Effects of estrogen via estrogen receptors on parvalbumin levels in cardiac myocytes of ovariectomized rats

Acta Histochemica 114 (2012) 46–54

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Effects of estrogen via estrogen receptors on parvalbumin levels in cardiac myocytes of ovariectomized rats Wimon Wirakiat a , Wandee Udomuksorn b , Surapong Vongvatcharanon c , Uraporn Vongvatcharanon a,∗ a b c

Department of Anatomy, Faculty of Science, Prince of Songkla University Hat-Yai, Songkhla 90112, Thailand Department of Pharmacology, Faculty of Science, Prince of Songkla University Hat-Yai, 90112, Thailand Department of Oral Surgery, Faculty of Dentistry, Prince of Songkla University Hat-Yai, 90112, Thailand

a r t i c l e

i n f o

Article history: Received 30 November 2010 Received in revised form 7 February 2011 Accepted 10 February 2011

Keywords: Estrogen Estrogen receptor Parvalbumin Ovariectomy Cardiac myocyte Calcium-binding protein Rat

a b s t r a c t The study investigated the effects of estrogen on parvalbumin (PV) levels in cardiac myocytes of ovariectomized rats, which is a model system for postmenopausal woman. Parvalbumin acts as a relaxing factor in cardiac myocytes. Adult female Wistar rats, 12 weeks old, were randomly divided into 5 groups of 10: sham-operated (SHAM), ovariectomized (OVX), and OVX receiving estrogen replacement of 10 ␮g/kg (Es10), 20 ␮g/kg (Es20) and 40 ␮g/kg (Es40). After 10 weeks, serum estrogen levels were measured and the ␣ and ␤ estrogen receptors in cardiac myocytes were investigated by immunohistochemistry. PV levels were examined by immunohistochemistry and Western blot analysis. Cardiac myocytes of all animals showed strong staining intensities for ␣ immunoreactive (Es ␣-ir), but weak staining for ␤ immunoreactive (Es ␤-ir) estrogen receptors. The Es ␣-ir was reduced in the cardiac myocytes of the OVX groups, but increased in the Es10, Es20 and Es40 groups. We therefore suggest that estrogen effects are mediated via Es ␣ receptors rather than Es ␤ receptors in female rat hearts. Estrogen and PV immunoreactive (PV-ir) levels and the intensity of the PV band observed in the OVX group were less than those of the SHAM group. In the Es10, Es20 and Es40 groups, the increased intensity of the PV-ir and PV bands correlated with the increased estrogen levels. The low PV levels in cardiac myocytes induced by low estrogen were restored by estrogen replacement therapy. Therefore a reduction of PV may lead to diastolic dysfunction in menopause. © 2011 Published by Elsevier GmbH.

Introduction Several human studies have demonstrated that pre-menopausal women are relatively protected from cardiovascular diseases when compared with age-matched men and postmenopausal women (Lloyd-Jones et al., 1999). Estrogen is known to mediate protective effects on the cardiovascular system. It has been shown to affect

Abbreviations: ABC reagent, avidin–biotin–peroxidase complex; AEC, aminoethyl carboazole; CIA, chemiluminescence immunoassay; DPX, dibutyl phthalate in xylene; EDL, extensor digitorum longus muscle; EGTA, 2aminoethoxylethane-NNN N -tetra-acetic acid; Es10, OVX groups that received estrogen replacement of 10 ␮g/kg; Es20, OVX groups that received estrogen replacement of 20 ␮g/kg; Es40, OVX groups that received estrogen replacement of 40 ␮g/kg; Es ␣, estrogen receptors ␣; Es ␣-ir, estrogen receptors ␣ immunoreactivity; Es ␤, estrogen receptors ␤; Es ␤-ir, estrogen receptors ␤ immunoreactivity; NCX, sodium–Ca2+ exchange protein; OVX, ovariectomized; PAP, Papanicolaou; PMSF, phenyl methyl sulfonyl fluoride; PV, parvalbumin; PV-ir, parvalbumin immunoreactivity; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM, standard error of mean; SERCA, sarcoplasmic reticulum Ca2+ –ATPase; SHAM, sham-operated; TBS, Tris–phosphate buffer; TESPA, 5-aminopropyltrithoxysilane. ∗ Corresponding author. E-mail address: [email protected] (U. Vongvatcharanon). 0065-1281/$ – see front matter © 2011 Published by Elsevier GmbH. doi:10.1016/j.acthis.2011.02.004

