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Cardiovascular Research 2006 72(2):303-312; doi:10.1016/j.cardiores.2006.08.003
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Copyright © 2006, European Society of Cardiology

Mechanical stress enhances serotonin 2B receptor modulating brain natriuretic peptide through nuclear factor-{kappa}B in cardiomyocytes

Yao-Jen Lianga, Ling-Ping Laia, Bao-Wei Wangb, Shiow-Jen Juangc, Che-Ming Changd, Jyh-Gang Leud and Kou-Gi Shyud,e,*

aInstitute of Pharmacology, National Taiwan University, Taipei, Taiwan
bDepartment of Medical Education and Research, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
cDepartment of Family Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
dDepartment of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
eGraduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan

* Corresponding author. Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan. Tel.: +886 2 2833 2211x2094; fax: +886 2 2836 5775. Email address: shyukg{at}ms12.hinet.net

Received 9 May 2006; revised 31 July 2006; accepted 1 August 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Result
 4. Discussion
 References
 
Objective: Serotonin via serotonin 2B receptors (SR2BR) regulates cardiac embryonic development and adult heart functions. However, the role of SR2BR in the failing heart due to pressure overload is not well understood.

Methods: Wistar rats of aortic banding and neonatal cardiomyocyte with mechanical stretch were used as cardiomyopathy models.

Results: After two weeks of aortic banding surgery, serum serotonin, mRNA and protein expression of SR2BR increased significantly. The selective SR2BR antagonist, SB215505 (SB), significantly reduced the increase in heart weight, decreased heart wall thickness, left ventricular mass and the expression of the brain natriuretic peptide (BNP) but did not attenuate the up-regulation of SR2BR protein expression in rats after aortic banding for three weeks. After following in vitro mechanical stretch of cardiomyocytes and incubation with serotonin 1 µM, the level of SR2BR and BNP protein increased time-dependently. When transfected by specific siRNA of SR2BR or pretreated with caffeic acid phenethyl ester in cardiomyocytes, the increase of nuclear factor-{kappa}B (NF-{kappa}B) translocation and BNP protein induced by serotonin incubation plus mechanical stretch were both reversed.

Conclusions: SR2BR expression is involved in pressure-induced cardiomyopathy and its downstream signaling may involve NF-{kappa}B to modulate BNP expression in cardiomyocyte.

KEYWORDS Serotonin (5-HT) 2B receptors; Hypertrophy; Cardiomyopathy; Mechanical stretch; Myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Result
 4. Discussion
 References
 
Cardiac hypertrophy is often accompanied by cardiac remodeling characterized by cardiomyocyte loss, interstitial fibroblasts, and collagen deposition, leading to decreased compliance and an increased risk for heart failure [1,2]. The complex process that causes cardiac hypertrophy is related to increased hemodynamic overload and multiple gene expression. Despite the importance of this response in myocardial pathophysiology, the biochemical initiators of hypertrophy remain poorly understood.

Serotonin (5-hydroxytryptamine, 5-HT) is widely distributed in mammals, mediating complex effects in the nervous and other systems. Although several effects attributable to serotonin have been known for more than a century [3], its role in cardiovascular regulation is still not well understood [4]. At present, it is known that serotonin exerts complex alterations in the cardiovascular system, including hypertension, changes vessel motion, and changes heart rhythm [5]. These effects can be explained by the existence of serotonin receptor subtypes that mediate different biological actions [6].

The latest addition to the serotonin 2 receptor class is the serotonin 2B receptor (SR2BR). It is expressed in embryonic [7] and adult [8] cardiovascular tissues from the rat, mouse, and man. Recent data showed genetic evidence that serotonin via SR2BR regulates cardiac development and functions [5,6]. It was suggested that this receptor subtype might be involved in cardiovascular diseases [9]. Transgene mice with a SR2BR gene ablation show embryonic and neonatal death due to lack of trabeculae in the heart [10]. Overexpression of SR2BR in the mouse heart induces mitochondrial proliferation associated with hypertrophy [11]. In vitro studies also suggested that serotonin induces hypertrophy in cultured adult rat ventricular myocytes [12]. Those studies strongly indicated that SR2BR may participate in cardiac hypertrophy but the interplay between SR2BR and the hypertrophy marker of brain natriuretic peptide (BNP) expression [13] is not yet clearly identified.

