S727 phosphorylation is reperfusion independent (Fig. 1B). Similar to STAT1, phosphorylation of STAT3 Y705 and S727 was present at basal levels in the nonrisk area, indicating that the injury had only affected the left ventricle, as expected. These data indicated I/R in vivo-induced STAT activation may contribute to infarct development. Reperfusion injury induces rapid activation of the ATM-␥H2AX pathway Figure 1. Time course of in vivo reperfusion after ischemia in rat hearts. Rats were sham operated or underwent 25 min ischemia, followed by the periods of reperfusion indicated. 20 ␮g protein from each heart (n⫽4 per time point, risk and nonrisk areas) were separated on SDS-PAGE gels and probed with antibodies for A) total STAT1, phospho STAT1Y701, and phospho STAT1S727, B) Total STAT3, phospho STAT3Y705, and phospho STAT3727. Equal protein loading was confirmed by probing with an antibody (Ab) to GAPDH. post test for multiple comparisons. A P value ⬍0.05 denotes a statistical significantly difference.

RESULTS STATs are phosphorylated in a time-dependent manner after I/R injury in vivo Using an in vivo coronary occlusion model, we performed a time course of I/R injury on the rat myocardium to assess the dynamics of STAT phosphorylation over time. Animals were subjected to either sham operation (no LAD occlusion), 25 min of ischemia, or 25 min of ischemia followed by reperfusion for 5 to 120 min. As expected, the mean arterial blood pressure fell during the ischemic insult, but remained constant throughout the reperfusion time, whereas heart rate remained constant during both ischemia and reperfusion (data not shown). As shown in Fig. 1A, STAT1 tyrosine phosphorylation (Y701) rather than STAT1 serine phosphorylation E1406

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H2AX has been shown to be activated by hypoxia and reperfusion in solid tumors (18). The ischemic insult failed to activate H2AX, suggesting that ischemia does not lead to large-scale DNA damage (Fig. 2). In contrast, reperfusion rapidly induced ␥H2AX in the risk area, showing for the first time that H2AX phosphorylation occurs in a reperfusion-dependent manner in cardiac tissue. This phosphorylation elicits time-dependent kinetics similar to STAT1 and 3 phosphorylation, highlighting concomitant activation of DNA damage and JAK-STAT pathways. Minimal ␥H2AX activation occurs in the nonrisk area, highlighting the fact that double-strand DNA breaks are confined to areas of tissue suffering the greatest reperfusion-induced injury.

γ

Figure 2. 20 ␮g protein from each heart (n⫽4 per time point, risk and nonrisk areas) were separated on SDS-PAGE gels and levels of DNA damage were assessed by probing for the phosphorylated form of histone-2AX (H2AX). Equal protein loading was confirmed by immunoblotting for GAPDH.

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STAT1 and STAT3 tyrosine phosphorylation is induced by ROS ROS generation is responsible for intracellular damage and has been shown to lead to the onset of apoptosis (7). To verify that tempol had reduced the concentration of ROS within the intracellular environment, we measured the cellular concentration of malondialdehyde (MDA). MDA is a marker of lipid peroxidation that occurs as a result of the damaging effects of ROS (17). We found that injection of tempol reduced the concentration of MDA to sham levels compared with the vehicle control (P⫽0.002), confirming that free radical concentration had been successfully inhibited during I/R (Fig. 3A). Comparing the sham-operated to the vehicle and tempoltreated animals in the nonrisk area, no statistically significant difference in the MDA levels (P⫽⬎0.2) was observed, indicating that I/R injury does not effect the nonrisk area (Fig. 3A). The decrease in MDA levels is coupled with decreased tyrosine phosphorylation of both STAT1 and STAT3 (Fig. 3B). Densitometry revealed that tyrosine phosphorylation of STAT1 was reduced by an average of 70 ⫾ 5% compared with the vehicle control, while STAT3 was decreased 45 ⫾ 7% (Fig. 3C). Immunohistochemical staining of tissue sections from the risk area confirms that I/R-induced STAT phosphorylation, which was predominantly nuclear, was indeed reduced by free radical scavenging (Fig. 4). These data imply that STAT1 and STAT3 phosphorylation in I/R can be attenuated by

