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Endocrinology, doi:10.1210/en.2007-0859
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Endocrinology Vol. 149, No. 3 1205-1213
Copyright © 2008 by The Endocrine Society

Physiological Concentration of 17β-Estradiol on Sympathetic Reinnervation in Ovariectomized Infarcted Rats

Tsung-Ming Lee, Mei-Shu Lin and Nen-Chung Chang

Department of Medicine (T.-M.L.), Cardiology Section, Taipei Medical University and Chi-Mei Medical Center, Tainan 710, Taiwan; Department of Pharmacy (M.-S.L.), National Taiwan University Hospital, Taipei 100, Taiwan; and Department of Medicine (N.-C.C.), Cardiology Section, Taipei Medical University and Hospital, Taipei 110, Taiwan

Address all correspondence and requests for reprints to: Prof. Nen-Chung Chang, Cardiology Section, Department of Medicine, Taipei Medical University and Hospital, 252, Wu-Hsing Street, Taipei, 110, Taiwan. E-mail: ncchang{at}tmu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
17β-Estradiol (E2) has been shown to exert antiarrhythmic effect after myocardial infarction; however, the mechanisms remain unclear. This study was performed to determine whether E2 exerts beneficial effects through attenuated sympathetic hyperreinnervation after infarction. Two weeks after ovariectomy, female Wistar rats were assigned to coronary artery ligation or sham operation. Twenty-four hours after coronary ligation, rats underwent one of five treatments: 1) sc vehicle treatment (control), 2) sc E2 treatment, 3) sc E2 treatment + tamoxifen (a potent estrogen receptor antagonist), 4) bosentan (an endothelin receptor blocker), or 5) sc E2 treatment + bosentan and followed for 4 wk. Myocardial endothelin-1 and norepinephrine levels at the remote zone revealed a significant elevation in control infarcted rats, compared with sham-operated rats, which is consistent with sympathetic hyperinnervation after infarction. Sympathetic hyperinnervation was blunted after giving the rats either E2 or bosentan, assessed by immunohistochemical analysis of tyrosine hydroxylase, growth-associated protein 43 and neurofilament, and Western blotting and real-time quantitative RT-PCR of nerve growth factor. Arrhythmic scores during programmed stimulation in E2-treated infarcted rats were significantly lower than in control-infarcted rats. Addition of bosentan did not have additional beneficial effects, compared with rats treated with E2 alone. The beneficial effect of E2 on sympathetic hyperinnervation was abolished by tamoxifen. Our data indicated that E2 has a role for sympathetic hyperinnervation after infarction, probably through an endothelin-1-depedent pathway. Chronic administration of E2 after infarction may attenuate the arrhythmogenic response to programmed electrical stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPIDEMIOLOGICAL STUDIES SHOWED that women appear to have a lower incidence of arrhythmia-induced sudden cardiac death than men (1). Sex steroid-induced changes in autonomic tone might have an effect on the sex-related differences in cardiac arrhythmias (2). The exact pathophysiologic mechanisms by which estrogen influences the occurrence of cardiac arrhythmias are far from being understood. Recent studies have shown that estrogen attenuates sympathetic overflow (3), which can play an important role in pathogenesis of antiarrhythmia (4). However, the underlying mechanism of estrogen-related antiarrhythmia remains unknown.

Endothelin (ET)-1 has been shown to play a critical role in the pathogenesis of sympathetic innervation in the development of the sympathetic nervous system in the heart (5). ET-1 is a key regulator of nerve growth factor (NGF) induction in the heart. NGF is a trophic factor that is critical for the differentiation, survival, and synaptic activity of the peripheral sympathetic and sensory nervous systems (6). Levels of NGF expression within innervated tissues roughly correspond to innervation density (7). Deletion of a single copy of the NGF gene results in a 50% reduction in sympathetic neurons (8), whereas overexpression of NGF in the heart results in cardiac hyperinnervation and hyperplasia in stellate ganglia neurons (9). Furthermore, administration of ET receptor blockers attenuates NGF expression in cardiomyocytes and reduces sympathetic neurite extension (5). These results demonstrate the importance of ET-NGF pathway in the regulation of sympathetic innervation.