Moreover the role of SR2BR in the failing heart is still unclear. In addition to the up-regulation of SR2BR both were found in lungs of pulmonary hypertension patients [14] and in arteries of hypertensive deoxycorticosterone acetate-salt rats [15]. We hypothesized that SR2BR expression played a role in the hypertrophic myocardium due to excess of pressure overload. The purpose of this study was to investigate SR2BR changes in models of hypertensive cardiomyopathy and the possible relationship between stress-induced SR2BR and BNP expression in the cardiomyocyte.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Result
 4. Discussion
 References
 
2.1. Rat model of abdominal aortic constriction
On the day of surgery, rats weighing 250 to 300 g were anesthetized with isoflurane and the aorta was exposed via abdominal midline incision. A polyethylene catheter (PE10) was put on the surface of abdominal aorta distal to the renal arteries. Then the needle and aorta were tightly constricted with 6–0 silk. After the procedure, the abdominal wound was sutured and the rats were left to recover. All rats were randomly divided into four groups: sham, banding, banding treated with vehicle (DMSO) and SB215505 (1 mg/kg/day) (Sigma-Aldrich, St. Louis, USA). The solutions were administered intraperitoneally after one week of aortic banding till the rats were sacrificed. The investigations conform to the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Assessment of cardiac hypertrophy and function
Cardiac function of rats after aortic banding was evaluated noninvasively by echocardiography performed with an Acuson Sequoia 512 machine using a 15-MHz probe at the day of sacrifice. Left ventricular percental fractional shortening, left ventricular end-diastolic dimension, left ventricular end-systolic dimension, interventricular septum thickness, left ventricular posterior wall thickness and left ventricular mass were calculated. The sonographer was blinded to the randomization of rats. A technician blinded to the design of the study performed the following experiments.

2.3. In vivo hemodynamic monitor
Three weeks after the surgery, the animals were anesthetized with isoflurane and their carotid arteries, and left ventricle were cannulated with polyethylene catheters to measure mean arterial pressure and left ventricular end-diastolic pressure, respectively. Heart rate was measured through a Grass model tachograph preamplifier. After the measurement of hemodynamic data, the rats were sacrificed to remove the heart to perform the following experiments.

2.4. RNA isolation and reverse transcription (RT) polymerase chain reaction
Total RNA was isolated from rat heart, stomach or neonatal cardiomyocytes using the single-step acid guanidinium thiocyanate/phenol/chloroform extraction method. The cDNA produced by RT was used to generate SR2BR cDNA probes by polymerase chain reaction (PCR) as described previously [16]. The primers for SR2BR were 5'-AGTAAGGCTCCGAAGTT-3' and 5'-CTATACCCGTGCGTTG-3'. The PCR products were run on 2% agarose gel for DNA fragment size verification, then eluted and served as a probe in the Northern blot analysis.

2.5. Northern blot analysis
RNA samples (20 µg/lane) were applied to 1.2% agarose gels with 2.2 M formaldehyde. After electrophoresis, gels were transblotted onto Nytrans membranes. SR2BR cDNA probes were labeled with [32P] dCTP using the random primer labeling method [17]. Following overnight hybridization at 65 °C, membranes were washed twice in 2x saline/sodium citrate for 5 min each. Membranes were then exposed to Kodak-film.