a wide variety of processes in the heart and vasculature to reduce cardiovascular risk (Roeters van Lennep et al., 2002). As estrogen levels decrease during and after menopause, an increase in cardiovascular disease and cardiovascular risk factors has been reported (Peters et al., 1999; Kuh et al., 2005; Antonicelli et al., 2008). The incidence of diastolic heart failure, which is identified by an abnormality in the left ventricular relaxation, seems to be most affected by the postmenopausal state (Tresch and McGough, 1995; Yildirir et al., 2001). It has been reported that left ventricular function changes in the postmenopausal state and estrogen replacement improve the left ventricular diastolic functions in postmenopausal women (Yildirir et al., 2001; Alecrin et al., 2004). However, the mechanism through which menopause or estrogen exerts its effects on the diastolic function is still unknown. Parvalbumin (PV) is a low molecular weight (12 kDa) calciumbinding protein that is present in cardiac myocytes of many species including: male and female rats (Vongvatcharanon et al., 2006, 2010a), mice, chickens, rabbits and pigs (Vongvatcharanon et al., 2008). It has been implicated in the relaxation of cardiac myocytes by removing Ca2+ from troponin C and then transferring the Ca2+ ions to the sarcoplasmic reticulum (Coutu et al., 2003). Therefore, any change in PV levels may affect the relaxation of cardiac

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myocytes. Some factors that can affect alterations in the PV levels of cardiac myocytes have been studied. It was shown that aging and alcohol administration reduced PV levels, whereas swimming exercise increased PV levels in rat cardiac myocytes (Vongvatcharanon et al., 2010b,c). However, the effects of sex hormones, especially estrogen, on PV levels have not been studied. The ovariectomized rat has been widely used as an animal model of menopause (Bossé and Di Paolo, 1995; McClung et al., 2006; Sotiriadou et al., 2006; Moran et al., 2007). Thus, this study aimed to investigate the effects of ovariectomy and different doses of estrogen replacement on the levels of PV in female rat cardiac myocytes. Most biological effects of estrogen are mediated via estrogen receptors and it has been reported that the heart contains both estrogen ␣ receptors (Es ␣) and estrogen ␤ receptors (Es ␤). We have therefore investigated the effects of different doses of estrogen replacement on both sets of estrogen receptors of the female rat heart after ovariectomy. Results from this study may help explain the fundamental mechanisms of estrogen effects on diastolic function and the pathology of diastolic dysfunction in menopause.

positive controls for the study of estrogen receptors as they have both ␣ and ␤ estrogen receptors (Pelletier et al., 2000).

