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Cardiovascular Research 2006 69(4):836-844; doi:10.1016/j.cardiores.2005.11.031
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Copyright © 2005, European Society of Cardiology

Estrogen improves cardiac recovery after ischemia/reperfusion by decreasing tumor necrosis factor-{alpha}

Yi Xu1, Ivan A. Arenas1, Stephen J. Armstrong, Wayne C. Plahta, Han Xu and Sandra T. Davidge*

232 HMRC Departments of Obstetrics/Gynecology and Physiology, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2

* Corresponding author. Tel.: +1 780 492 1864; fax: +1 780 492 1308. Email address: sandra.davidge{at}ualberta.ca

Received 28 July 2005; revised 4 November 2005; accepted 25 November 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Estrogen has cardioprotective effects on ischemia/reperfusion (I/R). Tumor necrosis factor alpha (TNF{alpha}) is an inflammatory cytokine with depressor effects on myocardial function and has been suggested to mediate I/R injury. Whether cardiac TNF{alpha}levels are influenced by estrogen status is unknown. We investigated the effect of estrogen on TNF{alpha}levels and TNF{alpha}receptors in the ischemic heart and its role in estrogen modulation of I/R injury.

Methods: Hearts were isolated from ovariectomized Sprague-Dawley female rats that were treated with either estrogen or placebo for 4 weeks. Working heart preparations were subjected to global, no-flow ischemia (25 min) followed by reperfusion (40 min).

Results: I/R increased TNF{alpha}levels in coronary effluent and in the left ventricle (LV) of estrogen-deficient rats, which were decreased by estrogen replacement. Moreover, estrogen improved functional recovery (55.0 ± 5.0% vs. 22.0 ± 7.0%, P<0.05), decreased LV apoptosis, and reduced myocardial necrosis. To further evaluate the role of TNF{alpha}in I/R injury, a selective TNF{alpha}inhibitor (etanercept) was used in vitro before the ischemic insult. TNF{alpha}inhibition improved functional recovery (39 ± 4.4% vs. 22.0 ± 7.0%, P<0.05) and reduced apoptosis and myocardial necrosis in estrogen-deficient animals but did not have a summative protective effect in the hearts of estrogen-replaced animals.

Conclusions: These data indicate that estrogen modulates cardiac expression of TNF{alpha}and TNF{alpha}receptors. Moreover, the cardioprotective effects of estrogen are in part mediated by regulation of TNF{alpha}levels in the ischemic heart.

KEYWORDS Cytokines; Inflammation; Ischemia; Reperfusion; Hormones

This article is referred to in the Editorial by J.A. Moolman (pages 777–780) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The incidence of cardiovascular disease and its associated mortality increase in women after menopause [1]. Indeed, mortality after a myocardial infarction (MI) and post-MI complications are more prevalent in older women [2–5]. Estrogen is known to have multiple effects on the heart [6], and some studies have suggested that estrogen is cardioprotective against myocardial ischemia [7–10]. We found that in a model of aging in female rats, estrogen deficiency was associated with enhanced susceptibility to ischemia/reperfusion (I/R) injury [11], which was improved by estrogen replacement [11]. However, the mechanisms by which estrogen elicits cardioprotection are unclear.

Inflammation is an important mediator of both injury and repair in the post-ischemic heart [12]. Tumor necrosis factor alpha (TNF{alpha}) is a proinflammatory cytokine involved in the pathogenesis of cardiovascular disease. TNF{alpha} acts on two receptors TNFR1 (p55) and TNFR2 (p75), both of which exist on the surface of cardiac myocytes [13]. Interactions between both receptors play an important role on determining the cellular effects of TNF{alpha} such as apoptosis or cell survival [14,15].

Cardiac myocytes and other cardiac cells have been shown to produce TNF{alpha} [16]. Moreover, some studies have suggested that increased cardiac TNF{alpha} levels after an ischemic event contributes to myocardial injury [17–19]. In fact, TNF{alpha} can induce apoptosis of cardiac myocytes in culture [15], or elicit direct negative inotropic effects on cardiac function [20]. On the other hand, other studies have also suggested that TNF{alpha} may have protective effects against I/R as indicated by increased apoptosis in TNFR1/TNFR2-deficient mice when compared with wild-type mice [21].

Estrogen deficiency and menopause have been associated with an increase in TNF{alpha} levels [22–24]. However, whether estrogen modulates the expression of TNF{alpha} or TNF{alpha} receptors in the ischemic heart is largely unknown. The main objectives of this study are to investigate the effect of estrogen on I/R injury and on cardiac TNF{alpha} expression, and to ascertain whether changes in cardiac TNF{alpha} play a role on the effects of estrogen in I/R injury. We hypothesized that estrogen will be associated with improvement in cardiac recovery after I/R, in part by reducing TNF{alpha} levels.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal model
This study was approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee and was in accordance with the Canadian Council on Animal Care and NIH guidelines. Female Sprague-Dawley rats were obtained from Charles River, Canada (Montreal, Quebec) and were housed in the facilities of the University of Alberta until experimentation at 12–15 months of age. This age was chosen since the animals have attained their state of reproductive senescence (i.e. similar to the postmenopausal state of women). Moreover, to decrease the variability of estrogen levels in these aged animals, we remove the ovaries at the time of the initial treatment. Then, we randomly assign the animals to different treatments.