2.6. Western blot analysis
Left ventricle heart tissue or stretched cardiomyocytes were homogenized in modified RIPA buffer (50 mmol/L tris [pH 7.4], 1% IGEPAL CA-630, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 µg/mL aprotinin, leupeptin, and pepstatin). Total or nuclear protein samples were separated by 10% SDS-PAGE and electroblotted to nitrocellulose membranes. The membranes were blocked, incubated with anti-BNP (Chemicon International, Inc., CA, USA), anti-SR2BR, anti-nerve growth factor-β (NGFβ), anti-nuclear factor-{kappa}B (NF-{kappa}B) or anti-nucleolin (Santa Cruz Biotechnology Inc., CA, USA) antibody, washed, and incubated with horseradish peroxidase-conjugated secondary antibody. Signals were visualized by chemiluminescent detection. The purity of nuclear and cytosolic extracts was examined by Western blotting with nucleolin and actin antibodies (Santa Cruz Biotechnology Inc., CA, USA).

2.7. Measurement of rat serum serotonin by enzyme-linked immunosorbent assay (ELISA)
Serum samples were collected from the rats after abdominal aortic constriction or sham operated for different time intervals (3 days, 1, 2, 3, or 4 weeks). Serum serotonin levels were measured using enzyme-linked immunosorbent assay kits (IBL Immuno Biological Lab., Hamburg, Germany). Supernates were diluted 1:32 before assay. The lowest limit of serotonin ELISA kit is 4.1 ng/mL. Both the intra-observer and inter-observer coefficient of variance were <10%. No cross-reactivity with other catecholamines was detected.

2.8. Immunohistochemistry
Slides were dried overnight at room temperature. Snap-frozen sections were postfixed in 4% paraformaldehyde for 20 min, treated in 3% hydrogen peroxide/PBS for 25 min, blocked in 5% normal rabbit serum for 20 min, blocked with biotin/avidin for 15 min each, and incubated with the following: primary antibody (anti-BNP or anti-SR2BR antibody) for 2 h at room temperature, biotinylated rabbit-anti-mouse IgG at 1:400 for 30 min, and Vector Elite ABC biotin–avidin–peroxidase complex for 30 min; sections were then developed with diaminobenzidine and diaminobenzidine enhancer (Vector, Vector Laboratories Inc., CA, USA), counterstained with hematoxylin, and mounted.

2.9. Primary cardiac myocyte culture
Cardiac myocytes were obtained from Wistar rats aged 2–3 days old by trypsinization, as previously described [18]. Cultured myocytes thus obtained were >95% pure as revealed by observation of contractile characteristics with a light microscope and stained with anti-desmin antibody. Enriched myocytes were then subjected to cyclical stretch.

2.10. In vitro cyclical stretch on cultured cardiomyocytes
The Flexcell FX-2000 strain unit, which has been characterized and described in detail elsewhere [18], consists of a vacuum unit linked to a valve controlled by a computer program (Flexcell International Corp., NC, USA). Cardiac myocytes cultured on the flexible membrane base were subjected to cyclical stretch produced by this computer-controlled application of sinusoidal negative pressure with a peak level of {cong}15 kPa at a frequency of 1 Hz (60 cycles per min) for various periods of time. Application of the vacuum results in maximum elongation of 20% to cells at the periphery of the wells with strain declining towards the center [19]. Serotonin (Sigma-Aldrich, St. Louis, USA) or caffeic acid phenethyl ester (Calbiochem, CA, USA) was added before 30 min of stretch.

2.11. RNA interference
Neonatal cardiac myocytes were transfected with 800 ng SR2BR annealed siRNA oligonucleotide or siRNA of green fluorescent protein (GFP) (Dharmacon Inc., Lafayette, CO, USA). SR2BR siRNA is a target-specific 21 nt siRNA according to a computer program provided by Dharmacon. The SR2BR targeted base sequences were sense: 5'-GGGAAGCAUUUGGCAGGUAUU; antisense: 5'-UACCUGCCAAAUGCUUCCCUUU and negative control, GFP siRNA was used: sense: 5'-GGCUACGUCCAGGAGCGCACC; antisense: 5'-UGCGCUCCUGGACGUAGCCUU. After overnight incubation, cells were stretched and subjected to analysis by Western blotting.

