B-type natriuretic peptide receptors in hypertrophied adult rat cardiomyocytes

Annales de Cardiologie et d’Angéiologie 59 (2010) 20–24

Article original

B-type natriuretic peptide receptors in hypertrophied adult rat cardiomyocytes Les récepteurs du peptide natriurétique de type B dans les cardiomyocytes hypertrophiés de rats adultes L. Nader a , L. Lahoud b , E. Chouery c , G. Aftimos d , P. Bois e , N.A. Farès a,∗ a

Laboratory of Physiology, Faculty of Medicine, University of Saint Joseph, BP 11-5076, Riad el solh, Beirut 1107 2180, Lebanon b National Institute of Pathology, Beirut, Lebanon c Laboratory of Medical Genetics, Faculty of Medicine, University of Saint Joseph, Beirut, Lebanon d National Institute of Pathology, Faculty of Medicine, University of Saint Joseph, Beirut, Lebanon e PBS CNRS UMR 6187, Laboratory of Cardiac Physiology and Physiopathology, Poitiers, France Received 8 September 2008; accepted 26 September 2009 Available online 17 October 2009

Abstract Brain natriuretic peptide (BNP) binds to three types of natriuretic peptide receptors, NPR-A, -B and -C (NPRs). The expression shape of BNP and NPRs seems to be an important modulator factor in the pathogenesis of cardiac hypertrophy. The aim of this study was to evaluate the expression of NPRs in an animal model of pressure overload hypertrophy. Left ventricular hypertrophy was induced by chronic abdominal aortic banding in adult male Wistar rats. After six weeks, NPRs gene expression was evaluated with RT-PCR, BNP plasma concentration and BNP positive myocytes were measured with ELISA and immunohistochemistry techniques respectively. NPR-A and NPR-C mRNA expression was significantly increased in left ventricular hypertrophied cardiomyocytes by 1.6-fold and 2.1-fold respectively (P < 0.01). Abdominal aortic banding increased significantly BNP plasma concentration (630 ± 8 pg/ml vs 106 ± 4 pg/ml; P < 0.01). The percentage of BNP positive cells in normal myocardial tissue were 40% while in the hypertrophied one it raised to 80%. The data suggest that in our left ventricular hypertrophy model, the NPR-A and NPR-C receptors were increased in association to the increased BNP level. This relationship may amplify beneficial paracrine/autocrine effects of BNP on cardiac remodelling in response to hemodynamic overload. © 2009 Elsevier Masson SAS. All rights reserved. Keywords: NPRs; BNP; Cardiac hypertrophy; Cardiomyocytes

Résumé Le peptide natriurétique de type B (BNP) possède trois types de récepteurs peptidiques : NPR-A, -B et -C (NPRs). Le degré d’expression du BNP et des NPRs semble être un facteur modulateur important dans la pathogenèse de l’hypertrophie cardiaque. Notre objectif est d’évaluer le profil d’expression des NPRs sur un modèle animal d’hypertrophie par surcharge de pression. L’hypertrophie ventriculaire gauche a été induite par la sténose de l’aorte abdominale chez des rats Wistars mâles adultes. Après six semaines, nous avons mesuré l’expression des gènes des NPRs par la technique RT-PCR ainsi que la concentration plasmatique du BNP et le pourcentage de myocytes BNP-positifs à l’aide des techniques Elisa et immunohistochimiques respectivement. L’expression de l’ARNm des NPR-A et NPR-C est significativement augmentée dans les cardiomyocytes hypertrophiés (1,6 et 2,1 fois respectivement ; p < 0,01). La concentration plasmatique de BNP a augmenté significativement en réponse à la surcharge de pression (630 ± 8 pg/ml versus 106 ± 4 pg/ml ; p < 0,01). Le pourcentage de cellules BNP-positives dans le myocarde normal est de 40 %, alors qu’il atteint 80 % dans le myocarde hypertrophié. Ces résultats laissent suggérer que la surexpression des récepteurs NPR-A et NPR-C dans notre modèle expérimental suit celle du BNP. Cette corrélation pourrait amplifier les effets bénéfiques paracrine–autocrine du BNP dans le remodelage cardiaque en réponse à une surcharge hémodynamique. © 2009 Elsevier Masson SAS. Tous droits réservés. Mots clés : NPRs ; BNP ; Hypertrophie cardiaque ; Cardiomyocytes ∗

Corresponding author. E-mail address: [email protected] (N.A. Farès).