To investigate the effects of estrogen deficiency/replacement on cardiac TNF{alpha} levels and functional recovery after I/R, rats were treated with either a placebo pellet (Placebo, Innovative Research of America; n=20) or an estrogen pellet (estrogen, 1.5 mg/pellet, 60-day release, Innovative Research of America; n=20), which results in maximal serum estrogen levels (~80 pg/mL) similar to that of intact cycling rats. Following anesthesia of the rats using sodium pentobarbital (~60 mg/kg body weight), hearts were excised and blood was collected from the thoracic cavity. Serum was obtained by centrifugation and samples were snap-frozen (–80 °C) for subsequent measurement of estrogen (University of Alberta Hospital Laboratories).

2.2 Isolated working heart preparation
Hearts were perfused for 10 min in retrograde Langendorff mode with Krebs-Henseleit solution (37 °C) containing 120 mmol/l NaCl, 25 mmol/l NaHCO3, 5 mmol/l glucose, 4.7 mmol/l KCl, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, and 2.5 mmol/l CaCl2 (pH 7.4, gassed with 95% O2–5% CO2) and against a constant perfusion pressure of 60 mm Hg. The hearts were switched to antegrade working perfusion mode and perfused in a closed recirculating system at 37 °C. The perfusate (100 ml) was a Krebs-Henseleit solution with the addition of 1.2 mmol/l palmitate prebound to 3% BSA (fraction V), 0.5 mmol/l lactate, and 100 mU/l insulin. In this mode, the buffer entered the cannulated left atrium at a pressure equivalent to 11.5 mm Hg (15.6 cm H2O), and passed to the left ventricle (LV) from which it was spontaneously ejected through the aortic cannula against a pressure equivalent to 80 mm Hg (afterload).

After an equilibration of 10 min, hearts were paced at 300 beats/min with an electrical stimulator (S-88; Grass Instruments, Quincy, MA) via two silver electrodes attached to the right atrium. Heart rate and aortic systolic and diastolic pressures were recorded using a Grass 7D polygraph (Grass Instruments, Quincy, MA). Cardiac output and aortic flow were measured using a Transonic flowmeter (Raytech Instrument Co., Vancouver, BC, Canada). Cardiac function (systolic pressure x cardiac output) and coronary flow were calculated as described previously [25]. The measurements of the cardiac function were carried out every 10 min during baseline and reperfusion. Any heart that showed any cardiac disturbance (ventricle arrhythmia and fibrillation) during the baseline perfusion was excluded from this study. Hearts used were perfused by working mode according to the protocol described below.

2.3 Experimental protocol
Hearts were subjected to I/R using the following protocol (Fig. 1): perfusion for a 50-min stabilization period, global ischemia (no-flow, 25 min) followed by reperfusion (40 min). To further investigate the role of TNF{alpha} on I/R injury, in a subset of experiments we evaluated the effects of acute TNF{alpha} inhibition with etanercept (Immunex Corporation, Thousand Oaks, CA) in hearts from estrogen depleted (n=10) or estrogen-replaced animals (n=10). Etanercept is composed of the extracellular ligand-binding portion of the human 75 kilodalton (p75) TNFR2, which binds and inactivates TNF{alpha} For these experiments, etanercept (30 µg/ml) was added to the perfusate 10 min before the onset of ischemia and remained throughout the reperfusion period [26].


Figure 1
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  		Fig. 1 Ischemia-reperfusion (I/R) protocols. Hearts were prepared (Prep) during a 10-min Langendorff perfusion. Hearts were then switched into working mode and perfusion for a 50-min stabilization period (Baseline), global ischemia (no-flow, 25 min) followed by reperfusion (40 min). Etanercept (30 µg/ml) was added to the perfusate 10 min before the onset of ischemia and remained throughout the reperfusion period. Drugs were added as indicated (arrows).

 
At the end of the experiments, ventricles were divided into a right ventricular free wall portion and a left ventricular septal portion containing both right and left septa. Cardiac tissue samples were blotted dry, weighed, immersed in liquid nitrogen, and stored at –80 °C until extraction. We randomly selected (n=4) frozen LV tissues from each group for TUNEL staining, DNA laddering and Western blot analysis.