2.12. Statistical analysis
All results were expressed as mean±S.E.M. Statistical significance was evaluated using analysis of variance (GraphPad Software Inc., San Diego, CA, USA). The Dunnett's test was used to compare multiple groups to a single control group. Tukey–Kramer comparison was used for pairwise comparisons between multiple groups after the ANOVA p<0.05 was considered as significant.


   3. Result
Top
 Abstract
 1. Introduction
 2. Methods
 3. Result
4. Discussion
 References
 
3.1. Adult rat heart tissue and neonatal cardiac myocyte express SR2BR
Total RNA was isolated from normal two-month-old rat heart or neonatal cardiomyocytes. After RT-PCR, the cDNA product was run on 2% agarose gel. As shown in Fig. 1A, neonatal cardiomyocyte and adult rat heart both expressed SR2B receptors. Rat stomach tissue served as positive control. The size of the PCR products for SR2BR was 240 bp and the cDNA sequence was confirmed by a DNA sequencer. These data indicated that SR2BR expressed basal level in normal non-treated Wistar rat cardiomyocyte and heart tissue.


Figure 1
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  		Fig. 1 SR2BR are expressed in untreated rat cardiomyocyte (CM), heart tissue (H). Effects of aortic constriction on SR2BR mRNA. (A) RT-PCR detection of SR2BR mRNA in cardiomyocytes and heart tissue. Stomach tissue (S) served as positive control. M=100 base-pair DNA marker. (B) Northern blot analysis for SR2BR mRNA (n=6). Values were normalized to that of the sham group. Left ventricular tissues from rats treated for different (0, 3d, 1w, 2w, 3w, 4w) aortic banding durations. *p<0.01 vs. sham.

 
3.2. Northern blot and Western blot analysis for SR2B receptor after aortic banding
After abdominal aortic constriction or sham operation for different time intervals (3 days, 1, 2, 3 and 4 weeks), we sacrificed rats and collected heart tissues for further study. SR2BR mRNA (Fig. 1B) and protein (Fig. 2A) levels both were up-regulated time-dependently as compared to sham-operated group. SR2BR mRNA and protein level both increased significantly from 2 weeks (2.8-fold vs. sham; 2.7-fold vs. sham) to 4 weeks (3.5-fold vs. sham; 3.2-fold vs. sham) after banding. We further examined hypertrophy marker expression in this banding model. Also BNP protein expression significantly increased at 2 weeks (3.0-fold vs. sham group) and NGF-β protein expression decreased from 1 week (0.7-fold vs. sham group) of aortic banding surgery. These data indicated that SR2BR mRNA and protein expression were both up-regulated in the heart after aortic constriction.


Figure 2
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  		Fig. 2 Increased SR2BR and BNP protein expression in left ventricular tissue and increased serum serotonin after aortic banding treatment. (A) Aortic banding increased SR2BR protein expression. Values were obtained from six independent experiments and were normalized to GAPDH. The SR2BR and BNP protein amount were increased at 2, 3 and 4 weeks as compared to the baseline (sham group). NGF-β decreased from 1 to 4 weeks. *,+,#p<0.05 vs. sham. (B) Serum serotonin level of banding rats. Serum serotonin increased from 1 to 4 weeks. *p<0.01 vs. sham.

 
3.3. Abdominal aortic constriction surgery increases serum serotonin
Serotonin levels in the serum were determined by ELISA. Sham rats had basal levels of serotonin in serum (42.1±5.0 ng/mL). The serotonin levels were time-dependently increased from 3 days (60.9±12.0 ng/mL) to 4 weeks (202.3±19.7 ng/mL) after aortic banding surgery (Fig. 2B). Serum serotonin level was significantly different from the first week (86.7±15.1 ng/mL) to its highest concentration after four weeks. These data demonstrated that aortic constriction increased circulating serum serotonin level in rats.

