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Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis

PAUL A. TOWNSEND^*,1, TIZIANO M. SCARABELLI, EVASIO PASINI, GIANLUCA GITTI, MARTA MENEGAZZI, HISANORI SUZUKI, RICHARD A. KNIGHT^*, DAVID S. LATCHMAN^* and ANASTASIS STEPHANOU^*,2

^* Medical Molecular Biology Unit, Institute of Child Health, University College London, London, England;
Biochemical Section, Department of Neuroscience, University of Verona, 37134, Italy; and
Division of Cardiology, St. John Hospital and Medical Center, Detroit, Michigan, USA

² Correspondence: Medical Molecular Biology Unit, 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 STAT-1 plays a critical role in promoting apoptotic cell death in cardiac myocytes after ischemia/reperfusion (I/R) injury. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea extract (GTE), has recently been reported to inhibit STAT-1 activity in noncardiac cells. In the present study we examined whether EGCG or green tea reduced STAT-1 phosphorylation and protected the isolated perfused rat heart against I/R-induced apoptotic cell death.

PRINCIPAL FINDINGS

1. EGCG enhances cell survival and reduces STAT-1 phosphorylation, Fas expression, and caspase activation in cultured myocytes exposed to simulated I/R injury
We first assessed the proportion of cell death in primary cultures of neonatal cardiac myocytes treated with EGCG. Myocytes were exposed to 4 h of simulated ischemia, followed by 16 h reoxygenation. EGCG was added to the myocytes 4 h before simulated ischemia injury and the levels of apoptotic cell death were assessed by annexin V. Preischemic incubation of neonatal cardiac myocytes with EGCG significantly reduced apoptotic cell death in a dose-dependent manner (Fig. 1 A).

View larger version (13K): [in this window] [in a new window]   Figure 1. EGCG protects neonatal cardiac myocytes from I/R-induced apoptosis. A) Neonatal cardiac myocytes were treated with or without EGCG (10 or 100 µM) before exposure to simulated I/R. Apoptotic cell death was assessed by the annexin V assay. Results are from three independent experiments. *P < 0.05 vs. cells treated with no EGCG. EGCG reduces STAT-1 phosphorylation after I/R. B) Neonatal cardiac myocytes were treated with or without EGCG (10 or 100 µM) before exposure to simulated I/R. Western blot was performed using specific antibodies to phosphorylated STAT-1 tyrosine-701 (ST1-701) or phosphorylated STAT-1 serine-727 (ST1-727) or unphosphorylated total STAT-1 (ST1). Similar results were observed in three independent Western blot experiments.

Since EGCG has been reported to reduce STAT-1 phosphorylation in noncardiac cells, we next determined by Western blot analysis whether EGCG is also able to modulate STAT-1 phosphorylation in cardiac myocytes. As shown in Fig. 1B , STAT-1 phosphorylation levels on tyrosine 701 and serine 727 were enhanced in myocytes subjected to simulated I/R. However, cardiac myocytes treated with EGCG showed reduced STAT-1 phosphorylation levels on tyrosine 701 and serine 727 in a dose-dependent manner. These data demonstrate that EGCG is able to inhibit STAT-1 701 and 727 phosphorylation as well as protect cardiac myocytes against I/R-induced apoptotic cell death.

One mechanism accounting for STAT-1-mediated apoptosis in cardiac cells undergoing simulated I/R injury is enhanced expression of death receptor Fas. Therefore, we tested whether the decrease in STAT-1 phosphorylation detected in myocytes pretreated with EGCG affects Fas expression. Consistent with our earlier findings, exposure of cardiac myocytes to simulated I/R resulted in enhanced expression of death receptor Fas. Pretreatment of cultured cardiac myocytes with EGCG before I/R stimulation significantly attenuated I/R-induced Fas receptor expression. Furthermore, cardiac myocytes treated with EGCG reduced the enhanced levels of active caspase-3 after simulated I/R. Thus, the cardioprotective effect of EGCG also results in reduced processing and activation of the downstream effector caspase-3 in cardiac cells exposed to I/R.