Materials and methods

The immunohistochemistry protocols for the study of estrogen ␣ and ␤ receptor were modified from Stenberg et al. (2003). The heart and ovary sections were deparaffinized, rehydrated and incubated sequentially with 0.1 M Tris–phosphate buffer (TBS) for pH 7.6 for 5 min. These steps were followed by placing the sections in a microwave oven at 700 W for antigen retrieval using 0.01 M citric buffer for 10 min. Samples were allowed to cool for 20 min before being washed in TBS. Non-specific blockings were performed for 60 min with 10% (v/v) normal horse serum (Vector Laboratories, Burlingham, CA, USA) for estrogen ␤ receptor staining and 10% (v/v) normal goat serum (Zymed, San Francisco, CA, USA) for estrogen ␣ receptor staining. The sections were then incubated overnight at 4 ◦ C with mouse anti-estrogen receptor ␣ antibody (Chemicon, Temecula, CA, USA) or rabbit anti-estrogen receptor ␤ antibody (Chemicon) at a dilution 1:200 in TBS. After washing in TBS, the sections were incubated with biotinylated secondary goat anti-mouse antibody (anti-mouse IgG, Vector Laboratories, Burlingham, CA, USA) or horse anti-rabbit IgG (Vector Laboratories), at a dilution of 1:200 in TBS for 2 h at room temperature. After an additional wash in TBS, the sections were incubated with an avidin–biotin–peroxidase complex using ABC reagent (Vector Laboratories, prepared according to the manufacturer’s instructions) for 2 h at room temperature. After further washing in TBS, the labeling was visualized using aminoethyl carboazole (AEC) (Vector Laboratories, prepared according to the manufacturer’s instructions) and sections were incubated in the reagent for 5 min. Finally, the sections were counterstained with hematoxylin (Sigma–Aldrich, St. Louis, MO, USA) for 5 min, dehydrated in a graded series of alcohol, cleared in xylene and mounted and coverslipped with DPX (Boehringer Ingelheim Bioproducts, Heidelberg, Germany). Coded sections were examined by light microscopy by an observer unaware of the experimental protocols. Images were captured with an Olympus DP-11 digital camera (Olympus, Tokyo, Japan). Controls for non-specific labeling were performed in which either the first or secondary antibody was excluded. None of these controls showed any labeling.

Animals Adult female Wistar rats (12 weeks) weighing 200–250 g were obtained from the Southern Laboratory Animal Facility, Prince of Songkla University, Thailand. The rats were maintained at 22 ◦ C with a 12/12-dark/light cycle (light on at 06.00 am). Standard commercial food pellets and filtered tap water were available ad libitum. The experimental protocols described in the present study were approved and guided by the Animal Ethics Committee of the Prince of Songkla University for the care and use of experimental animals. The rats were randomly divided into 5 groups (n = 10 per group); sham-operated (SHAM), ovariectomized (OVX), OVX groups that received 10 weeks of estrogen replacement of 10 ␮g/kg (Es10), 20 ␮g/kg (Es20) and 40 ␮g/kg (Es40). Body weights of all groups were recorded weekly. Ovariectomy procedure Ovariectomies were performed under aseptic conditions. The rats were anesthetized by an intraperitoneal injection of 40 mg/kg of pentobarbital sodium (Sigma–Aldrich, St. Louis, MO, USA). For the ovariectomized group, the ovaries were exteriorized, ligated and removed via bilateral lumbar incisions. The wounds were then sutured. For the SHAM groups, the rats were anesthetized and incisions were made followed by visualization of the ovaries through the incisions in the abdominal cavity prior to wound closure by suture. Two weeks after surgery, the hormone treatment in the OVX groups that received estrogen replacement or olive oil (SHAM groups) was started. The ovariectomized and hormone replacement procedures were modified from the work of Chu et al. (2006). Sample preparation Rats were anesthetized by intraperitoneal injection with 75 mg/kg of sodium pentobarbital (Sigma–Aldrich, St. Louis, MO, USA). Blood samples were taken from the thoracic aorta to determine estrogen levels using a chemiluminescence immunoassay (CIA) kit (LKE2 10261, DPC, Gwynedd, UK). The heart and uterus were then removed and weighed. Only the left and right ventricles were further used in this study. The extensor digitorum longus (EDL) muscle was removed and used as a positive control for the study of parvalbumin levels owing to its high parvalbumin content (Heizmann et al., 1982). Some of the removed ovaries were used as

Tissue preparation Hearts, EDL muscle and ovaries were fixed in 10% formalin and processed for embedding in paraffin wax. Serial 50-␮mthick sections were cut and mounted on TESPA-coated slides (Sigma–Aldrich, St. Louis, MO, USA). Evaluation of estrous cycle To determine the stages of the rat estrous cycle, vaginal smears were taken. The samples containing cells were placed on untreated glass microscope slides, air-dried prior to staining and then stained with the Papanicolaou (PAP) stain. These methods of vaginal smear and Papanicolaou stain were adopted from Hubscher et al. (2005). Immunohistochemistry for estrogen ˛ and ˇ receptors