3.4. Changes in hemodynamic parameters and ventricular weights
Physical parameters after aortic constriction surgery and SR2BR antagonist (SB215505, i.p. from 1 to 3 weeks) treatment were evaluated in this rat model. Body weight was not significantly different between sham, banding of three weeks and banding rat treated with SB215505 or vehicle (DMSO) (Table 1). Heart weight, left ventricular weight and the ratio of heart weight and body weight were significantly reduced with SB215505 treatment. Left ventricular weight corrected for body weight was significantly lower in the SB treated group than in the vehicle group. Mean arterial pressure increased significantly after banding. Mean arterial pressure decreased in banding rats treated with SR2B receptor antagonist, but did not reach statistical significance as compared to banding rats without treatment. Heart rate was significantly higher in the banding group than in the sham-operated group or vehicle-injected rat. SB215505 did not attenuate heart rate significantly.


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  		Table 1 Hemodynamic data and ventricular weight

 
3.5. Left ventricular function change after banding and treatment with SR2B receptor antagonist
Echocardiographic assessments of ventricular morphology and function were performed after three weeks of abdominal aortic constriction. As shown in Table 2, interventricular septum thickness (IVST) (sham: 1.1±0.1 mm vs. banding: 2.2±0.2 mm), left ventricular posterior wall thickness (LVPWT) (sham: 1.3±0.2 mm vs. banding: 2.2±0.2 mm), and left ventricular mass (LV mass) (sham: 652±38 mg vs. banding: 1425±63 mg) significantly increased in the rat with banding compared with the sham-operated group. SB significantly reduced the increased IVST, LVPWT and LV mass of banding group. Left ventricular end-diastolic dimension, end-systolic dimension and fraction shortening were similar among the four groups.


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  		Table 2 Echocardiographic assessment of left ventricular geometry and function

 
3.6. Effects of SR2BR antagonist on BNP and NGF-β in the pressure-overloaded heart
We evaluated the effect of SR2BR subtype selective antagonist (SB215505, SB) treatment on pressure-overloaded heart. Aortic banding with SB treatment did not attenuate SR2BR protein expression (Fig. 3A), but reduced BNP protein expression in left ventricular tissues. NGF-β protein expression was reversed by SB treatment after aortic constriction. Immunohistochemical staining confirmed the previous finding from Western blots. Intensity of BNP immunoreactivity in the left ventricular myocardium increased at 3 weeks after induction of aortic banding (Fig. 3B). Banding with SB treatment decreased the immunohistochemical labeling of BNP but not in the ventricular tissue of vehicle-treated rat. Immunoreactivity of SR2BR in the left ventricular myocardium was similar between sham, banding with or without SB treatment and vehicle treatment. These results demonstrated that SR2BR antagonist treatment did not inhibit up-regulation of SR2BR protein in the left ventricle but reduced the BNP and reversed the NGF-β expression after aortic constriction, respectively.


Figure 3
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  		Fig. 3 SB215505 (SB) inhibits BNP protein expression induced by pressure overload in heart. (A) Representative Western blot analysis for SR2BR, BNP and NGF-β. Treatment with SB significantly attenuated banding-induced BNP protein expression but not SR2BR. Reduced expression of NGF-β protein by aortic banding was reversed after SB treated. Vehicle treated with DMSO did not show any effect (n=6). *p<0.01 vs. sham; +p<0.05 vs. B3w (B). Representative SR2BR and BNP immunohistochemical stainings of left ventricular free wall. At six random x200 fields, increased immunoreactive signal for BNP after 3 weeks banding (B3w) and attenuated by SB treatment (B3w+SB). Banding treated with vehicle (B3w+Ves) showed similar effects as sham operated (sham). SR2BR immunoreactive signal increased after banding but B3w did not show different immunoreactivity between B3w+SB and B3w+Ves groups.