To test further whether this effect of EGCG on Fas expression was mediated at the transcriptional level, we examined Fas promoter reporter activity in transient transfection experiments in cardiac myocytes exposed to simulated I/R before treatment with or without EGCG. EGCG reduced I/R- or STAT-1-induced enhancement in Fas reporter activity in cardiac myocytes exposed to simulated I/R or transfected with a STAT-1 expression vector. Hence, EGCG prevented not only STAT-1 activation, but also I/R / STAT-1-mediated enhancement of Fas promoter activity, a pathway known to promote apoptosis in cardiac cells exposed to simulated I/R injury.

2. Inhibition of STAT-1 plays a role in the protective effects of EGCG
To demonstrate that inhibition of STAT-1 is necessary for the cardioprotective effects of EGCG, we exposed cardiac myocytes to interferon- (IFN-), a potent activator of STAT-1 and a promoter of apoptotic death in cardiac cells, to determine whether increasing doses of IFN- are able to overcome the protective effects of EGCG in cardiac myocytes. IFN- treatment enhanced levels of apoptosis in cardiac myocytes. However, cardiac myocytes pretreated with EGCG, then exposed to IFN-, showed significantly reduced apoptotic cell death. In contrast, increasing doses of IFN- were able to overcome the protective effects of EGCG and increase the levels of apoptotic death in a dose-dependent manner. EGCG was able to abolish the enhanced STAT-1 phosphorylation on tyrosine 701 and serine 727 in cardiac myocytes exposed to IFN-; an increasing concentration of IFN- was able to overcome the inhibitory effects of EGCG on STAT-1 phosphorylation. Thus, these experiments show that another agent that activates the STAT-1 pathway is able to overcome the protective effects of EGCG, suggesting that inhibition of STAT-1 by EGCG is involved in its cardioprotective properties.

3. GTE oral administration and EGCG infusion minimize infarct size, reduce myocyte apoptosis, and promote postischemic recovery of cardiac function in the I/R rat heart
To further validate these in vitro findings, we evaluated the extent of myocardial infarction and apoptotic cell death in hearts of rats receiving either oral supplementation with GTE for 7 days or pretreatment with EGCG added to the perfusion buffer. The percentage of infarction was significantly reduced after oral intake of GTE and infusion of EGCG, respectively. In agreement with our previous studies, TUNEL-positive staining, detected only during reperfusion, was consistently colocalized with caspase-3-positive labeling. One wk oral intake of GTE produced a significant reduction in these markers of apoptotic cell death in cardiac myocytes after I/R.

To verify whether the enhanced myocyte survival observed in the ex vivo setting was associated with a recovery in cardiac function, we evaluated the hemodynamic effects induced by GTE oral intake and EGCG infusion in rat hearts exposed to 35 min of regional ischemia, followed by 2 h of reperfusion. Consistent with reports using the same experimental model of I/R injury, I/R control hearts showed a progressive rise in diastolic pressure (dP) and a rapid decline in systolic pressure (sP), which recovered poorly during reperfusion (Fig. 2 A). Oral supplementation with GTE (Fig. 2B ), as well as preischemic infusion of EGCG (Fig. 2C ), which had no effect on the hemodynamics of isolated hearts during aerobic perfusion, significantly reduced the progressive rise in dP seen during I/R. In either case, the recovery in dP started after 15 min of ischemia (both P values <0.05 vs. I/R control) and improved throughout reperfusion. In addition, a concurrent recovery in sP, which became significant after 10 min of reperfusion (P values <0.05 vs. I/R control) and was progressively ameliorative with increasing reperfusion times, was recorded after either treatment. Hence, rat hearts receiving 1 wk oral treatment with GTE, as well as 30 min EGCG infusion before undergoing I/R, exhibited a significant reduction in cell death, which was associated with the recovery in cardiac function during the postischemic phase. GTE or EGCG infusion also reduced STAT-1 tyrosine 701 and serine 727 phosphorylation in isolated rat hearts exposed to I/R. Rats on GTE supplementation also showed reduced Fas and active caspase-3 expression compared with control animals not receiving GTE.