0003-3928/$ – see front matter © 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ancard.2009.09.009

L. Nader et al. / Annales de Cardiologie et d’Angéiologie 59 (2010) 20–24

1. Introduction The natriuretic peptides (NPs) are a family of hormones that affect the cardiovascular and endocrine systems through their actions on diuresis, natriuresis, vasorelaxation and aldosterone and renin inhibition [1]. The NPs are potent endogenous inhibitors of hypertrophy. There are three members: atrial natriuretic peptide (ANP), brain or B-type natriuretic peptide (BNP) and C-type (CNP). In the heart, ANP expression is confined to the atria, BNP is expressed in both the atria and ventricles and the extent of CNP expression in the heart is unclear. Following their secretion, the N-terminals of NPs are cleaved to release the biologically active C-terminal derived peptide and exert their effects by binding to the natriuretic peptide receptors (NPRs), also known as guanylyl cyclase receptors. There are three receptor subtypes: NPR-A, NPR-B and NPR-C. NPRA is responsible for most of the physiological actions of BNP [2]. Binding to these receptors induces guanylyl cyclase activity and subsequent conversion of guanosine triphosphate (GTP) to the second messenger guanosine 3’, 5’-cyclic monophosphate (cGMP) [3]. NP signaling is terminated either through binding to NPR-C which internalizes and degrades NPs thus removing them from the circulation or through cleavage by neutral endoperoxidase [4–5]. BNP is mainly secreted in response to increases in ventricular wall stress like in ventricular hypertrophy and it is detectable at high concentrations in a number of circumstances which appears in cardiac ischemia and severe heart failure [6]. In addition, the expression of ANP and BNP is significantly increased in animal models of chronic hemodynamic overload [7]. Because NP signalling modulates cardiac growth and fibrosis, we investigated the effect of abdominal aorta banding in rats on the expression rate of NPRs genes expression in myocardial tissue at the level of isolated ventricular cardiomyocytes. 2. Materials and methods 2.1. Animals Male Wistar rats (n = 40) weighing 250–300 g were housed in an air-conditioned room with a 12:12-h light-dark cycle, and fed standard rat chow. During ketamine hydrochloride (75 mg/kg; Rotexmedica, Germany) and ilium xylazil (10 mg/kg; Troy laboratories PTY limited, Australia) intraperitoneal anesthesia, the abdominal aorta was exposed and a titanium clip with an inner diameter of 0.6 mm was placed (H group, n = 20) (Weck Closure Systems, Research Triangle Park, NC). Sham-operated animals (N group, n = 20) underwent the same procedures except for the placement of the clip. During the operation, the animals were kept on a surgical thermostatically controlled table at 38 ± 1 ◦ C. Animals were fed with ab libitum access to food and water. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and by the university ethics committee. The duration of pressure overload was six weeks; after that, the rats were sacrificed to remove the heart for immunohistochemical