 
3.7. Decreased SR2BR and BNP protein expression after SR2BR siRNA transfection in cardiomyocytes with in vitro mechanical stretch
To examine the possible relationship between SR2BR up-regulation and BNP expression in the cardiomyocyte, we used an in vitro mechanical stretch model. Cardiomyocytes obtained from neonatal Wistar rats were cultured on flexible membranes. After serotonin incubation (add 30 min before stretch) and different stretch duration, protein was extracted from cardiomyocyte for further analysis. Mechanical stretch without serotonin incubation did not increase SR2BR protein (Fig. 4A, upper). Serotonin 1 µM alone without the stretch did not show any effect during incubation time intervals. We tested the expression of SR2BR during mechanical stretch for 18 h at various serotonin concentrations (0, 1, 3 and 5 µM). SR2BR protein expression significantly increased at 1, 3 and 5 µM (data not shown). We added 1 µM serotonin into each culture dish 30 min before stretch for the subsequent experiments. As shown in Fig. 4B, control cardiomyocytes (0h) express baseline SR2BR protein and SR2BR increased after serotonin incubation and mechanical stretch from 6 h to 24 h (p<0.01 vs control). Mechanical stretch for 24 h did not show a different result compared to 18 h. BNP protein expression was similar to expression of SR2BR protein, which also increased. Mechanical stretch without serotonin incubation induced BNP protein significantly from 6 h (1.4-fold to control) to 24 h (2.7-fold to control). Cardiomyocytes treated with serotonin and stretch had a higher level of BNP protein.


Figure 4
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  		Fig. 4 Effects of SR2BR and BNP on neonatal cardiomyocyte during different mechanical stretch duration in vitro. SR2BR protein expressed in rat neonatal cardiomyocytes was measured after serotonin (SR) treatment with or without mechanical stretch by Western blot analysis (n=6). (A) Neonatal cardiomyocytes incubated with serotonin at different duration (0, 2 h, 4 h, 6 h, 18 h, 24 h). Treatment with serotonin alone did not up-regulate SR2BR in cardiomyocytes. Mechanical stretch for different time without serotonin incubation did not significantly induce SR2BR protein expression. (B) Treatment with mechanical stretch at different duration and added SR 30 min before stretch treatment. SR2BR and BNP protein increased from 6 h to 24 h. *p<0.01 vs. control (0 h).

 
3.8. Up-regulation of BNP protein involved in SR2BR downstream signaling via the NF-{kappa}B pathway
In order to identify the relationship between SR2BR, BNP expression and NF-{kappa}B pathway, we pre-transfected cells with SR2BR siRNA overnight or added caffeic acid phenethyl ester (NF-{kappa}B inhibitor, 15 µg/mL) half an hour before stretch for 18 h with serotonin incubation. Stretch and serotonin incubation increased NF-{kappa}B translocation into the nucleus. Caffeic acid phenethyl ester, or siSR2BR alone or together treatment significantly reduced the translocation (Fig. 5A). Caffeic acid phenethyl ester did not significantly reduce the increase of SR2BR protein but inhibited the increase of BNP protein. siSR2BR not only inhibited the increase of SR2BR expression but also attenuated the increase of down stream BNP expression. The siGFP as a negative control had no effect on SR2BR expression (Fig. 5B). These results strongly suggest that SR2BR signaling was involved in NF-{kappa}B translocation in cardiomyocytes and blockade of SR2BR or NF-{kappa}B translocation can attenuate the increase of BNP in vitro.