2.4 Measurement of TNF{alpha} and lactate dehydrogenase activity
Frozen LV tissue samples were solubilized by homogenization in lysis buffer containing phenylmethylsulphonylfluoride and leupeptin (Sigma Chemical Co, Oakville, Ontario, Canada), and protein concentration was determined by comparison with BSA standards using BCA Protein Assay (Pierce Biotechnology Inc., Rockford, IL). TNF{alpha} protein expression was determined in LV tissue and coronary effluent using commercially available ELISA kits (Biosource International, Camarillo, CA). Coronary effluent (1 ml/each time point) was collected at 50 and 10 min before ischemia and then 5, 10, 20, 30, 40 min after ischemia. Lactate dehydrogenase (LDH; Sigma Chemical Co., Oakville, Ontario, Canada) activity was spectrophotometrically measured in samples from the coronary effluent at 50 and 10 min before ischemia and then 5, 10, 20, 30, 40 min after ischemia and expressed as units of activity released per gram heart weight as described [27,28].

2.5 TUNEL staining
The TUNEL staining assay was performed as previously described with the TdT apoptosis detection kit (Roche Molecular Biomedicals/Boehringer Mannheim Co., Laval, QC) with some modifications [29]. Frozen LV tissues were fixed and placed in optimal cutting temperature media (Tissue-Tek, Sakura Finetechnical Co., Torrance, CA), then cut on a cryostat into 6-µm-thick sections. Negative controls were performed using serial sections that were not stained with TdT. Lung tissue was used for positive controls [29]. The TUNEL reaction was incubated for 1 h at 37 °C to induce DNA strand breaks. The tissue was blocked with 3% skim milk in phosphate-buffered saline (PBS) with 0.5% Tween 20 detergent for 30min.

The fluorescent label, FITC-conjugated Alexa Fluor 488 streptavidin antibody (Molecular Probes, Eugene, OR) was applied on each section for 30 min, diluted 1:200 in PBS milk solution, in order to detect ligated biotinylated dNTPs in apoptotic nuclei. Nuclei were then labeled with a blue fluorescent medium DAPI (4',6 diamidino-2-phenylindole; Vectashield H-1200, Vector Laboratories, Burlingame, CA), diluted 1:333 in PBS solution. Sections were incubated with the DAPI solution for 5 min and then sequentially rinsed in PBS. Samples were viewed at 40 x with a fluorescent microscope (Olympus IX81F-2, Olympus American Co., Melville, NY). Images were captured with a camera (Roper Scientific, Cascade Photometrics, Tucson, AZ) and assembled in slidebook software (Slidebook 4, Intelligent Imaging Innovations, Inc., Santa Monica, CA). Three transverse sections through the heart were analyzed of which 5 sample images were collected per animal. The number of TUNEL-positive cardiomyocyte nuclei in the left ventricle wall was manually counted. Stained cellular debris was eliminated from counting. The investigator was blinded to the group's identity.

2.6 DNA fragmentation by agarose gel electrophoresis
Myocardial DNA was extracted from 70 to 100 mg of the frozen LV samples (n=4, each) using an apoptotic ladder kit (Roche Diagnostics, Laval, Quebec, Canada). The extracted DNA (4 µg) was loaded on a 1% agarose gel containing ethidium bromide. Gels were run at 100 V in TBE buffer (0.04 mol/l Tris, 0.04 mol/l boric acid, 2 mmol/l EDTA, pH 8.0). The gels were photographed under UV light using a Fluor-S MultiImager (Bio-rad, Mississauga, Ontario, Canada).

2.7 Western blot analyses
Samples were resolved by SDS polyacrylamide gel electrophoresis as previously described [30]. After electrophoresis, the proteins were transferred onto nitrocellulose membranes, and membranes were stained with the antibodies against TNFR1 (1:100), TNFR2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), and Caspase-3 (1:2000, Cell Signaling, Beverly, MA). Then, we used peroxidase-conjugated avidin secondary antibody for visualization. To control for differences in protein concentration between samples or loading errors, blots were stripped and reprobed for {alpha}-actin expression.

2.8 Statistics
Data are presented as mean+SE. Either one-way or two-way ANOVA was used to compare differences among groups when appropriate. Post hoc analysis was performed with the Tukey or Student-Newman-Keuls tests. Student's t-test was used to compare two groups. Significance was taken at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of Estrogen on body weight, and left/right ventricular weight
There were no significant differences in body weight (BW) among groups (Table 1). LV weight (LVW) and LVW/BW ratio were lower in the estrogen-replaced group compared with estrogen deficient animals (Table 1). There were no differences in right ventricular weight (RVW) and right ventricular weight/body weight (RVW/BW) among groups.