Immunohistochemistry for parvalbumin This immunohistochemical method has been described previously (Vongvatcharanon et al., 2010b,c). Briefly, heart and EDL sections were dewaxed, rehydrated in graded alcohol, incubated with 0.3% (v/v) Triton-X 100 for 30 min and washed in 0.01 M Tris–phosphate buffer (TBS) pH 7.6. After washing, the sections were incubated in 3% (v/v) H2 O2 in methanol for 30 min and 10% (v/v) normal horse serum (Vector Laboratories, Burlinghame, CA,

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Table 1 Rate of weight gain, organ weights, and plasma estrogen levels in sham-operated (SHAM), ovariectomized (OVX), and ovariectomized rats supplemented with 10 ␮g/kg (Es10), 20 ␮g/kg (Es20) and 40 ␮g/kg (Es40) estrogen. Parameter

SHAM

Weight gain (g/month) Uterine weight (g) Heart weight (g) Uw/Bw (× 100) Hw/Bw (× 100) Estrogen (pg/ml)

3.07 0.73 0.83 0.27 0.31 39.85

*

OVX ± ± ± ± ± ±

1.13 0.29 0.20 0.11 0.03 4.71

18.19 0.11 0.80 0.04 0.29 18.80

Es10 ± ± ± ± ± ±

1.67* 0.03* 0.08 0.01* 0.03 1.15*

5.45 0.67 0.79 0.28 0.33 41.97

Es20 ± ± ± ± ± ±

1.12 0.5 0.07 0.03 0.03 2.60

9.37 0.70 0.76 0.29 0.31 71.84

Es40 ± ± ± ± ± ±

1.71 0.10 0.07 0.04 0.02 5.25*

8.88 0.80 0.69 0.34 0.29 201.47

± ± ± ± ± ±

1.47 0.12 0.05 0.05 0.02 20.88*

Significant difference from SHAM, P < 0.05, n = 10.

USA) for 60 min. The sections were then incubated with mouse anti-PV antibody (Parv-19, Sigma–Aldrich, St. Louis, MO, USA) at a dilution of 1:1000 in TBS for 48 h at 4 ◦ C. After washing in TBS, the sections were incubated with biotinylated secondary horse anti-mouse antibody (anti-mouse IgG, Vector Laboratories), at a dilution of 1:200 in TBS for 2 h at room temperature. After an additional wash in TBS, the sections were incubated with an avidin–biotin–peroxidase complex (Vector Laboratories, prepared according to manufacturer’s instructions) for 2 h. After a further wash, immunoreactive sites were visualized using aminoethyl carboazole (AEC) (Vector Laboratories, prepared according to the manufacturer’s instructions) and the sections were incubated in this reagent for 5 min. Finally, the sections were counterstained with hematoxylin (Sigma–Aldrich, St. Louis, MO, USA) for 5 min, dehydrated through a graded alcohol series, cleared with xylene and mounted and coverslipped with DPX (Boehringer Ingelhen Bioproducts, Heidelberg, Germany). Negative controls were performed by omitting the primary antibodies. None of the controls showed any labeling. Coded sections were examined by an observer unaware of the identity of the sections (“blinded”) using light microscopy. Western blot analysis The Western blot analysis method has been described previously (Vongvatcharanon et al., 2010b,c). Briefly, ventricles and EDL were homogenized using a Polytron homogenizer (Kinematica, Lucerne, Switzerland) in lysis buffer (10 mM, Tris pH 8.0, 40 mM sodium fluoride, 5 mM MgCl2 , 100 ␮M sodium orthovanadate, 10 mM EGTA, 1% Triton-X 100, 0.5% sodium deoxycholate, 1 mM PMSF, 20 ␮g/ml leupeptin, and 20 ␮g/ml aprotinin) and then centrifuged at 14,000 × g for 10 min at 4 ◦ C to remove cellular debris. The protein concentration of the supernatant was determined using a Lowry protein assay (BioRad Laboratories, Hercules, CA, USA), performed according to the manufacturer’s instruction. 10 ␮g of protein from each heart from all treatment groups and 0.5 ␮g of protein of EDL were separated by SDS–PAGE in a 12% (w/v) polyacrylamide gel. After electrophoresis protein was transferred onto a nitrocellulose membrane (Amersham, Piscataway, NJ, USA). After blocking with 5% non-fat dried milk in TBS (150 mM NaCl, 100 mM Tris–HCl, pH 8), the membranes were treated with mouse anti-PV antibody (Parv-19, Sigma–Aldrich, USA) at a dilution of 1:1000 for the primary antibody, in 2.5% non-fat dried milk in TBS for 2 h, followed by horseradish peroxidase conjugated horse anti-mouse IgG (Cell Signaling, Danvers, MA, USA) dilution 1:10,000 as secondary antibody in 2.5% non-fat dried milk in TBS for 2 h. The PV protein bands (12 kDa) were detected using an enhanced chemiluminescence system (Amersham, Piscataway, NJ, USA) and visualized by exposure to X-ray hyperfilm (Amersham, Piscataway, NJ, USA). Following development, the film was scanned using a BioRad GS-700 scanner (BioRad; Hercules, CA, USA) driven by Volume Analysis software, and quantified using Molecular Analysis software (version 4).