Figure 5
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  		Fig. 5 Mechanical stretch (MS) plus serotonin incubation induces NF-{kappa}B translocation and inhibition of SR2BR reduced translocation and BNP expression. (A) Western blot (n=5) shows NF-{kappa}B p65 in nuclear extracts from treated cardiomyocytes. The translocation was reversed by pretreatment with caffeic acid phenethyl ester (CAPE), siRNA of SR2BR (siSR2BR) or both. Western blot (n=6) in total protein lysates, caffeic acid phenethyl ester also significantly decreased BNP protein but not SR2BR. siSR2BR inhibited NF-{kappa}B translocation, SR2BR and BNP protein. (B) Transfection with siSR2BR significantly reduced the SR2BR up-regulation induced by stretch for 18 h. The siGFP had no effect on stretch induced SR2BR and BNP expression in neonatal cardiomyocytes. *p<0.01 vs. control. +p<0.05 vs. MS.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Result
 4. Discussion
References
 
The present study showed that SR2BR mRNA and protein levels can be up-regulated in the heart tissue under aortic constriction. The results also demonstrated that decrease of SR2BR in cardiomyocytes by siRNA transfection attenuated BNP protein expression through the NF-{kappa}B pathway during mechanical stretch. We suggest that up-regulation of SR2BR protein after aortic banding can at least partially increase binding of serotonin and then subsequently increase the signaling from the receptor to the myocyte. Accumulating evidence shows that serotonin does not only modulate a variety of behavioral functions but also plays a role in cardiovascular regulation [20,21]. It has been clearly demonstrated that SR2BR contribute to cardiac hypertrophy [22] and pulmonary hypertension [23]. This study used in vivo aortic banding and in vitro mechanical stretch models for identifying relationship between SR2BR signaling and BNP protein expression in cardiomyocytes.

The effects of serotonin in man have been studied since the 19th century. In clinical studies, patients with congestive heart failure have been reported to show increased serotonin plasma levels and serotonin activity [24]. The carcinoid heart syndrome also had higher levels of serotonin in serum and 5-hydroxyindoleacetic acid in urine [25]. On the other hand, many humoral factors are elevated as well as blood pressure after aortic constriction, such as angiotensin II, endothelin-1 [26]. In this study, serum serotonin was shown to be 2.5-fold elevated starting fourth week of aortic banding rat. An increase of subtype SR2BR may play a crucial role in the pressure overload. Prior studies have demonstrated that enhanced serotonin levels or short-term agonist exposure results in the desensitization of serotonin receptor in vitro [27,28]. In the present study, high levels of serotonin in rat serum induced by long-term aortic constriction did not show any desensitization of SR2BR in cardiac hypertrophy process, although other signaling pathways of SR2BR may be further studied for desensitization.

Although serotonin has been under scruting for an extensive period of time, the subtype of SR2BR in the heart and its mechanism in cardiovascular regulation are still not well understood. RT-PCR reveals that rat heart tissue and cardiomyocytes consistently express basal level of SR2BR. SR2BR has been recognized as a Gq-coupled receptor at the endothelium of vessels, aortic valve cells and cardiomyocyte [5,8,9]. Nebigil et al. showed that overexpression of SR2BR in the mouse heart induces mitochondrial proliferation associated with hypertrophy [11]. Recent studies implicate that activation of SR2BR is a limiting step in the development of pulmonary hypertension [14]. In this study, we used an aortic constriction model to induce blood pressure elevation and heart weight increase. The hemodynamic and echocardiographic data certainly support a pressure overload. The elevated BNP and decreased NGF-β protein support the presence of cardiac hypertrophy. We also demonstrated that SR2BR mRNA and protein were up-regulated in pressure overload induced cardiac hypertrophy. Past studies have shown increased SR2BR expression in thoracic arteries from hypertensive deoxycorticosterone acetate-salt rats [15]. Pulmonary artery SR2BR expression is increased during pulmonary hypertension in mice [14]. Although many studies noted a relationship between hypertension and SR2BR up-regulation in arteries, little is known about the change of SR2BR expression in the heart. The present study suggests that increased SR2BR gene expression in the heart is dependent on time after aortic banding.