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  		Table 1 Characteristics of animal model

 
3.2 Effects of estrogen on TNF{alpha} release, TNF{alpha} LV content and TNF{alpha} receptors expression after I/R
TNF{alpha} levels in the coronary effluent were not significantly different among groups during aerobic perfusion (Fig. 2A). However, I/R was associated with a marked increase in TNF{alpha} levels in the coronary artery effluent of estrogen deficient animals, which was decreased in estrogen-replaced animals (Fig. 2A). Moreover, TNF{alpha} protein content in LV after I/R was also reduced in estrogen-replaced animals (Fig. 2B). Estrogen replacement was associated with increased ventricular expression of TNFR1 (Fig. 3A, P<0.05), whereas TNFR2 expression was reduced compared with estrogen-depleted animals (Fig. 3B).


Figure 2
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  		Fig. 2 Effects of ischemia/reperfusion on TNF{alpha} content in coronary effluent and left ventricle. Perfused isolated hearts from estrogen deficient (placebo; n=8) and estrogen-replaced (E2; n=8) animals were subjected to global ischemia (25 min) followed by reperfusion (40 min). (A) TNF{alpha} levels in the coronary effluent were evaluated by a high sensitivity ELISA at 10 and 50 min before ischemia and at 5, 10, 20, 30 and 40 min of reperfusion. (B) TNF{alpha} LV content was evaluated by Western blot at the end of reperfusion. Values are means ± SE. *P<0.05 vs. placebo.

 

Figure 3
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  		Fig. 3 Left ventricular protein expression of (A) TNF{alpha} Receptor1 (TNFR1) and (B) TNF{alpha} Receptor2 (TNFR2) in hearts from estrogen deficient (placebo; n=4) and estrogen-replaced (E2; n=4) animals after ischemia/reperfusion. Bars represent means ± SE. *P<0.05 vs. placebo.

 
3.3 Role of estrogen and TNF{alpha} on functional recovery following I/R
The baseline values of cardiac work (Fig. 4A) and cardiac output (Table 2) were significantly lower in the estrogen deficient compared with estrogen-replaced animals. The baseline function was confirmed in a series of hearts not subjected to ischemia.


Figure 4
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  		Fig. 4 Effects of ischemia/reperfusion on cardiac work. Perfused isolated hearts were subjected to global ischemia (25 min) followed by reperfusion (40 min). (A) Cardiac work and (B) Recovery (percentage of baseline) is shown for estrogen-replaced (E2; n=8) and estrogen deficient (placebo; n=8) animals without and with in vitro etanercept (Placebo+Etan, n=5; E2+Etan, n=8). Values are means ± SE. Different letters indicate values that are statistically significant among groups (P<0.05).

 

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  		Table 2 Hemodynamic parameters during baseline and reperfusion periods of ischemia/reperfusion protocol

 
After ischemia, cardiac function was depressed in all groups. Estrogen replacement improved cardiac compared with estrogen deficient (placebo) animals (% of recovery: 55.0 ± 5.0 and 22.0 ± 7.0, respectively, P<0.05; Fig. 4A and B). TNF{alpha} inhibition increased functional recovery in estrogen deficient animals (39 ± 4.4%, Fig. 4A and B), but had no additional effect in estrogen-replaced animals.

During reperfusion, peak systolic pressure (PSP) and cardiac output were significantly reduced from baseline in hearts from all animals (Table 2). However, PSP and cardiac output were greater in the estrogen-replaced groups compared to estrogen deficient animals. Moreover, in estrogen deficient but not in estrogen-replaced animals TNF{alpha} inhibition was associated with improved PSP and cardiac output (Table 2).

Coronary flow was not different between groups during aerobic perfusion. After I/R coronary flow was significantly depressed in estrogen deficient animals compared with estrogen-replaced groups. TNF{alpha} inhibition in estrogen deficient animals improved coronary flow to similar levels seen in estrogen-replaced animals (Table 2).

3.4 Role of estrogen and TNF{alpha} on LDH release
Lactate dehydrogenase (LDH) assay was used to determine the extent of cell death after ischemia and throughout the reperfusion period. LDH cardiac release increased after I/R in hearts from all animals (Fig. 5). However, LDH in coronary effluent of hearts from estrogen-treated animals was approximately half of that observed in the estrogen-deficient animals. TNF{alpha} inhibition also decreased LDH levels by about one-third in estrogen-deficient hearts, but when given to those that had received estrogen-treatment there was no summative effect observed (Fig. 5).


Figure 5
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  		Fig. 5 Effects of ischemia/reperfusion on Lactate dehydrogenase (LDH) release from hearts subjected to global ischemia (25 min). LDH in the coronary effluent was evaluated by a specific bioassay at 10 and 50 min before ischemia and at 5, 10, 20, 30 and 40 min of reperfusion. LDH release is shown for estrogen-replaced (E2; n=8) and estrogen deficient animals (placebo; n=8) without and with in vitro etanercept (Placebo+Etan, n=5; E2+Etan, n=8). Values are means ± SE. Different letters indicate values that are statistically significant among groups (P<0.05).