Increased sympathetic nerve density after myocardial injury has been shown to be responsible for the occurrence of lethal arrhythmias and sudden cardiac death in humans (10). During a chronic stage of myocardial infarction (MI), a regional increase of sympathetic nerves (neural remodeling) was commonly observed (11). Increased sympathetic nerve activity plays an important role in generation of ventricular arrhythmia and sudden cardiac death (10, 12) because sympathetic nerve activation exerts significant effects on electrophysiological properties such as automaticity, refractoriness, and conduction velocity of myocardial cells (13, 14). An enhanced spatial inhomogeneity in cardiac innervation might amplify the spatial inhomogeneity of these electrophysiological properties and facilitate the initiation of arrhythmias. Thus, nerve sprouting has been shown to be an important contributing factor for the occurrence of ventricular arrhythmias and sudden cardiac death in healed stages of MI in animals (15, 16) and humans (10). Estrogen is implicated in sympathetic nerve remodeling because chronic administration of estrogen could regulate neurotrophic systems by reducing ET-1 levels (17). Estrogen treatment has been shown to modulate ventricular remodeling after infarction (18, 19, 20). However, estrogen was administered before experimental MI in previous studies. Because treatment before acute MI is a virtual impossibility in most clinical situations, there has been a great deal of interest in agents that do not require pretreatment (adjunctive treatment). Thus, we assessed whether adjunctive administration of estrogen after infarction can result in attenuated sympathetic hyperinnervation in a rat MI model, in which enhanced myocardial levels of ET-1 play a crucial role (21) and the role of ET-1 in nerve sprouting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Procedures for animal care, surgery, and euthanasia were approved by our institutional review committee for animal experiments. Female Wistar rats that weighed 120–150 g (around 8 wk old) were fed a normal sodium diet, with a sodium content of 0.32 wt% and offered tap water ad libitum before the study. They were kept in cages, five per cage, in a standard light/dark room at a constant temperature (22 ± 1 C) and humidity. Ovariectomies (OVX) were performed through dorsal incisions under ketamine anesthesia in 8-wk-old rats. Two weeks after OVX, the animals received sc implants of slow-release 17β-estradiol (E2) pellets (0.5 mg; Innovative Research of America, Sarasota, FL) 24 h after inducing MI. We previously demonstrated that E2, administered before coronary ligation, can provide cardioprotection against infarct size and arrhythmias (22, 23). To avoid the confounding effect, the E2 pellets were implanted sc in the neck area 24 h after MI, at a time when drugs can exert maximum benefits (24). The sustained release pellets were designed to maintain constant E2 concentrations instead of the fluctuating E2 levels that occur in intact rats during the estrous cycle. Confirmation of estrogen status was determined by uterus weight and plasma E2 levels. The non-OVX (intact) group and the control OVX rats received placebo pellets. Recently E2 has been shown to attenuate ET-1 production via an estrogen receptor-mediated pathway (25). To examine the estrogen receptor effect, a dose of 10 mg/kg · d of tamoxifen, a potent estrogen receptor antagonist, was administered. The dose of tamoxifen used in the present study was previously shown to normalize the exaggerated cerebral artery response to norepinephrine in the ovariectomized female rat (26). To further confirm the role of chronic ET-1 activation in the progression of sympathetic innervation, we included an OVX infarcted group treated with bosentan (10 mg/kg · d; Actelion Pharmaceuticals Ltd., Allschwil, Switzerland), a nonspecific endothelin receptor blocker. The therapeutic efficacy of this dose, without hypotensive effects, has been previously demonstrated (27). The drugs were dissolved in drinking water, and the concentration was adjusted for the daily water intake and body weight to obtain the target dosage. Thus, together, seven experimental groups were studied: sham group and infarction groups (intact, control OVX, OVX + E2, OVX + E2 + tamoxifen, OVX + bosentan, and OVX + E2 + bosentan). The study duration was designed to be 4 wk because the majority of the myocardial remodeling process in the rat (70–80%) is complete within 3 wk (28).

Experimental MI
To create the model, rats were anesthetized with ketamine (90 mg/kg, ip). After adequate anesthesia they were intubated with a 14-gauge polyethylene catheter and ventilated with room air using a small animal ventilator (model 683; Harvard Apparatus, Boston, MA) as described previously (21). The heart was exposed via a left-sided thoracotomy, and the anterior descending artery was ligated using a 6–0 silk between the pulmonary outflow tract and the left atrium. The ligation of the anterior descending artery was confirmed by paling the left ventricular (LV) free wall before the thoracotomy was closed. Sham rats underwent the same procedure except the suture was passed under the coronary artery and then removed. Mortality in the animals with MI was approximately 50% within the first 24 h. None of the sham-operated animals died.

Echocardiogram
At 28 d after operation, rats were lightly anesthetized with ip injection of ketamine HCl (25 mg/kg). Echocardiographic measurements were done with a HP Sonos 5500 system with a 15–6L (6–15 MHz, SONOS 5500; Agilent Technologies, Palo Alto, CA) probe as described previously (29). M-mode tracing of the LV was obtained from the parasternal long-axis view to measure LV end-diastolic diameter dimension and LV end-systolic diameter dimension, and fractional shortening (percent) was calculated. After this, the hearts quickly underwent hemodynamic measurement after systemic heparinization.