a single bolus injection of tempol, which may allow tempol to protect against cell death on the onset of reperfusion. Activated STAT1 contributes to infarct development, in vivo Although activated STAT1 is induced after I/R injury and has been implicated in promoting apoptosis (11– 14), it was unclear whether this activation contributed to infarct development, in vivo. To test this, rats were subjected to I/R injury with injection of the potent STAT1 activator, IFN-␥. IFN-␥ was introduced to allow continual activation of STAT1 for up to 120 min (postischemia), at which point the infarct area can be measured. Baseline values of mean arterial blood pressure (MAP), heart rate, and pressure rate index (PRI) were not significantly different between groups (data not shown) compared with sham-operated animals; 25 min of LAD occlusion followed by 2 h of reperfusion did not cause significant alterations in MAP, heart rate, or pressure rate index compared with their respective controls. Neither IFN-␥ or IFN-␥ plus tempol had any significant effect on MAP, HR or PRI (data not shown). The mean values for the risk area were similar in all animal groups studied and ranged from 48 ⫾ 2% to 51 ⫾ 4% (data not shown) compared with sham-operated animals; 25 min of LAD occlusion, followed by 2 h of reperfusion, caused a significant increase in infarct size. Administration of IFN-␥ (25 ␮g/kg) 5 min before reperfusion caused a significant increase (by 30%) in myocardial infarct size

Figure 3. Rats were sham operated or underwent 25 min of ischemia and 30 min of reperfusion with infusion of saline or 100 mg/kg tempol, after which risk and nonrisk areas were dissected. A) The MDA lipid peroxidation assay was carried out using equal amounts of tissue (n⫽5); results are expressed in ␮mol MDA/g protein (*P⬍0.001) compared with I/R. B) 20 ␮g protein from each group (n⫽5) were immunoblotted for pSTAT1Y701, pSTAT3Y705, pSTAT3S727, total STAT1, and total STAT3. C) Densitometric analysis of pSTAT1Y701and pSTAT3Y705 normalized to total STAT and expressed as % change from vehicle (*P⬍0.05).


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Figure 4. Tissue was fixed in 10% formaldehyde, mounted on wax blocks, cut into 4 ␮m sections, followed by immunohistochemical staining using pSTAT1Y701 and pSTAT3Y705 antibodies. A) Tyrosine phosphorylation of STAT3 is significantly decreased on tempol treatment. B) The same result for tyrosine phosphorylated STAT1.

compared with vehicle (Fig. 5A); IFN-␥-induced activation of STAT1 in the risk area was confirmed by Western blot (Fig. 5B). In the presence of IFN-␥, tempol no longer had any affect on STAT1 phosphorylation; strikingly, IFN-␥ completely abrogated the cardioprotective affect of tempol. This strongly implicates STAT1 activation as a main target in tempol-mediated cardioprotection

DISCUSSION In the present study we show for the first time significant changes in levels of phosphorylated STAT1 and STAT3 in the infracted area in the in vivo myocardium exposed to I/R injury. The free radical scavenger, tempol, was shown to reduce STAT1 and STAT3 phosphorylation. Previous studies have demonstrated that administration of tempol in the same manner decreases infarct size (8). Taken together, these data imply that the activation of STAT1 and 3 are important functions within the infarct area and may contribute to cardiac remodeling. Furthermore, the decrease in infarct size may be partly due to the inhibition of the JAK-STAT pathway by tempol. The molecular pathway resulting in cell death after free radical generation is complex and not fully understood. Our studies also suggest that free radical production may trigger distinct kinases that are responsible for STAT1 or SATAT3 phosphorylation and that by reducing STAT phosphorylation by using tempol treatment, the extent of myocardial damage is reduced, which could in part explain the reduction in infarct size previously reported (8). STAT1 has been shown to induce apoptosis after I/R in vitro, whereas STAT3 was found to elicit opposite and thus protective effects (11, 12). It is paradoxical, therefore, that both STAT1 and STAT3 phosphorylation was reduced by tempol treatment in vivo. Although there is less overall activated STAT3, it may still demonstrate its protective effect. High levels of serine-phosphorylated E1408