 
3.5 Role of estrogen and TNF{alpha} on apoptosis, DNA fragmentation and caspase expression in the LV
Cardiac myocytes with TUNEL-positive nuclei were observed in all groups (Fig. 6). The percentage of TUNEL-positive myocytes after I/R was reduced in estrogen treated animals compared with estrogen deficient animals (7.3 ± 0.9% and 23.5 ± 2.33% respectively, P<0.05; Fig. 6A and B). TNF{alpha} inhibition was associated with decreased apoptosis only in estrogen-depleted animals (13.5 ± 0.8%; Fig. 6A and B). DNA degradation observed using the apoptosis ladder kit confirmed results from the TUNEL assay (data not shown). Similar results were obtained for Caspase-3 cleavage products expression at p17 and p20, which were significantly reduced in estrogen-replaced animals. Moreover, TNF{alpha} inhibition reduced caspase-3 cleavage but only in estrogen deficient animals (Fig. 7A–D).


Figure 6
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  		Fig. 6 Percentage of TUNEL-positive nuclei cells in left ventricles from estrogen deficient (placebo; n=4), and estrogen-replaced (E2; n=4) animals without and with in vitro etanercept (Placebo+Etan, n=4; E2+Etan, n=3). Panel A: Representative images of the TUNEL staining assay from each group are shown. Panel B: Bar graphs represent the percentage of TUNEL-positive nuclei cells in left ventricles from each group. Bars represent means ± SE. Different letters indicate values that are statistically significant among groups (One-way ANOVA, P<0.05).

 

Figure 7
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  		Fig. 7 Caspase-3 (p32) cleavage products (p20 and 17) expression in left ventricles from estrogen deficient (placebo; n=4), and estrogen-replaced (E2; n=4) animals without and with in vitro etanercept (Placebo+Etan, n=4; E2+Etan, n=3). (A) Caspase-3 immunoblot, (B) Bar graph depicting Caspase-3 p32 expression, (C) Bar graph depicting Caspase-3 p20 expression, (D) Bar graph depicting Caspase-3 p17 expression. E2 (n=4) group vs. placebo (n=4) group. Bar graphs are mean ± SE. Different letters indicate values that are statistically significant among groups (one-way ANOVA, P<0.05).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
These data indicate that estrogen status influences the expression of TNF{alpha} and TNF{alpha} receptors in the heart. Estrogen replacement reduced TNF{alpha} levels in LV myocardium and decreased the release of this cytokine after I/R, which was accompanied by improved functional recovery and a decrease in markers of tissue injury and apoptosis.

Moreover, estrogen replacement was associated with increased TNFR1 but decreased TNFR2 expression in the ischemic myocardium.

Previous studies have suggested that estrogen may have inhibitory effects on TNF{alpha} production [22,23]. Liao et al. [22] demonstrated that an increase of TNF{alpha} after cerebral I/R corresponded to a decline in circulating estrogen levels in normal cycling female rats. Estrogen attenuated endotoxin-induced TNF{alpha} expression and neuronal injury, suggesting that the down-regulation of TNF{alpha} expression is involved in estrogen mediated neuroprotection. Regarding the myocardium, a previous study showed that serum TNF{alpha} levels correlate with the severity of myocardial injury after an acute myocardial infarction [17], and some studies in animal models have shown benefits from TNF{alpha} inhibition after I/R [12,18,31]. Therefore, we sought to investigate whether some of the cardioprotective effects of estrogen after I/R are mediated by modulation of TNF{alpha}.

In this study, hearts from estrogen deficient animals subjected to I/R have a marked increase in TNF{alpha} levels in the perfusate after 5 min of reperfusion, and interestingly, TNF{alpha} levels further increased after longer periods of reperfusion. Thus, although ischemia is a powerful stimulus for the production of TNF{alpha} in the heart [32], results from this study indicate that in estrogen deficient animals, reperfusion also contributes to induce TNF{alpha} production. The apparent reduction in TNF{alpha} levels in the coronary effluent at 10 min of reperfusion is an unexpected finding, but it likely results from a dilution of TNF{alpha} in the circulating perfusate. Importantly, estrogen replacement was associated with significantly lower levels of TNF{alpha} after I/R. Moreover, in estrogen-replaced animals reperfusion was not associated with a further increase in TNF{alpha} levels. Likewise, TNF{alpha} expression in LV after I/R was also decreased in estrogen-replaced animals compared with estrogen deficient rats. Altogether these observations indicate that estrogen decreases TNF{alpha} levels after I/R.