Hemodynamics and infarct size measurements
Hemodynamic parameters were measured in lightly anesthetized rats via the left carotid artery at the end of the study as described previously (21). A polyethylene Millar catheter was inserted into the carotid artery and connected to a transducer (SPR-407, TX) to measure arterial blood pressure and LV end-diastolic pressure. After the arterial pressure measurement, the heart was rapidly excised and suspended for retrograde perfusion with a Langendorff apparatus. At completion of the electrophysiological tests, both atria were trimmed off, the right ventricle was weighed and the LV was rinsed in cold physiological saline, weighed, and immediately frozen in liquid nitrogen after obtaining a coronal section of the LV for infarct size estimation. A section, taken from the equator of the LV, was fixed in 10% formalin and embedded in paraffin for determination of infarct size. Each section was stained with hemotoxylin and eosin and trichrome. The areas of scar and nonscar regions were measured the tracings by computerized planimetry (Image Pro Plus; Media Cybernetics, Silver Spring, MD) at the same midpapillary slice of each heart. The infarct size was determined according to the method of Pfeffer and Braunwald (30): the lengths of scar for the endocardial and epicardial surfaces were summed as endocardial and epicardial circumferences. With respect to clinical importance, only rats with large MI (>30%) were selected for analysis.

Perfusion of isolated hearts
Each heart was perfused with a modified Tyrode’s solution (containing, in millimoles, NaCl, 117.0; NaHCO3, 23.0; KCl, 4.6; NaH2PO4, 0.8; MgCl2, 1.0; CaCl2, 2.0; and glucose, 5.5) equilibrated at 37 C and oxygenated with a 95% O2-5% CO2 gas mixture. The perfusion medium was maintained at a constant temperature of 37 C with a constant flow at 4 ml/min. Epicardial electrograms were recorded by an atraumatic unipolar electrode and placed on the epicardial surface of the right atrium and anterior LV wall 2 mm below the circumflex artery. A bipolar pacing electrode was placed near the apex of the heart on the anterior epicardial surface of the right ventricle. Atrial and ventricular epicardial electrocardiograms were continuously recorded.

Spontaneous and induced arrhythmias
After completing the perfusion of isolated hearts, the hearts were observed for 20 min to allow stabilization of contraction and rhythm. Because the residual neural integrity at the infarcted site is one of the determinants of the response to electrical induction of ventricular arrhythmias (31), only rats with the infarcted area of the LV totally replaced by scar tissue were included. The protocol for pacing and an arrhythmia scoring system were used as previously described (21). When multiple forms of arrhythmias occurred in one heart, the highest score was used. The experimental protocols were typically completed within 10 min.

Real-time RT-PCR of NGF
Real-time quantitative RT-PCR was performed from samples obtained from the remote zone with the TaqMan system (Prism 7700 sequence detection system; Applied Biosystems, Courtaboeuf, France) as previously described (21). For NGF, the primers were 5'-TCCACCCACCCAGTCTTCCA-3' (sense) and 5'-GCCTTCCTGCTGAGCACACA-3' (antisense). For glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) the primers were 5'-CTTCACCACCATGGAGAAGGC-3' (sense) and 5'-GGCATGGACTGTGGTCATGAG-3' (antisense). Standard curves were plotted with the threshold cycles vs. log template quantities. For quantification, NGF expression was normalized to the expressed housekeeping gene GAPDH. Reaction conditions were programmed on a computer linked to the detector for 40 cycles of the amplification step. Experiments were replicated three times and results expressed as the mean value.

Western blot analysis of NGF
Samples obtained from the remote zone were homogenized with a kinematic polytron blender in 100 mM Tris HCl (pH 7.4), supplemented with 20 mmol/liter EDTA, 1 mg/ml pepstatin A, 1 mg/ml antipain, and 1 mmol/liter benzamidin. Homogenates were centrifuged at 10,000 x g for 30 min to pellet the particulate fractions. The supernatant protein concentration was determined with the BCA protein assay reagent kit (Pierce Endogen, Rockford, IL). Twenty micrograms of protein were separated by 10% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. The nitrocellulose membrane was then rinsed with a blocking solution of 5% nonfat dry milk and incubated with rabbit polyclonal anti-NGF antibodies (Chemicon, Temecula, CA) at 1:500 dilution. Antigen-antibody complexes were detected with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium chloride (Sigma, St. Louis, MO). Films were volume integrated within the linear range of the exposure using a scanning densitometer. Relative abundance was obtained by normalizing the density of NGF protein against that of β-actin. Experiments were replicated three times and results expressed as the mean value.

