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* 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
2Correspondence: Department of Medical Molecular Biology, Institute of Child Health, University College London, 30 Guilford St., London, WC1N 1EH, UK. E-mail: a.stephanou{at}ich.ucl.ac.uk
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 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 in order to assess the dynamics of STAT1 and STAT3 tyrosine or serine phosphorylation over time. As shown in Fig. 1
A, 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.
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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).
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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 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 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 µg/kg) 5 min before reperfusion caused an increase of 30% in myocardial infarct size compared with vehicle (Fig. 2
A); 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 tempol-mediated cardioprotection.
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/R-induced 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, 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 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.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-6188fje
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