Statistical analysis Data are expressed as a mean ± SEM. The statistical evaluation of the data was performed using the non-parametric two dependent ttest and the Mann–Whitney test for post hoc analysis to determine significant differences between means. Differences among means were considered significant when P < 0.05. Results The rate of body weight gain per month was significantly higher in ovariectomized rats compared to the SHAM groups (P < 0.05) during the treatment period. However, estrogen replacement brought them back in line with the SHAM groups (Table 1). Ovariectomy significantly reduced uterine weight and the uterine-to-body weight ratio compared to the matched SHAM counterpart and the uterine weight returned to that of the SHAM group after 10 weeks of 10 ␮g/kg, 20 ␮g/kg and 40 ␮g/kg estrogen replacement therapy (Table 1). No differences were found in the heart and heart-tobody weight ratio between the SHAM, OVX and estrogen treatment groups (Table 1). In the OVX groups estrogen levels were significantly reduced, whereas they were similar in the Es10 group and significantly increased in the Es20 and Es40 groups (P < 0.05) compared to the SHAM group (Table 1). Cell types in PAP stained vaginal smears of all four stages Vaginal smears of the SHAM group exhibited four stages of the estrous cycle: proestrus, estrus, metestrus and diestrus. The proestrus smear had a predominance of nucleated epithelial cells (Fig. 1A). The nucleated epithelial cells were oval shaped, with a blue-purple stained nucleus and light pink stained cytoplasm. The estrous smear had a predominance of cornified cells (Fig. 1B). The cornified cells were anucleated with a pink stained cytoplasm. The metestrus smear showed densely packed leukocytes and nucleated epithelial cells (Fig. 1C). The leukocytes had a blue-purple stained nucleus and contained a small amount of cytoplasm. The diestrus smear had only scattered nucleated epithelial cells (Fig. 1D). As determined by vaginal cytology, all SHAM groups were cycling randomly, while the OVX group was persistently in diestrus. The Es10 and Es20 groups exhibited 3 stages of the estrous cycle: proestrus, estrus and diestrus, whereas, the Es40 group showed only proestrus and estrus (Table 2). estrogen levels were highest at proestrus The (47.34 ± 5.64 pg/ml), reduced in estrus (43.94 ± 9.04 pg/ml) and metestrus (38.44 ± 16.52 pg/ml) and lowest in the diestrus stage (22.19 ± 4.50 pg/ml). Estrogen receptor ˛ immunoreactivity A strong, estrogen receptor ␣ immunoreactivity (Es␣-ir) was identified in the nuclei of granulosa cells, thecal cells and stromal

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Fig. 1. Micrograph of vaginal smears using the PAP method in all four stages: proestrus (A), estrus (B), metestrus (C) and diestrus (D). Scale bars = 50 ␮m. N, nucleated epithelial cells; L, leukocytes; C, cornified cells.

cells of the rat ovary (Fig. 2A). The intensity of the Es␣-ir in the cardiac myocytes from the left ventricular wall of the SHAM group was strong (Fig. 2B) but reduced in the OVX group (Fig. 2C) and then increased with increasing estrogen level replacements (Es10, Es20 and Es40) (Fig. 2D, E and F).

of the OVX and was similar to that of the SHAM groups (Fig. 4C). In the Es20 and Es40 groups, the intensity of PV-ir was increased compared to that of the OVX and SHAM group (Fig. 4D and E).