Immunohistochemical studies of tyrosine hydroxylase, growth-associated factor 43, and neurofilament
To investigate the spatial distribution and quantification of sympathetic nerve fibers, analysis of immunohistochemical staining for tyrosine hydroxylase, growth-associated factor 43 (a marker peptide for neuronal regeneration and outgrowth), and neurofilament (a marker for sympathetic nerve) (32) was performed on LV muscle from the remote regions (>2 mm outside the infarct). Papillary muscles were excluded from the study because a variable sympathetic innervation has been reported (33). Paraffin-embedded sections were performed at a thickness of 5 µM. Tissues were incubated with tyrosine hydroxylase antibodies (1:200; Chemicon), anti-growth-associated factor 43 (1:400; Chemicon), and antineurofilament (1:1000; Chemicon) in 0.5% BSA in PBS overnight at 37 C. Immunostaining was performed using a standard immunoperoxidase technique (N-Histofine simple stain MAX PO kit; Nichirei Co., Tokyo, Japan). Isotype-identical directly conjugated antibodies served as a negative control.

The density of tyrosine hydroxylase-labeled nerve fibers was qualitatively estimated from 10 randomly selected fields at a magnification of x400. The nerve density was measured on the tracings by computerized planimetry (Image Pro Plus; Media Cybernetics, Silver Spring, MD) as described previously (34). The value was expressed as the ratio of tyrosine hydroxylase-labeled nerve fiber area to total area. The slides were coded so that the investigator was blinded to the rat identification.

Measurement of plasma E2, tissue ET-1, and tissue norepinephrine
Blood samples (1 ml) were withdrawn from the LV into EDTA-coated tubes for plasma samples. Blood samples were separated by centrifugation at 3000 x g for 10 min at 4 C. E2 levels were measured with the use of a RIA kit (Diagnostic Products Corp., Los Angeles, CA). To measure tissue ET-1 concentrations, the samples from the remote zone were homogenized with a polytron homogenizer for 60 sec in 10 vol of 1 mol/liter acetic acid containing 10 µg/ml pepstatin and immediately boiled for 10 min at 4 C. ET-1 was measured by immunoassay (R&D System Inc., Minneapolis, MN).

Although cardiac reinnervation has been shown in immunohistochemical staining of tyrosine hydroxylase, growth-associated factor 43, and neurofilament, it did not imply that the nerves are functional. Thus, to examine the sympathetic nerve function after administering E2, we measured LV norepinephrine levels from the remote regions and the border zone 4 wk after infarction. Because the samples were collected after perfusing with modified Tyrode’s solution during electrophysiological study, catecholamine in blood was eliminated. The myocardiums were minced and suspended in a 0.4 N perchloric acid with 5 mmol/liter reduced glutathione (pH 7.4), homogenized with a polytron homogenizer for 60 sec in 10 vol. Samples were immediately centrifuges at 3000 x g for 10 min, and the supernatant was stored at –70 C until further analysis. The supernatant protein concentration was determined with the BCA protein assay reagent kit (Pierce Endogen). Total norepinephrine was measured using a commercial ELISA kit (Noradrenalin ELISA; IBL Immuno-Biological Laboratories Co., Hamburg, Germany).

Statistical analysis
Results were presented as mean ± SD. Statistical analysis was performed using the SPSS statistical package (version 10.0; SPSS, Chicago, IL). Differences among the groups of rats were tested by a one-way ANOVA. Subsequent analysis for significant differences between the two groups was performed with a multiple comparison test (Scheffé’s method). Electrophysiological data (scoring of programmed electrical stimulation-induced arrhythmias) were compared by a Kruskal-Wallis test followed by a Mann-Whitney test. The significant level was assumed at value of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight, E2 levels, uterus weight, and infarct size
Body weight was significantly higher in infarcted rats treated with control OVX and OVX + bosentan (Table 1Go) despite there being no difference in weight among any of the groups at the start of the study. The finding that body weights were negatively related to E2 status was consistent with previous observation (35). MI did not affect body weight.


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  		TABLE 1. Morphometry, hemodynamics, E2, ET-1, and NE concentrations at the end of study

 
OVX animals replaced with an E2 pellet had an average plasma estradiol level similar to that seen at proestrus in the cycling rat (average 95 ± 38 pg/ml). In contrast, control OVX animals had low levels of estrogen (average 19 ± 10 pg/ml), similar to that found in postmenopausal women.