In this study, estrogen deficient rats had slightly enlarged hypertrophic hearts and reduced aerobic function compared with estrogen-replaced animals. These observations agree with our previous studies indicating that estrogen deficiency is associated with alterations in aerobic cardiac function. Moreover, in the present study, the reduction in TNF{alpha} levels after I/R by estrogen-replacement was accompanied by greater cardiac recovery and a decrease in markers of myocardial damage, suggesting that TNF{alpha} may in part mediate the detrimental effects of estrogen deficiency after I/R. In fact, TNF{alpha} inhibition in estrogen deficient rats was associated with a two-fold improvement in cardiac work, as well as a decrease in cell necrosis and markers of apoptosis. However, TNF{alpha} inhibition did not have any additive beneficial effect when given to estrogen-replaced animals, indicating that part of the protective effect of estrogen is achieved by modulating the production of TNF{alpha}.

In agreement with previous observations showing that TNF{alpha} is able to induce apoptosis in cardiac myocytes [15], estrogen replacement or TNF{alpha} inhibition in estrogen deplete animals were associated with a decrease in the percentage of TUNEL-positive nuclei cells and reduced caspase-3 cleavage products expression, consistent with lower apoptotic rates in ventricles from these animals compared with estrogen-deficient animals. However, TNF{alpha} inhibition was not associated with a further decrease in apoptosis in estrogen-replaced animals supporting the hypothesis that TNF{alpha} may mediate the effects of I/R in estrogen deficient animals. Moreover, both estrogen replacement or etanercept treatment in estrogen deficient animals were also associated with decreased levels of LDH, a marker of cell necrosis, suggesting that this cytokine is involved in inducing cell necrosis during the acute phase of myocardial infarction. Hence, higher levels of TNF{alpha} in estrogen deficient animals have contributed to greater tissue damage compared with estrogen-replaced animals.

One of the mechanisms by which TNF{alpha} could contribute to ischemic injury is by inducing endothelial dysfunction [33,34], which may result in coronary spasm and decreased flow during reperfusion [18,34]. Interestingly, we found that hearts from estrogen-deficient animals had decreased coronary flow after I/R compared to animals treated with estrogen, which was improved by estrogen replacement or TNF{alpha} inhibition. These observations may suggest that part of the protective effect of TNF{alpha} inhibition or estrogen replacement may have resulted from increasing coronary flow.

To our knowledge a role of estrogen on the modulation of cardiac TNF{alpha} receptors has not been reported. TNFR1 contains a death domain and it is thought to mediate the pro-apoptotic effects of TNF{alpha} [35]. However, signaling via TNFR1 can also mediate survival pathways by activation of NF-kappaB [35]. In this study, TNFR1 expression was decreased in LV homogenates from estrogen deficient rats compared with estrogen treated animals. This suggests that activation of this receptor may have protective effects after I/R. Indeed, downregulation of TNFR1 in the coronary microcirculation is associated with poor recovery after I/R [36]. In contrast, TNFR2 expression was decreased in estrogen-replaced compared with estrogen deficient rats. Signaling via this receptor activates known pro-survival pathways such as NF-kB and Inhibitor of Apoptosis (IAP) [37]. However, when co-activated with TNFR1, TNFR2 has been shown to facilitate the pro-apoptotic effects of TNF{alpha} by increasing the degradation of TNFR-associated factor 2 (TRAF2) that is required for TNF{alpha} activation of anti-apoptotic signals [38]. Results from the present study suggest that the combined effect of TNFR2 downregulation with a concomitant upregulation of TNFR1 may have a protective effect against I/R injury in the heart.

In summary, these data indicate that estrogen status influences the expression of TNF{alpha} and TNF{alpha} receptors in the ischemic heart. Moreover, higher levels of this cytokine after I/R are associated with reduced cardiac tolerance to ischemia. Therefore, these observations suggest that estrogen may have cardioprotective effects by inhibiting the expression of cardiac TNF{alpha} and modulating TNF{alpha} receptors expression.


   Acknowledgements
 
We thank Dr. Dan Pehowich and Ms. Bonnie Winkler-Lowen for their technical assistance. This work was supported by a grant from the Canadian Institute of Health Research. I.A. Arenas is supported by the Alberta Heritage Foundation for Medical Research (AHFMR). S.T. Davidge is the Canadian Chair in Women's Cardiovascular Health and a Senior Scholar of AHFMR.