Estrogen receptor ˇ immunoreactivity

Levels of PV protein were examined in EDL, the SHAM, OVX, Es10, Es20 and Es40 groups (Fig. 5A). A significant decrease of PV was observed in the OVX (3.98 ± 1.13, 31.51%) compared to that of the SHAM groups (11.57 ± 1.62, 100%) (P < 0.05). In the Es10, the PV level was almost similar to that of the SHAM group (12.81 ± 5.31, 95.95%). The PV levels were increased in the Es20 (17.92 ± 5.52, 152.16%) and the Es40 (16.48 ± 5.07, 145.34%) compared to that of the SHAM group (Fig. 5B).

A strong intensity of the estrogen receptor ␤ immunoreactivity (Es␤-ir) was identified in granulosa cells, thecal cells and stromal cells of the rat ovary (Fig. 3A). The intensity of Es␤-ir in the cardiac myocytes from the left ventricular wall of all groups was very weak when compared with the intensity of Es␣-ir (Fig. 3B–F). Furthermore, the intensity of Es␤-ir was slightly reduced in the OVX groups (Fig. 3C) compared to that of the SHAM group. The intensity of Es␤-ir was slightly increased in the Es10, Es20 and Es40 treated groups compared to that of the OVX and was almost similar to that of the SHAM group. Parvalbumin immunoreactivity Parvalbumin immunoreactivity (PV-ir) was identified in the cardiac myocytes of the SHAM, OVX, Es10, Es20 and Es40 groups (Fig. 4A–E). However, the intensity of PV-ir was reduced in the OVX group (Fig. 4B) compared with that of the SHAM group (Fig. 4A). In the Es10, the intensity of PV-ir was increased compared to that Table 2 Identity of estrous cycle stages in all treatment groups. Group/estrous cycle

Proestrus

Estrus

Metestrus

Diestrus

SHAM OVX Es10 Es20 Es40

+ − + + +

+ − + + +

+ − − − −

+ + + + −

Parvalbumin protein levels

Discussion Our data have demonstrated that in the OVX group, the estrogen levels, uterine weight and uterine-to-body weight ratio were decreased, whereas the rate of weight gain was increased. Furthermore, the examination of cell types in the PAP stained vaginal smears in the OVX group showed that all the animals had stopped cycling and remained in the diestrus stage that also exhibited the lowest levels of estrogen. In all the estrogen replacement groups (Es10, Es20 and Es40), the estrogen levels, uterine weight, uterineto-body weight ratio were increased, but the rates of weight gains were decreased when compared with the OVX group. All these data confirmed the success of the ovariectomy procedure and hormone replacement therapy. It has been suggested that estrogen is involved in the modulation and distribution of body fat mass and that estrogen deficiency leads to an elevation of body fat mass (Raso et al., 2009). This explains the increase of body weight in the OVX group. In the OVX group, without input from estrogen, the epithelium of the uterus did not proliferate, thus the size of uterus was reduced (Hubscher et al.,

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Fig. 2. Micrographs showing estrogen receptor ␣ immunoreactivity (red-brown color) in granulosa cells (G), thecal cells (T) and stromal cells (S) from rat ovaries (A), and in the cardiac myocytes from the left ventricular wall of the SHAM (B), OVX (C), Es10 (D), Es20 (E) and Es40 (F). Scale bars = 40 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