The weight of the uterine horns and body were significantly less in control OVX (30 ± 17 mg/g body weight) than sham-operated (60 ± 13 mg/g body weight) rats. There was significant increase in uterine weight in OVX rats treated with E2 pellets than those of intact rats. MI had no effect on uterus weight. Tamoxifen treatment significantly decreased uterine weight, compared with rats treated with E2 alone, indicating an effective dose as an estrogen antagonist.

Mean infarct size was similar among the infarcted groups. We found no significant differences in mortality between the infarcted groups throughout the study. E2 had little effect on cardiac gross morphology in the sham-operated rats (data not shown). Four weeks after infarction, the infarcted area of the LV was very thin and was totally replaced by fully differentiated scar tissue. The weight of the LV inclusive of the septum remained essentially constant 4 wk among the infarcted groups.

Hemodynamics
In sham rats, LV end-diastolic pressure (LVEDP), LV end-systolic pressure, +dP/dt, and –dP/dt were not affected by E2 status (data not shown). Heart rate was significantly higher in infarcted rats treated with control OVX than in the rats treated with intact, OVX + E2, OVX + E2 + tamoxifen, and OVX + E2 + bosentan (Table 1Go). LVEDP was similarly increased in the infarcted groups, compared with sham.

Myocardial ET-1 levels
Compared with sham animals, tissue levels of ET-1 from the remote regions were significantly increased in intact animals (1.4 ± 0.6 vs. 2.8 ± 1.4 pg/mg tissue in infarcted rats; P < 0.05, Table 1Go). In OVX rats treated with E2, the levels of ET-1 were significantly lower than those without being administered E2. Tamoxifen administration significantly increased ET-1 levels, compared with rats treated with OVX + E2 alone.

Myocardial norepinephrine levels
LV norepinephrine levels were significantly up-regulated 2.1-fold at the remote zone in the intact rats than in sham-operated rats (2.38 ± 0.21 vs. 1.15 ± 0.42 µg/g protein, P < 0.0001). Expression was region dependent with a significant increase at the remote zone (2.38 ± 0.17 µg/g protein), compared with that at the border zone (1.77 ± 0.25 µg/g protein, P = 0.002) after infarction in the intact group. Compared with rats treated with control OVX, either E2 or bosentan administration significantly decreased LV norepinephrine levels at both border zone and remote regions. However, addition of bosentan did not further attenuate LV norepinephrine levels, compared with rats treated with E2 alone. The beneficial effect of E2 on LV norepinephrine levels was reversed by administering tamoxifen.

Echocardiographic data
After 4 wk of E2 intervention, LV structure and function were evaluated in vivo by echocardiographic analysis (Table 2Go). Compared with sham-operated hearts, infarcted hearts showed structural changes such as increased LV diastolic and systolic diameters. Functional abnormalities accompanied structural remodeling of the LV after MI. The combination of impaired regional function and LV cavity enlargement resulted in a substantial decrease in fractional shortening (42 ± 3% in sham vs. 14 ± 4% in MI hearts; P < 0.0001). Modulation of hormonal status by OVX or OVX + E2 or drug intervention by tamoxifen or bosentan had no effect on echocardiographic indices of cardiac function in infarcted hearts.


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  		TABLE 2. Echocardiographic findings

 
Immunohistochemical analyses
The tyrosine hydroxylase-immunostained nerve fibers appeared to be oriented in the longitudinal axis of adjacent myofibers (Fig. 1Go, upper panel). The tyrosine hydroxylase-positive nerve area fraction was significantly larger in control infarcted rats than that in sham group (Fig. 1Go, lower panel). Infarcted rats in the E2- or bosentan-treated group show smaller nerve area fractions in the remote regions than control OVX rats (0.10 ± 0.05% in E2, 0.09 ± 0.04% in bosentan vs. 0.23 ± 0.08% in control OVX group, P < 0.001, respectively). Similar to tyrosine hydroxylase results, growth-associated factor 43-positive (Fig. 2Go) and neurofilament-positive (data not shown) nerve area fractions were significantly attenuated in the E2-treated infarcted rats, compared with control OVX rats. Addition of bosentan did not further attenuate expression of tyrosine hydroxylase, growth-associated factor 43, and neurofilament, compared with rats treated with E2 alone. The attenuated expression of E2-related tyrosine hydroxylase, growth-associated factor 43, and neurofilament can be reversed to the levels similar to the control OVX rats after adding tamoxifen. These morphometric results mirrored those of norepinephrine contents.