   Notes
 
1 Both authors have contributed equally to this work. Back

Time for primary review 47 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Wenger N.K. Clinical characteristics of coronary heart disease in women: emphasis on gender differences. Cardiovasc Res (2002) 53:558–567.[Free Full Text]
  2. Hochman J.S., Tamis J.E., Thompson T.D., Weaver W.D., White H.D., Van de Werf F., et al. Sex, clinical presentation, and outcome in patients with acute coronary syndromes. Global use of strategies to open occluded coronary arteries in acute coronary syndromes IIb investigators. N Engl J Med (1999) 341:226–232.[Abstract/Free Full Text]
  3. Becker R.C., Terrin M., Ross R., Knatterud G.L., Desvigne-Nickens P., Gore J.M., et al. Comparison of clinical outcomes for women and men after acute myocardial infarction. The thrombolysis in myocardial infarction investigators. Ann Intern Med (1994) 120:638–645.[Abstract/Free Full Text]
  4. Long-term effects of intravenous thrombolysis in acute myocardial infarction: final report of the GISSI study. Gruppo Italiano per lo Studio della Streptochi-nasi nell'Infarto Miocardico (GISSI). Lancet (1987) 2:871–874.[Medline]
  5. Lee K.L., Woodlief L.H., Topol E.J., Weaver W.D., Betriu A., Col J., et al. Predictors of 30-day mortality in the era of reperfusion for acute myocardial infarction. Results from an international trial of 41,021 patients. GUSTO-I investigators. Circulation (1995) 91:1659–1668.[Abstract/Free Full Text]
  6. Dubey R.K., Jackson E.K. Cardiovascular protective effects of 17beta-estradiol metabolites. J Appl Physiol (2001) 91:1868–1883.[Abstract/Free Full Text]
  7. Lee T.M., Chou T.F., Tsai C.H. Differential role of K(ATP) channels activated by conjugated estrogens in the regulation of myocardial and coronary protective effects. Circulation (2003) 107:49–54.[Abstract/Free Full Text]
  8. Node K., Kitakaze M., Kosaka H., Minamino T., Funaya H., Hori M. Amelioration of ischemia- and reperfusion-induced myocardial injury by 17beta-estradiol: role of nitric oxide and calcium-activated potassium channels. Circulation (1997) 96:1953–1963.[Abstract/Free Full Text]
  9. Zhai P., Eurell T.E., Cotthaus R., Jeffery E.H., Bahr J.M., Gross D.R. Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats. Am J Physiol Heart Circ Physiol (2000) 279:H2766–H2775.[Abstract/Free Full Text]
  10. Beer S., Reincke M., Kral M., Lie S.Z., Steinhauer S., Schmidt H.H., et al. Susceptibility to cardiac ischemia/reperfusion injury is modulated by chronic estrogen status. J Cardiovasc Pharmacol (2002) 40:420–428.[CrossRef][ISI][Medline]
  11. Xu Y., Armstrong S.J., Arenas I.A., Pehowich D.J., Davidge S.T. Cardioprotection by chronic estrogen or superoxide dismutase mimetic treatment in the aged female rat. Am J Physiol Heart Circ Physiol (2004) 287:H165–H171.[Abstract/Free Full Text]
  12. Frangogiannis N.G., Smith C.W., Entman M.L. The inflammatory response in myocardial infarction. Cardiovasc Res (2002) 53:31–47.[Abstract/Free Full Text]
  13. Torre-Amione G., Kapadia S., Lee J., Bies R.D., Lebovitz R., Mann D.L. Expression and functional significance of tumor necrosis factor receptors in human myocardium. Circulation (1995) 92:1487–1493.[Abstract/Free Full Text]
  14. Ashkenazi A., Dixit V.M. Death receptors: signaling and modulation. Science (1998) 281:1305–1308.[Abstract/Free Full Text]
  15. Krown K.A., Page M.T., Nguyen C., Zechner D., Gutierrez V., Comstock K.L., et al. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest (1996) 98:2854–2865.[ISI][Medline]
  16. Kapadia S., Lee J., Torre-Amione G., Birdsall H.H., Ma T.S., Mann D.L. Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration. J Clin Invest (1995) 96:1042–1052.[ISI][Medline]
  17. Hirschl M.M., Gwechenberger M., Binder T., Binder M., Graf S., Stefenelli T., et al. Assessment of myocardial injury by serum tumour necrosis factor alpha measurements in acute myocardial infarction. Eur Heart J (1996) 17:1852–1859.[Abstract/Free Full Text]
  18. Gurevitch J., Frolkis I., Yuhas Y., Lifschitz-Mercer B., Berger E., Paz Y., et al. Anti-tumor necrosis factor-alpha improves myocardial recovery after ischemia and reperfusion. J Am Coll Cardiol (1997) 30:1554–1561.[Abstract]
  19. Kubota T., Bounoutas G.S., Miyagishima M., Kadokami T., Sanders V.J., Bruton C., et al. Soluble tumor necrosis factor receptor abrogates myocardial inflammation but not hypertrophy in cytokine-induced cardiomyopathy. Circulation (2000) 101:2518–2525.[Abstract/Free Full Text]