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study or for enzymatic digestion to recover cardiomyocytes for RT-PCR. 2.2. Cell dissociation Ventricular myocytes were enzymatically dissociated by retrograde perfusion of 10 rat hearts from each group, as previously described in other studies [8]. Briefly, animals were injected with heparin and anesthetized; the heart was quickly removed via thoracotomy and transferred to an ice-cold Tyrode solution. The aorta was cannulated and the heart then mounted on the Langendorff apparatus and successively perfused (at 37 ◦ C) with the following oxygenated solutions: five minutes with the Tyrode solution to recover its spontaneous activity; four minutes with a nominally Ca2+ -free Tyrode solution, and about 20 minutes with the same solution complemented with 0.05% collagenase (Sigma, type V), 0.06 mM CaCl2 and 0.1% bovine serum albumin (BSA). When the heart was flaccid, it was rinsed with Kraft Brüh (KB) solution for two minutes. The ventricles were cut off, chopped into small pieces and gently stirred in KB medium. After that, the isolated cells were filtered and centrifuged at 250 rpm for 10 minutes in Percoll gradient (40%) in order to keep only intact cardiomyocytes, and to remove KB solution and undesirable cells. The composition of the Tyrode was (in mM): NaCl, 140; KCl, 5; CaCl2 , 1.8; MgCl2 , 1.8; HEPES, 10; D-Glucose, 10; pH 7.4 (with Tris). The KB solution contained (in mM): KCl, 70; Kglutamate, 5; KH2 PO4 , 20; MgSO4 , 5; CaCl2 , 0.08; EGTA, 0.5; creatine, 5; Na2 ATP, 5; taurine, 20; HEPES, 10; D-Glucose, 10; pH 7.2 (with KOH). 2.3. Total RNA purification and cDNA amplification Total RNA was isolated from freshly isolated ventricular cells with Trizol Reagent (Invitrogen, Life technologies, Carlsbad, CA, USA) (1 ml to 20 × 106 cells), then vortexed. The extraction of total RNA was pursued by the addition of chloroform. The total RNA were rushed to isopropanol and then washed with 75% ethanol (Sigma). The cap of total RNA obtained was resuspended in a volume of RNase-free water. The solution of total RNA was incubated at 65 ◦ C for five minutes. The integrity of the total RNA was checked by electrophoresis on an agarose gel. 2.4. Quantitation with real-time PCR Quantitative Real-time PCR analyses were performed with 7500 Real-time PCR system and the SYBR Green PCR Master mix (Applied Biosystems, Foster city, CA, USA). Following extraction of RNA, the next step is synthesis of cDNA from total RNA by reverse transcription (RT). The test of RT consists of total RNA, Random primers, dNTP and the SuperScript II Reverse Transcriptase kit (Invitrogen, Life technologies, Carlsbad, CA, USA) and RNAsin. The primers were: NPR-A: 5’-TTATCTGGAGGAGAAGCGCAA-3’, 5’GCAGAATCTGGTAAAGCAAGG-3’; NPR-B: 5’-TGGTCAGAGGCCGTATTTCC-3’, 5’-TCGTTCAGTTGTCCGGTC3’; NPR-C: 5’-GCTTCCATGATGCCATCCTC-3’, 5’-TGAG-

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CACTTCATGCAAAGCC-3’; TBP: 5’-GCTGGCCCATAGTGATCTTT-3’, 5’-CTTCACACGCCAAGAAACAGT-3’; ACTB: 5’-CCGCATCCTCCTCTTCTCT-3’, 5’-GCCGCAAGATTCCATACC-3’. All mRNAs were quantitated three times in separate analyses; the results were normalized to the content of TATA Box Protein (TBP) within the same sample [9–11]. The volume of cDNA solution obtained after reverse transcription is mixed with a volume of PCR mix that includes for a final volume of 20 ␮l PCR: 4 ␮l DEPC-treated water (H2 O DEPC), 10 ␮l SYBR Green and 0.5 ␮l of each primer R and F. The PCR reaction is carried out also in the absence of cDNA (blank H2 O) to check the specificity of the response and insure the lack of any contamination.

Fig. 1. Left ventricular expression of mRNA for natriuretic peptide receptors (NPRs) in normal (n = 10) and hypertrophied (n = 10) rats. *: P < 0.01.

2.5. Plasma BNP measurements At the time of sacrifice and under anesthesia, 1 ml of blood was collected from the inferior vena cava of each rat in both groups and immediately centrifuged at 3000 revolutions per minute for 15 minutes. The plasma supernatant was recovered and kept at −80 ◦ C until the date of BNP concentration measurement by the ELISA technique using a specific BNP kit (IBL immunobiological laboratories, Hamburg). 2.6. Immunohistochemistry After sprawl, deparaffinization and drying of the 3 ␮m thick left ventricle sections of hearts, primary antibody antiBNP (Bachem, Switzerland) diluted 1:600 was incubated for 32 minutes at room temperature and then was applied the biotinylated anti-rabbit immunoglobulins (secondary antibody) for five minutes. Evaluation of immunohistochemistry findings was carried out by a pathologist blind to hemodynamic parameters and plasma BNP values. To quantify the immunostaining, BNP positive myocytes were counted in 20 random high-power fields (× 400) in each specimen, and the percentage of positive cells on total count of myocytes was calculated. 2.7. Statistics The average results (± SE) are statistically compared using One Way Anova Test followed by Mauchly test. Post-hoc analyses were done by Bonferroni method. All statistics were done with SPSS Software. P < 0.05 was considered significant.