Figure 1
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  		FIG. 1. Upper panel, Immunohistochemical staining for tyrosine hydroxylase from the remote regions (magnification, x400). Tyrosine hydroxylase-positive nerve fibers (brown color) are located between myofibrils and are oriented parallel direction as that of the myofibrils. Myocytes are not stained and appear pale in this view. A, Sham. B, Intact infarcted rat treated with vehicle. C, Control (Ctl) OVX infarcted rat treated with vehicle. D, OVX infarcted rat treated with E2. E, OVX infarcted rat treated with E2 + tamoxifen (TAM). F, OVX infarcted rat treated with bosentan. G, OVX infarcted rat treated with E2 + bosentan. Bar, 50 µm. Lower panel, Nerve density area fraction (percent) at the remote zone. Each column and bar represents mean ± SD. The number of animals in each group is indicated in parentheses. Bars with different superscript letters differ at P < 0.05.

 

Figure 2
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  		FIG. 2. Immunohistochemical staining for growth-associated protein 43 from the remote regions (magnification, x400). A, Sham. B, Intact infarcted rat treated with vehicle. C, Control (Ctl) OVX infarcted rat treated with vehicle. D, OVX infarcted rat treated with E2. E, OVX infarcted rat treated with E2 + tamoxifen. F, OVX infarcted rat treated with bosentan. G, OVX infarcted rat treated with E2 + bosentan. Bar, 50 µm. Bars with different superscript letters differ at P < 0.05.

 
Western blot, and real-time RT-PCR of NGF
Western blot shows that NGF levels were significantly up-regulated 2.2-fold at the remote zone in the intact rats than sham-operated rats (P < 0.0001, Fig. 3Go). Compared with control OVX rats in either E2- or bosentan-treated rats, NGF levels were significantly lower at the remote zone. Addition of bosentan did not further attenuate expression of NGF, compared with rats treated with E2 alone. The attenuated expression of E2-related NGF can be reversed to the levels after adding tamoxifen, similar to the control OVX rats.


Figure 3
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  		FIG. 3. Western blot analysis of NGF (molecular mass, 13 kDa) showing the effect of E2 on immunorecognition of NGF in homogenates after MI from the remote zone. A significantly reduced amount of NGF had taken place in the OVX groups treated with E2, compared with control (Ctl). Relative abundance was obtained by normalizing the density of NGF protein against that of β-actin. Results are mean ± SD of three independent experiments. TAM, Tamoxifen. The number of animals in each group is indicated in parentheses. Bars with different superscript letters differ at P < 0.05.

 
PCR amplification of the cDNA revealed that the NGF mRNA levels showed a 2.3-fold up-regulation at the remote zone in the intact rats, compared with the sham-operated rats (P < 0.0001, Fig. 4Go). In E2 or bosentan-treated infarcted rats, the NGF mRNA levels were significantly decreased than those in the control OVX rats. NGF protein expression was in parallel to the mRNA level changes, implying that the NGF production is a critical regulation step for nerve sprouting.


Figure 4
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  		FIG. 4. LV NGF mRNA levels. Each mRNA was normalized to an mRNA level of GAPDH. TAM, Tamoxifen. Each column and bar represents mean ± SD. The number of animals in each group is indicated in parentheses. Bars with different superscript letters differ at P < 0.05.

 
Electrophysiological stimulation
To further elucidate the physiological effect of attenuated sympathetic reinnervation, ventricular pacing was performed. Arrhythmia score in sham-operated rats was very low (0.3 ± 0.3, Fig. 5Go). In contrast, ventricular tachyarrhythmias consisting of ventricular tachycardia and ventricular fibrillation were inducible by programmed stimulation in intact infarcted rats. Either E2 or bosentan treatment significantly decreased the inducibility of ventricular tachyarrhythmias, compared with those in the control OVX group. Addition of bosentan did not further attenuate arrhythmia score, compared with rats treated with E2 alone. The beneficial effect of E2 on arrhythmic score was reversed by administering tamoxifen.


Figure 5
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  		FIG. 5. Inducibility quotient of ventricular arrhythmias by programmed electrical stimulation 4 wk after MI. TAM, Tamoxifen. The number of animals in each group is indicated in parentheses. Bars with different superscript letters differ at P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our present results clearly show for the first time that despite the late administration of estrogen at 24 h after coronary ligation, chronic treatment with physiological concentrations of E2 leads to attenuated sympathetic innervation, probably through an estrogen receptor-dependent ET-1 pathway. Furthermore, addition of bosentan attenuated sympathetic innervation and did not have additional beneficial effects, compared with rats treated with E2 alone, confirming the critical role of ET-1 in sympathetic innervation. These results were consistent with the beneficial effects of E2, as documented structurally by reduction in cardiac nerve sprouting, molecularly by myocardial NGF protein and mRNA levels, biochemically by tissue ET-1 and norepinephrine levels, and electrophysiologically by improvement of fatal ventricular tachyarrhythmias. Our results confirmed the notion that estrogen has significant neuroprotective properties and may be a potential therapeutic agent in neuroinjury in females. Indeed, our findings were consistent with a recent study showing that estrogen reduced NGF protein content in sympathetic vascular targets, which may lead to decreased sympathetic innervations to the heart (36).