2005), and this can explain the decrease in uterine weight and the uterine-to-body weight ratio of the OVX group. Examination of cell types from the vaginal smears showed that in the SHAM group, four stages of estrous cycle were identified: proestrus, estrus, metestrus and diestrus, whereas in the OVX group, only diestrus was observed. Our results were similar to those of Hubscher et al. (2005). In the Es10 and Es20, a metestrus stage was not found. In the metestrus stage during the last 6-h, the epithelium of the uterus degenerated and leukocytes infiltrated the thin vaginal epithelium owing to a decline in estrogen secretion and passed into the vaginal canal. In nature, the estrogen levels fluctuate, whereas, with daily treatment a constant level of estrogen may lead to a continuous proliferation of the uterine epithelium and no infiltration of the leukocytes. Therefore, metestrus was not detected in the estrogen replacement groups. This was clearly shown in the Es40 group, where a daily constant high level of estrogen was used, and neither the metestrus nor diestrus stages were identified. The prolonged constant high estrogen level may lead to an abnormal proliferation of the uterine epithelium. Therefore, the 40 ␮g/kg of estrogen replacement may not be recommended for use over a long period.

Estrogen has been shown to mediate gene transcription through the activation of two receptors: estrogen receptors ␣ (Es ␣) and ␤ (Es ␤). Both estrogen receptors ␣ and ␤ immunoreactivities (Es ␣-ir and Es ␤-ir, respectively) were identified in the female rat cardiac myocytes. This result was similar to that described by Jankowski et al. (2001) where both estrogen receptor ␣ and ␤ mRNA were detected in the male and female heart tissue. Jankowski et al. (2001) also reported that in the adult rat ventricles, the mRNA of estrogen receptors ␣ was elevated, whereas the mRNA of the estrogen receptors ␤ was dramatically reduced. According to our data we found a strong intensity of Es ␣-ir and a very weak intensity of Es ␤-ir in the adult female rat ventricles. This also correlates well with the work of Jankowski et al. (2001). Furthermore, they showed that a decrease of the mRNA of estrogen receptors ␣ was reversed by estrogen replacement (25 ␮g/kg). Our data showed a decrease of Es ␣-ir intensity in the OVX group and an increased intensity of the Es ␣-ir in the estrogen replacement groups. In contrast, the intensity of Es ␤-ir in the ventricle of all treatment groups was very weak, thus a slight decrease of Es ␤-ir in the OVX group was observed. Therefore, we suggest that in the adult female rat heart ventricles, Es ␣ plays a more important role than Es ␤.

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Fig. 3. (A) Micrograph showing strong estrogen receptor ␤ immunoreactivity (red-brown color) in the granulosa cells (G), thecal cells (T) and stromal cells (S) from rat ovaries and weak estrogen receptor ␤ immunoreactivity of the cardiac myocytes from the left ventricular wall of the SHAM (B), OVX (C), Es10 (D), Es20 (E) and Es40 (F) groups. Scale bars = 40 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Our data have demonstrated that in the OVX group, estrogen and Es ␣ levels were decreased and this was correlated with a decrease of PV levels in the female rat cardiac myocytes. In contrast, in the estrogen replacement groups, estrogen and Es ␣ levels were increased and this was associated with an increase of PV levels. It has been demonstrated that after estrogen binding and dimerization, an estrogen receptor binds to DNA with a high affinity through their DNA-binding domain (DBD) at specific sites, termed ‘estrogen responsive element sites’ and this activated gene transcription (Osborne et al., 2000). Transcription is the initial process of protein synthesis, thus, a decrease in estrogen level and/or estrogen receptors may lead to a reduction of protein synthesis. This could explain the decline of PV in the OVX group and the increase of PV in the animals that received estrogen replacement. The effect of estrogen on other protein levels in cardiac myocytes has previously been reported such as by Bupha-Intr and Wattanapermpool (2006). This demonstrated a downregulation of sarcoplasmic reticulum Ca2+ –ATPase (SERCA) proteins, a reduction in the SERCA mRNA level, and suppressed maximum SERCA activity in the ovariectomized female rat heart. Estrogen supplementation prevented all these changes. According