View larger version (20K): [in this window] [in a new window]   Figure 2. Green tea extract (GTE) or EGCG reduces infarct size and apoptosis in the myocardium after I/R injury. A) Rats were given water supplemented with GTE (0.1%) or water alone (Ctrl) or isolated perfused hearts were infused with EGCG (100 µM). Hearts were removed and infarct size was assessed as the infarct risk ratio (by measuring the infarct area against the whole heart) after 35 min of ischemia + 2 h of reperfusion. *P < 0.05 vs. animals with no GTE in drinking water. GTE or EGCG reduced the levels of apoptosis after I/R injury. B) Hearts were isolated from control, I/R, or GTE supplemented in their drinking water (GTE+I/R) or receiving EGCG (100 µM) + I/R and sectioned for immunofluorescence analysis to assess % colocalization of TUNEL and active caspase-3 staining in cardiac myocytes (CM). *P < 0.01 vs. hearts exposed to I/R. GTE or EGCG (100 µM) enhances cardiac function after I/R. Systolic pressure (sP) and diastolic pressure (dP) were measured during ischemia/reperfusion (I/R) in the isolated rat heart. C) sP and dP in control hearts exposed to I/R. Solid triangles: sP; open square: dP.

CONCLUSIONS AND SIGNIFICANCE

In conclusion, we show for the first time that EGCG can mediate cardioprotection in the intact heart against I/R-induced apoptotic cell death and that one pathway may involve the inhibition of STAT-1 activation. Green tea is the most widely consumed beverage in the world. EGCG is the major polyphenol in green tea and has greater antioxidant activity than vitamins C and E. Protection of the ischemic myocardium against tissue injury and loss of cardiac myocytes continues to elude basic investigators and clinicians, and therefore a major objective is to identify effective strategies to treatment ischemic heart diseases. Thus, EGCG may be used as a therapeutic agent for the inhibition of the damaging effects of STAT-1 after I/R injury and so may reduce the loss of cardiac myocytes that eventually leads to end-stage heart failure (Fig. 3 ). Furthermore, its long history of consumption without adverse health effects makes it a good candidate for the treatment of ischemic heart disease and heart failure.

View larger version (13K): [in this window] [in a new window]   Figure 3. Hypothetical pathway for the inhibition of STAT-1 by EGCG Ischemia/reperfusion (I/R) injury leads to STAT-1 activation via phosphorylation of STAT-1 on tyrosine 701 and serine 727 that leads to enhanced STAT-1-induced proapoptotic genes and increase levels of cardiac myocyte cell death. Loss of cardiac myocytes eventually leads to heart failure. EGCG is able to inhibit STAT-1 activation by reducing the phosphorylation of STAT-1 tyrosine 701 and serine 727 after I/R in the intact isolated heart.

It was recently reported that green tea consumption may be linked to a reduced risk of myocardial infarction. GTE has also been shown to be neuroprotective in experimental I/R brain injury. Similarly, GTE given to stroke-prone spontaneously hypertensive rats prolonged the life span of these animals. These studies also indicate the beneficial effects of GTE in reducing the risk against brain infarction and in protecting the brain after cerebral I/R injury. STAT-1 is activated and plays a role in animal models of cerebral I/R injury. However, we cannot rule out that the active agent in GTE, EGCG, may have several pharmacological protective pathways other than just inhibiting the STAT-1 pathway. In addition, a protective function for factors other than EGCG in GTE cannot be excluded. It is clear, however, from our work that the activation of STAT-1 by IFN- can overcome the protective effect of EGCG, indicating that STAT-1 is a critical target for the protective effect of EGCG.

FOOTNOTES

¹ Current address: Department of Human Genetics, University of Southhampton, England, UK

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1716fje;

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