The beneficial effect of E2 on the anatomical and functional conditions of sympathetic reinnervation was supported by three lines of evidence. First, E2 administration attenuated ET-1 levels after infarction. The current results confirm our previous demonstration that the ET-1 level was significantly increased after infarction (21) and can be inhibited after administering E2. However, the beneficial effects of E2 were abolished by adding tamoxifen, implying the important role of the estrogen receptor in regulating ET-1 expression. This finding was consistent with the notion that estrogens inhibit ET-1 levels through an estrogen receptor-dependent pathway (37). Second, ET-1 inhibition ameliorates sympathetic hyperinnervation in chronically infarcted hearts. The involvement of ET-1 in E2-related sympathetic hyperinnervation after infarction was confirmed by administering bosentan. Addition of bosentan to E2-treated rats afforded no further beneficial effects on sympathetic hyperinnervation, indicating a common target of these two agents. And third, the severity of pacing-induced fatal arrhythmias was associated with the degree of sympathetic reinnervation. This model has the advantage of isolated preparations, with free of the influence from circulating hormones and hemodynamic reflexes. The finding was further supported by Cao et al. (10), showing that increased postinjury sympathetic nerve density may be responsible for the occurrence of ventricular arrhythmia and sudden cardiac death in animals and patients. In contrast to previous studies, the beneficial effects of E2 on sympathetic reinnervation were not related to the extent of ventricular remodeling. The electrophysiological differences shown in infarcted OVX rats with chronic MI do not appear to be related to infarct size or the extent of LV remodeling.

Mechanisms
In this study, we demonstrated attenuated sympathetic reinnervation in E2-treated hearts. The detailed mechanisms by which E2 affected sympathetic reinnervation are not fully defined. However, several factors can be excluded. One is hemodynamics: previous studies have shown a significantly reverse correlation between LVEDP and tyrosine hydroxylase-immunostained profiles (38). In this study, we found that there was similar LVEDP among the infarcted groups. Thus, factors other than blood pressure may contribute to the differences of sympathetic hyperinnervation after infarction. Another factor is the difference in infarct sizes: the degree of sympathetic reinnervation has been shown to be related to the infarct sizes (39). Successful fiber reinnervation appears dependent on repopulating sheaths with Schwann cells, which would be injured according to the sizes of infarction. This possibility was excluded in this study due to similar infarct sizes among the infarcted groups.

Despite a reduction in sympathetic reinnervation, the influence of E2 on chronic remodeling processes after MI remained sparse and revealed conflicting results (18, 19). E2 treatment has been shown to prevent ventricular remodeling assessed by diastolic as well as systolic dilatation after MI (18). However, other studies have shown that estrogen is detrimental at the time of MI or in the early post-MI period because it results in an increased infarct size or infarct expansion (19). The use of different species and the different doses and timing of distinct hormone administration may account for the conflicting results. In our study, E2 treatment was initiated in stable rats that survived an MI for 24 h. Our results were consistent with the notion that estradiol substitution did not alter the remodeling process after MI (20). These findings are of potential clinical relevance because worsening cardiac remodeling is an important negative predictor of morbidity and mortality (40). Besides, we (22, 23) and others (41) have shown that E2 limited the infarct size in models with temporary coronary occlusion followed by reperfusion. However, we used a model of permanent coronary occlusion in this study. In the absence of reperfusion, no therapeutic intervention can salvage ischemic cells, and these cells will die regardless of the treatment applied. Thus, it is not surprising that there were similar infarct sizes among the infarcted groups.

Other mechanisms
Although the present study suggests that the mechanisms of E2-induced neuroprotection are related to attenuated NGF expression, estrogen has been shown to have multifaceted effects on heart and other potential mechanisms need to be studied such as electrical remodeling and cardiac fibrosis. First, E2 might prevent fatal arrhythmias by directly inhibiting electrophysiological alterations (22, 23, 42, 43). We and others have shown that E2 treatment activated ATP-sensitive potassium channels (22, 23), inhibited calcium channels (43), and down-regulated Kv4.3 expression (42). All of the above ionic channels are important for the induction of abnormal automaticity and reentrant arrhythmias. Preventing ionic remodeling may be an upstream approach to antiarrhythmic therapy (44). Second, functional estrogen receptors are present on cardiac fibroblasts (45), and estrogen has been shown to inhibit the formation of collagen in noncardiac cells (46), thereby reducing the risk of isolated regional slowing of conduction and reentrant arrhythmias. Taken together, regardless of the relative importance of each of these factors, all of the E2-caused changes are compatible with our understanding of their protective effects against ventricular arrhythmias.