to Chu et al. (2006), OVX reduced sodium–Ca2+ exchange protein (NCX) and estrogen restored protein abundance to SHAM levels. The reduction of SERCA and NCX may affect the contraction and relaxation cycles of cardiac myocytes. In comparing the effects of a various doses of estrogen replacement in our study, 10 ␮g/kg of estrogen replacement seems to return the animals to a normal state for their heart and other organs such as the uterus. Treatment of OVX rats with 20 ␮g/kg and 40 ␮g/kg of estrogen produced normal estrogen levels, and an estrous cycle and PV level that were almost similar to those of the control groups. The 20 ␮g/kg and 40 ␮g/kg of estrogen replacement may provide a beneficial effect on the heart due to their ability to mediate an increase of PV levels in female rat cardiac myocytes. However, these may induce a negative effect on the uterus due to stimulation of the epithelial cells of the uterus to proliferate continuously. Long-term administration of 0.3 mg of conjugated equine estrogens has been reported to cause a five fold higher risk for endometrial cancer (Cushing et al., 1998). Furthermore, administration of a daily dose of 0.625 mg conjugated estrogen combined with 2.5 mg medroxyprogesterone acetate or placebo caused a significant increase in invasive breast cancer (Chlebowski et al., 2003).

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Fig. 4. Micrograph showing parvalbumin immunoreactivity (red-brown color) in the cytoplasm of cardiac myocytes from the left ventricular wall of the SHAM (A), OVX (B), Es10 (C), Es20 (D) and Es40 (E) groups. Scale bars = 40 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

It has been suggested that the risk of developing cancer is proportional to the duration of estrogen therapy. Woman who have used estrogen replacement for 5 years or less have no increased risk of breast cancer, long-term users exhibited a significant increase in risk of approximately 20–30% compared to women who have never used estrogen therapy (Kenemans and Stampfer, 1996). Thus it would be of interest to investigate the effects of long-term replacement therapy of 10 mg/kg estrogen on the heart and other organs of OVX rats. Due to PV being involved in mediating relaxation in cardiac myocytes (Coutu et al., 2003) a decrease of PV level in the OVX similar to that occurring at menopause may lead to a reduction of relaxation in the ovariectomized female rat cardiac myocytes. This may explain the pathology of diastolic dysfunction in menopause (Tresch and McGough, 1995; Yildirir et al., 2001). In addition, our data have shown that estrogen replacement restored the PV levels in ovariectomized female rat cardiac myocytes. This may explain the fundamental mechanism of estrogen replacement in improving left ventricular diastolic dysfunction in the menopausal woman. In hormone replacement therapy, chronic daily treatment of estrogen has been used, however, there are no reports on the effect

of an irregular dose of estrogen replacement on the cardiovascular system and other organs. This would be a similar situation to that occurring in the irregular doses of estrogen obtained while using contraceptive drugs. Thus, the effect of irregular dose of estrogen replacement on cardiovascular system and other organs will be further investigated. The ovariectomized rat seems to be a suitable model system to study the effect of sex hormones, especially estrogen, on many organs, however, in menopausal woman, aging is another factor that has to be considered due to physiological aging being correlated with decreased cardiac function (Rengo et al., 1991). Therefore, the effect of estrogen replacement on cardiovascular system in menopause may differ from that obtained in the ovariectomized model. In conclusion, ovariectomy, considered as a model for menopause, resulted in the decrease of estrogen receptors ␣ in cardiac myocytes and led to a reduction of PV levels in cardiac myocytes. 10 ␮g/kg estrogen replacement restored the estrogen receptors ␣ and PV levels to normal. Higher doses of estrogen replacement (20 ␮g/kg and 40 ␮g/kg) led to an increase of the PV level, however, it also increases the risk of abnormal functions of other organs, especially the uterus.

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Fig. 5. (A) Western blot showing bands of parvalbumin (PV) from EDL, ventricular walls (left and right) of the SHAM, OVX, Es10, Es20 and Es40 groups. (B) Histogram showing densitometric quantification of blots of parvalbumin in the SHAM, OVX, Es10, Es20 and Es40 groups. A.U., arbitrary units; n = 10; *significantly different at P < 0.05.

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