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STAT3 were seen during ischemia that were not further enhanced upon reperfusion, but were reduced upon introduction of tempol. In contrast, however, low-level serine phosphorylation of STAT1 was observed during ischemia but diminished upon the onset of reperfusion. Phosphorylation of STAT on S727 has been shown to enhance transcriptional activity of both STAT1 and STAT3 (9 –11). Thus, the serine phosphorylation of STAT3 rather than STAT1 further suggests that STAT3 is still functioning to protect the myocardium from I/R injury. The generation of STAT3 S727 mutant mice indicated that these animals were highly susceptible to doxorubicin-induced heart failure, signifying the importance of this residue (19). The reduction in phosphorylation at S727 after reperfusion suggests rapid activation of a distinct phosphatase as a possible feedback mechanism to inhibit full STAT transcriptional activity. Initiation of double-strand DNA breaks during apoptosis causes accumulation of the phosphorylated form of the H2AX (␥H2AX) through activation of the ataxia telangiectasia mutated (ATM) and ATM-related (ATR) kinases, leading to recruitment of DNA repair factors (20). Here we show for the first time that I/R injury rapidly induces the activation of ␥H2AX in the risk area whereas minimal ␥H2AX activation occurs in the nonrisk area. We recently demonstrated a role of STAT1 in the ATM-mediated pathway (21), and our present data may suggest that STAT1 may also modulate the ATM pathway that is known to modulate the apoptotic process via the ATM-p53 pathway. The present study suggests that during I/R injury, a balance exists between proapoptotic STAT1 and antiapoptotic STAT3 that determines the response of the cell to I/R injury. Indeed, STAT3 has been shown to protect cells from IFN-␥-induced apoptosis by abrogating the proapoptotic effects of STAT1 (23). Moreover, STAT3-deficient mice are more sensitive to I/R-in-

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affects of a natural antioxidant green tea are mediated through STAT1 (16), this highlights the role of STAT1 as a general molecular target for antioxidants in the ischemic myocardium. J.M. and S.B. are Ph.D. students funded by the British Heart Foundation, London, UK.

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Figure 5. Prolonged STAT1 activation with IFN-␥ overcomes the cardioprotective effects of tempol. A) Infarct size was measured in rats subjected to the surgical procedure alone (sham n⫽5) or LAD coronary artery occlusion (25 min) and reperfusion (2 h) with infusion (5 min prior to reperfusion) of either saline (I/R n⫽7), tempol (n⫽7), IFN-␥ (n⫽6), or tempol and IFN-␥ together (n⫽6). *P ⬍ 0.05 compared with I/R vehicle. B) Western blot analysis of STAT1 phosphorylation from heart tissues extracted from the experiment in panel A Similar blots were observed from 3 different hearts form each group.

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duced apoptosis and have larger infarct sizes. It was shown recently that granulocyte colony-stimulating factor (G-CSF) activates both STAT1 and STAT3 in cardiac myocytes (24). The protective effect of G-CSF in myocardial infarction is mediated, however, through STAT3, which was activated to a greater extent than STAT1. Similarly, we demonstrated that increasing the ratio of STAT3 to STAT1 in transfection experiments protected cardiac myocytes from hypoxia/reoxygenation-induced cell death (12). In the present study, injection of IFN-␥, a potent activator of STAT1, exacerbated cardiac damage as evidenced by increased infarct sizes. Furthermore, IFN-␥-induced STAT1 activation during reperfusion significantly reduced the cardioprotective effect of tempol, indicating that this antioxidant’s antiapoptotic effect is likely to be mediated partly through reduction in activated STAT1. Since we previously showed that the cardioprotective FREE RADICAL SCAVENGING IN I/R INJURY

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Received for publication March 30, 2006. Accepted for publication May 8, 2006.

McCORMICK ET AL.

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Free radical scavenging inhibits STAT phosphorylation after in vivo ischemia/reperfusion injury James McCormick,*,1 Sean P. Barry,*,1 Ahila Sivarajah,† Giorgio Stefanutti,§ Paul A. Townsend,‡ Kevin M. Lawrence,† Simon Eaton,§ Richard A. Knight,* Christoph Thiemermann,† David S. Latchman,* and Anastasis Stephanou*,2 *Department of Medical Molecular Biology, §Department of Surgery, Institute of Child Health, University College London, London, UK; †Centre for Experimental Medicine, Nephrology and Critical Care, The William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, Queen Mary, University of London, Charterhouse Square, London, UK; and ‡Human Genetics Division, Southampton General Hospital, University of Southampton, Southampton, UK To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-6188fje SPECIFIC AIMS We previously demonstrated that STAT1 plays an important role in promoting apoptotic cell death in cardiac myocytes after ischemia/reperfusion (I/R) injury. The mechanism and pathways of STAT activation after I/R are still unclear. Using an in vivo model, we demonstrate that STAT1 and STAT3 phosphorylation and myocardial infarction can be reduced by the free radical scavenger, tempol. Moreover, IFN␥, a potent activator of STAT1, not only enhances infarct size but can overcome the protective effects of tempol. The results presented here demonstrate a potential mechanism of action for tempol and indicate the therapeutic use of free radical scavengers for targeting STAT1 activation in postischemic insults.