  20. Yokoyama T., Vaca L., Rossen R.D., Durante W., Hazarika P., Mann D.L. Cellular basis for the negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian heart. J Clin Invest (1993) 92:2303–2312.[ISI][Medline]
  21. Kurrelmeyer K.M., Michael L.H., Baumgarten G., Taffet G.E., Peschon J.J., Sivasubramanian N., et al. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc Natl Acad Sci U S A (2000) 97:5456–5461.[Abstract/Free Full Text]
  22. Liao S.L., Chen W.Y., Chen C.J. Estrogen attenuates tumor necrosis factor-alpha expression to provide ischemic neuroprotection in female rats. Neurosci Lett (2002) 330:159–162.[CrossRef][ISI][Medline]
  23. Pacifici R., Brown C., Puscheck E., Friedrich E., Slatopolsky E., Maggio D., et al. Effect of surgical menopause and estrogen replacement on cytokine release from human blood mononuclear cells. Proc Natl Acad Sci U S A (1991) 88:5134–5138.[Abstract/Free Full Text]
  24. Kamada M., Irahara M., Maegawa M., Ohmoto Y., Takeji T., Yasui T., et al. Postmenopausal changes in serum cytokine levels and hormone replacement therapy. Am J Obstet Gynecol (2001) 184:309–314.[CrossRef][ISI][Medline]
  25. Xu Y., Clanachan A.S., Jugdutt B.I. Enhanced expression of angiotensin II type 2 receptor, inositol 1,4, 5-trisphosphate receptor, and protein kinase cepsilon during cardioprotection induced by angiotensin II type 2 receptor blockade. Hypertension (2000) 36:506–510.[Abstract/Free Full Text]
  26. Calabresi L., Rossoni G., Gomaraschi M., Sisto F., Berti F., Franceschini G. High-density lipoproteins protect isolated rat hearts from ischemia-reperfusion injury by reducing cardiac tumor necrosis factor-alpha content and enhancing prostaglandin release. Circ Res (2003) 92:330–337.[Abstract/Free Full Text]
  27. Engelman D.T., Watanabe M., Engelman R.M., Rousou J.A., Kisin E., Kagan V.E., et al. Hypoxic preconditioning preserves antioxidant reserve in the working rat heart. Cardiovasc Res (1995) 29:133–140.[CrossRef][ISI][Medline]
  28. Ma X.L., Lopez B.L., Liu G.L., Christopher T.A., Ischiropoulos H. Peroxynitrite aggravates myocardial reperfusion injury in the isolated perfused rat heart. Cardiovasc Res (1997) 36:195–204.[Abstract/Free Full Text]
  29. Kubota T., Miyagishima M., Frye C.S., Alber S.M., Bounoutas G.S., Kadokami T., et al. Overexpression of tumor necrosis factor- alpha activates both anti- and pro-apoptotic pathways in the myocardium. J Mol Cell Cardiol (2001) 33:1331–1344.[CrossRef][ISI][Medline]
  30. Xu Y., Arenas I.A., Armstrong S.J., Davidge S.T. Estrogen modulation of left ventricular remodeling in the aged heart. Cardiovasc Res (2003) 57:388–394.[Abstract/Free Full Text]
  31. Stamm C., Friehs I., Cowan D.B., Moran A.M., Cao-Danh H., Duebener L.F., et al. Inhibition of tumor necrosis factor-alpha improves postischemic recovery of hypertrophied hearts. Circulation (2001) 104:I350–I355.[ISI][Medline]
  32. Irwin M.W., Mak S., Mann D.L., Qu R., Penninger J.M., Yan A., et al. Tissue expression and immunolocalization of tumor necrosis factor-alpha in post-infarction dysfunctional myocardium. Circulation (1999) 99:1492–1498.[Abstract/Free Full Text]
  33. Plutzky J. Inflammatory pathways in atherosclerosis and acute coronary syndromes. Am J Cardiol (2001) 88:10K–15K.[ISI][Medline]
  34. Kupatt C., Habazettl H., Goedecke A., Wolf D.A., Zahler S., Boekstegers P., et al. Tumor necrosis factor-alpha contributes to ischemia- and reperfusion-induced endothelial activation in isolated hearts. Circ Res (1999) 84:392–400.[Abstract/Free Full Text]
  35. Liu Z.G., Hsu H., Goeddel D.V., Karin M. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell (1996) 87:565–576.[CrossRef][ISI][Medline]
  36. Cai D., Xaymardan M., Holm J.M., Zheng J., Kizer J.R., Edelberg J.M. Age-associated impairment in TNF-alpha cardioprotection from myocardial infarction. Am J Physiol Heart Circ Physiol (2003) 285:H463–H469.[Abstract/Free Full Text]
  37. Deveraux Q.L., Roy N., Stennicke H.R., Van Arsdale T., Zhou Q., Srinivasula S.M., et al. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. Embo J (1998) 17:2215–2223.[CrossRef][ISI][Medline]
  38. Li X., Yang Y., Ashwell J.D. TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2. Nature (2002) 416:345–347.[CrossRef][Medline]

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