Clinical implications
These data have important implications because they provide additional evidence of the critical importance of E2 in regulating ischemic tissue repair and suggest that modulation of sympathetic innervation may have important therapeutic implications. Premenopausal women are protected from cardiovascular disease, compared with their age-matched postmenopausal counterparts and age-matched men (1, 2), and it was generally accepted that ovarian hormones, including E2, play a major role in this cardioprotection. However, the validity of this perspective, including the efficacy of hormone replacement therapy in prolonging this cardioprotection, has been recently called into question. Prospective end point trials including the Heart and Estrogen/Progestin Replacement Study (47), the Estrogen Replacement and Atherosclerosis Study (17), and the recently terminated Women’s Health Initiative study (48) revealed controversial results on hormone replacement therapy in human vascular disease. In the guidelines of the American College of Cardiology/American Heart Association, hormone replacement therapy for secondary prevention of coronary events should not be given de novo to postmenopausal women after acute MI (49) because of an unexpected significant increase in vascular thrombosis during the first year after therapy initiation. Our study does not address acute thrombosis-related ischemic events. However, estrogen effects are not necessarily limited to vascular pathology because our results are consistent with the notion that estrogen effects on cardiac innervation represent another mechanism to explain beneficial effects in heart disease. In the specific setting of acute MI, however, there is evidence that estrogen may improve mortality (50). These findings may provide clues toward understanding the seemingly conflicting experimental, epidemiological, and clinical data on E2 in outcome after MI.

This study identifies a potential benefit of premenopausal endogenous estrogens and postmenopausal E2 replacement therapy: a reduction in ET-1. This reduction may be especially important in the presence of pathology where activation of ET-1 plays an etiological role such as hypertension, diabetic nephropathy, and atherosclerosis.

Study limitations
There are limitations in the translation of the results of this study to other species and to human physiology. First, only one time point after infarction was studied. The immunohistochemical and electrophysiological studies were not performed until 4 wk after infarction. The interaction between reinnervation and ventricular arrhythmias over the 4-wk period was not assessed. Nori et al. (51) have shown that reinnervation was started as early as 6 d after infarction in rats. Thus, it remains unclear whether E2 either inhibited development (late stage of infarction) or enhanced degeneration (early stage of infarction) of myocardial sympathetic nerve fibers. Krizsan-Agbas et al. (52) have demonstrated that estrogen induces rapid and extensive degeneration of rodent uterine myometrial sympathetic innervation, consistent with the E2-induced attenuation of sympathetic innervation. Future research should identify these early events and trace their progression after administering E2 after infarction. Second, our finding in rats cannot necessarily be extrapolated to humans with MI. The drug effect of permanent coronary occlusion in the rat model and late patency of the infarcted-related artery in most clinical settings on the development of sympathetic reinnervation may be different. Third, we induced estrogen deficiency by bilateral ovariectomy. Therefore, E2 plasma levels were low but still detectable, similar to the menopausal situation. We do not know whether additional blockade of estrogen receptors by specific estrogen receptor antagonists (e.g. ICI 182,780) may cause a detrimental effect on cardiac innervation. Our study does not provide information on the relative roles of the estrogen receptor-{alpha} or estrogen receptor-β pathways.

Conclusions
These data show that E2 plays an important role in the sympathetic reinnervation after infarction. Attenuated sympathetic reinnervation by E2 administration 24 h after coronary artery occlusion has benefits in not only anatomical structures but also arrhythmia susceptibility. E2 may provide a new strategy for the prevention of postinfarction ventricular arrhythmias. Further studies of the specific role of E2 in the myocardium may contribute to our understanding of their neuroprotective actions and the development of novel neuroprotective therapies.


   Footnotes
 
This work was supported by the Grants CMFHT 9501, CMFHR9602, CMFHR9603, and CM-TMU9601 from Chi-Mei Medical Center and Grant NSC 95-2314-B-384-009 from the National Science Council, Republic of China.

Disclosure Statement: All authors have nothing to disclose.

First Published Online November 29, 2007

Abbreviations: E2, 17β-Estradiol; ET, endothelin; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; LV, left ventricular; LVEDP, LV end-diastolic pressure; MI, myocardial infarction; NGF, nerve growth factor; OVX, ovariectomy.

Received June 27, 2007.

Accepted for publication November 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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