PRINCIPAL FINDINGS 1. STAT1 and STAT3 are phosphorylated in a timedependent manner after I/R injury in vivo Using an in vivo coronary occlusion model, we performed a time course of I/R injury on the rat myocardium in order to assess the dynamics of STAT1 and STAT3 tyrosine or serine phosphorylation over time. As shown in Fig. 1A, STAT1 tyrosine phosphorylation (Y701), rather than STAT1 serine phosphorylation (S727), was induced 15 min after reperfusion in the risk area (infarct area) compared with the nonrisk area and reached a maximum at 30 min. Levels of STAT1 Y701 phosphorylation decreased dramatically at 60 min of reperfusion and remained unchanged for up to 120 min. Thus, STAT1 activation occurred predominantly in the risk area, the main area of reactive oxygen species (ROS) generation, highlighting the role of activated STAT1 in the damaged myocardium. 0892-6638/06/0020-2115 © FASEB

In contrast to STAT1, STAT3 tyrosine 705 (Y705) phosphorylation was seen during ischemia and after the reperfusion phase, reaching a maximum at 30 min (Fig. 1A). Similarly, STAT3 S727 phosphorylation was observed during ischemia and after the reperfusion (Fig. 1B). 2. STAT1 and STAT3 phosphorylation is induced by ROS ROS generation is responsible for intracellular damage and has been shown to lead to the onset of apoptosis. To verify that tempol had reduced the concentration of ROS within the intracellular environment, we measured the cellular concentration of malondialdehyde (MDA). MDA is a marker of lipid peroxidation that occurs as a result of the damaging effects of ROS. We found that injection of tempol reduced the concentration of MDA to sham levels compared with the vehicle control, confirming that free radical concentration had been successfully inhibited during I/R (see Fig. 3A, full-length article). Comparing the sham-operated to the vehicle and tempol-treated animals in the nonrisk area, no statistically significant difference in the MDA levels (P⫽⬎0.2) was observed, indicating that I/R injury does not effect the nonrisk area (see Fig. 3A, full-length article). The decrease in MDA levels was associated with decreased tyrosine phosphorylation of both STAT1 and STAT3 (see Fig. 3B, full-length article). Immunohistochemical staining of tissue sections from the risk area 1

These authors contributed equally to this work. Correspondence: Department of Medical Molecular Biology, Institute of Child Health, University College London, 30 Guilford St., London, WC1N 1EH, UK. E-mail: [email protected] doi: 10.1096/fj.06-6188fje 2

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occlusion followed by 2 h of reperfusion did not cause significant alterations in MAP, heart rate, or pressure rate index compared with their respective controls. Neither IFN-␥ alone or IFN-␥ plus tempol had any significant effect on MAP, HR, or PRI (data not shown). Administration of IFN-␥ (25 ␮g/kg) 5 min before reperfusion caused an increase of 30% in myocardial infarct size compared with vehicle (Fig. 2A); IFN-␥ induced activation of STAT1 in the risk area was confirmed by Western blot (Fig 2B, lower panel). In the presence of IFN-␥, tempol no longer affected STAT1 phosphorylation, and IFN-␥ completely abrogated the cardioprotective affect of tempol. This strongly implicates STAT1 activation as a main target in tempolmediated cardioprotection.