SR2BR has been found as the only serotonergic receptor to be involved in cardiac proliferating cells and in neural crest-derived progenitors [29]. It has been proved that SR2BR are essential for isoproterenol-induced cardiac hypertrophy in SR2BR-knockout mice study [22]. We tried to use a selective drug to block SR2BR in order to identify its function in the pressure-overload heart. SB215505 exhibits at least 95-fold selectivity for SR2BR over SR2C receptor in the rat [30]. After 3 weeks of aortic banding and treatment of SB215505 for two weeks, SR2BR protein was not significantly reduced in heart tissue but BNP was decreased and NGF-β protein was increased. In addition, left ventricular mass, LV posterior wall thickness and interventricular septum thickness were all decreased by SB215505 treatment. We suppose that SB215505 did not influence the up-regulation of SR2BR but the blockade of SR2BR downstream signaling resulted in interrupting the hypertrophy progress.

To further determine the interaction of activated SR2BR and BNP protein expression, we used short interfering dsRNA technique to interfere with SR2BR mRNA in vitro. Research in the past few years has established physical forces to influence the structure and function of cardiomyocytes whereas excessive mechanical forces may lead to many pathophysiologic conditions and several alterations in gene expression [31]. In this study, mechanical stretch alone or serotonin incubation alone at different time intervals did not up-regulate SR2BR in rat neonatal cardiomyocytes. In previous studies, there was no strong evidence suggesting that cardiomyocytes can secrete serotonin by themselves. Therefore, adding serotonin and providing excess mechanical stretch may be similar to the serotonin in a patient who has a high serotonin level in the circulation and thereby hypertension. Mechanical stretch increased SR2BR and BNP protein expression in a time-dependent manner in cultured neonatal cardiomyocytes when incubated with serotonin. High levels of serotonin may be a cofactor of cardiac hypertrophy in cardiomyocytes of excess force treated. Cao et al. suggested that 100 µM serotonin suppress the atrial natriuretic peptide secretion in a nonbeating rat atrial model [32]. In this study, a lower concentration of serotonin (1 µM) in the treatment of cultured neonatal ventricular cardiomyocytes did not show any inhibition of BNP protein expression. On the other hand, Nebigil et al. showed that SR2BR activated NF-{kappa}B pathway via PI3 kinase/Akt in cardiomyocyte [33]. Liang and Gardner provided evidence that cardiomyocytes in mechanical stretch increased BNP promoter activity through activation of NF-{kappa}B [34]. Consistent with this concept, our results suggest that stretch can induce nuclear NF-{kappa}B translocation and BNP protein expression but both were partially inhibited by a reduced number of SR2BR. Transfection of specific siRNA for SR2BR not only decreased the number of SR2BR, but also inhibited the BNP protein expression. However, caffeic acid phenethyl ester (a NF-{kappa}B inhibitor) can decrease BNP protein expression but not SR2BR up-regulation. These results suggest that an increase of SR2BR number may augment BNP protein expression through NF-{kappa}B translocation. We added serotonin into culture medium and provided excessive mechanical force to set up an overload condition in vitro. Those data suggested that SR2BR activated by serotonin, at least partially participated in regulation of BNP protein expression via NF-{kappa}B pathway in vitro.

In summary, our data indicated that aortic banding in rats directly increases circulating levels of serotonin. The pressure overload hypertrophy in this model can be partially reversed by a SR2BR selective antagonist. Moreover, we have demonstrated clearly that SR2BR is time-dependently up-regulated in heart tissue from rat with aortic constriction and in cardiomyocytes with serotonin incubation during mechanical stretch. Serotonin activating its receptor on cardiomyocytes is known to play a key role in cardiomyopathy processes. However, in addition to SR2BR, other subtypes of serotonin receptors still need to be studied. This study suggests that SR2BR play a role in the BNP protein expression after circulating serotonin stimulation and that SR2BR expression is involved in pressure-induced cardiomyopathy and its downstream signaling may involve NF-{kappa}B pathway. These results may provide a model for the study insight of SR2BR and target on SR2BR may provide new meaningful therapies on hypertension-induced hypertrophic cardiomyopathy.


   Acknowledgement
 
This study was sponsored in part by Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.


   Notes
 
Time for primary review 19 days


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

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