Figure 1. Time course of STAT1 and STAT3 phosphorylation after ischemia/reperfusion injury. Protein lysates from heart were separated on SDS-PAGE gels and probed with antibodies for A) total STAT1, phospho STAT1Y701, and phospho STAT1S727 and B) total STAT3, phospho STAT3Y705, and phospho STAT3727. Equal protein loading was confirmed by probing with an antibody (Ab) to GAPDH.

confirms that I/R-induced STAT phosphorylation, which was predominantly nuclear, was indeed reduced by free radical scavenging (see Fig. 4, full-length article). These data imply that STAT1 and STAT3 phosphorylation in I/R can be attenuated by the free scavenging/antioxidant property of tempol, and this may allow tempol to protect against cell death on the onset of reperfusion. 3. Activated STAT1 contributes to infarct development, in vivo Although activated STAT1 is induced after I/R injury and has been implicated in promoting apoptosis, it was unclear whether this activation contributed to infarct development, in vivo. To test this, rats were subjected to I/R injury with injection of the potent STAT1 activator, IFN-␥. IFN-␥ was introduced to allow continual activation of STAT1 for up to 120 min (postischemia), at which point the infarct area can be measured. Baseline values of mean arterial blood pressure (MAP), heart rate, and pressure rate index (PRI) were not significantly different between groups (data not shown). Compared with sham-operated animals, 25 min of LAD 2116

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Figure 2. Prolonged STAT1 activation with IFN-␥ overcomes the cardioprotective effects of tempol. A) Infarct size was measured in rats subjected to the surgical procedure alone (sham n⫽5) or to LAD coronary artery occlusion (25 min) and reperfusion (2 h) with infusion (5 min before reperfusion) of either saline (I/R n⫽7), tempol (n⫽7), IFN-␥ (n⫽6), or tempol and IFN-␥ together (n⫽6). *P ⬍ 0.05 compared with I/R vehicle. B) Western blot analysis of STAT1 phosphorylation from hearts tissues extracted from the experiment of panel A. Similar blots were observed from 3 different hearts form each group.

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McCORMICK ET AL.

CONCLUSIONS AND SIGNIFICANCE In the present study we show for the first time significant changes in the levels of phosphorylated STAT1 and STAT3 in the infracted area in the in vivo myocardium exposed to I/R injury. The free radical scavenger, tempol, was shown to reduce STAT1 and STAT3 phosphorylation. Previous studies have demonstrated that administration of tempol in the same manner decreases infarct size. Taken together, these data imply that the activation of STAT1 and 3 are important functions within the infarct area and may contribute to cardiac damage. Furthermore, the decrease in infarct size may be partly due to the inhibition of the janus-kinase (JAK)-STAT pathway by tempol. The molecular pathway resulting in cell death following free radical generation is complex and not fully understood. Our studies suggest that free radical production may trigger distinct kinases, which are responsible for STAT1 or SATAT3 phosphorylation, and that by reducing STAT phosphorylation with tempol treatment, the extent of myocardial damage is reduced, which could explain in part the reduction in infarct size as reported. The present study suggests that during I/R injury a balance exists between proapoptotic STAT1 and antiapoptotic STAT3 that determines the response of the cell to I/R injury (Fig. 3). Indeed, STAT3 has been shown to protect cells from IFN-␥-induced apoptosis by abrogating the proapoptotic effects of STAT1. Moreover, STAT3-deficient mice are more sensitive to I/Rinduced apoptosis and have larger infarct sizes. It was recently shown that granulocyte colony-stimulating factor (G-CSF) activates both STAT1 and STAT3 in cardiac myocytes. The protective effect of G-CSF in myocardial infarction, however, is mediated through STAT3, which was activated to a greater extent than STAT1. Similarly, we previously demonstrated that increasing the ratio of STAT3 to STAT1 in transfection experiments protected cardiac myocytes from hypoxia/ reoxygenation-induced cell death. In the present study, injection of IFN-␥, a potent activator of STAT1, exac-


Figure 3. Overview of the JAK/STAT pathway activated after ischemic preconditioning (IP) or ischemia/reperfusion (I/R) injury. Free radical production (reactive oxygen species) is known to mediate the damaging effects of IR. The extent of cardiac myocyte damage after I/R as a result of cell death will depend on the balance and levels of activated STAT1 (pST1) or STAT3 (pST3) modulating pro- and antiapoptotic genes. Agents that act as ROS scavengers such as tempol may also modulate the functional activity of STATs.

erbated cardiac damage, as evidenced by increased infarct sizes. Furthermore, IFN-␥-induced STAT1 activation during reperfusion significantly reduced the cardioprotective effect of tempol, indicating that this antioxidant’s antiapoptotic effect is likely to be mediated partly through a reduction in activated STAT1. Since we previously showed that the cardioprotective affects of a natural antioxidant green tea are mediated through STAT1, this highlights the role of STAT1 as a general molecular target for antioxidants in the ischemic myocardium.

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