Table 1 Effect of abdominal aortic banding on heart weight. Values are means ± SE for n = 20 rats on each group.

Normal (n = 20) Hypertrophied (n = 20)

BW (g)

HW (mg)

HW/BW (mg/g)

496 ± 7 435 ± 4

1259 ± 10 1818 ± 18

2.53 ± 0.04 4.17 ± 0.08*

AAS: abdominal aortic stenosis; HW: heart weight; BW: body weight. * P < 0.001.

3.2. Effect of pressure overload on heart weight and BNP plasma concentration The ratio of heart weight to body weight was significantly increased, approximately 64%, (P < 0.001; Table 1) after six weeks in rats with abdominal aortic banding compared with the N group. Cardiac hypertrophy was accompanied, as expected, with a significant increase in BNP plasma concentration by about six fold compared to the N group (Fig. 2) (630 ± 8 pg/ml vs 106 ± 4 pg/ml; P < 0.01). 3.3. Effect of pressure overload on rate expression of BNP in left ventricular myocardium Immunohistochemistry with anti-BNP antibodies showed the presence of BNP granules in both normal and hypertrophied left ventricular myocardium biopsies. However, the immunoreactivity and the intensity of staining were more pronounced in

3. Results 3.1. Effect of pressure overload on expression of genes for natriuretic peptides In cardiomyocytes isolated from left ventricular myocardium of H group, the rate expression of the mRNA NPR-A was significantly increased (P < 0.01) by 1.6-fold and that of NPR-C mRNA by 2.1-fold compared to the N group (Fig. 1). On the other hand, the expression rate of NPR-B mRNA was similar to the N group (1.1-fold) (Fig. 1).

Fig. 2. BNP plasma concentration in normal and hypertrophied rats. Values are means ± SE for n = 20 rats on each group. *: P < 0.01.

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Fig. 3. Immunohistochemistry with antibrain natriuretic peptide antibodies in left ventricular myocardium biopsy from group N (n = 10) (× 40). BNP positive myocytes are stained in brown (grey in the figure).

Fig. 4. Immunohistochemistry with antibrain natriuretic peptide antibodies in left ventricular myocardium biopsy from group H (n = 10) (× 40). BNP positive myocytes are stained in brown (grey in the figure).

the H group in comparison to the N group; the percentage of BNP positive cells in normal myocardial tissue was estimated to be 40% (see Materials and Methods) (Fig. 3) while that in the hypertrophied myocardium was more significantly greater, 80% (Fig. 4). Furthermore, there was a significant correlation between the extent of myocardial fibrosis and the percentage of BNP positive myocytes in H group (data not shown). 4. Discussion In the present study, abdominal aorta banding in rats was used to investigate the correlation between the expression of myocardial BNP and NPRs gene regulation during left ventricular hypertrophy after pressure overload by abdominal aortic stenosis. The major findings were 160% and 210% increases in NPR-A and NPR-C gene expression, respectively in response to cardiac wall stress that follows the increase of BNP myocardial synthesis in the left hypertrophic ventricle. Hypertrophy and heart failure are almost always associated with an increased BNP concentration in the blood [12–14]

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putting this peptide as the biological marker of cardiac hypertrophy. In our experimental model, the plasma BNP concentration is elevated and correlates positively with left ventricular BNP synthesis in hypertrophied hearts where BNP granules positive cells invades left ventricular myocardium. This increase may be regarded as a tentatively compensatory mechanism aimed to reduce ventricular filling pressure, wall tension and unfavorable remodeling [15]. The finding of increased cardiac NPR-A mRNA expression is compatible with previous reports of increased NPR-A expression in hypertensive rats [16] and in rats in which the hearts were volume-overloaded by an aortocaval shunt [17,18]. It should be noted that steady-state mRNA levels were measured and that the results may reflect increased gene transcription, decreased mRNA degradation, or a combination of both mechanisms. NPR-A is expressed mainly in myocytes and fibroblasts [19] but is also found in cardiac endothelial cells [20,21]. Genetic ablation of the NPR-A gene in cardiac myocytes of mice results in cardiac hypertrophy as well as fibrosis from birth [22]. In addition, it was recently reported that targeted overexpression of a dominant-negative NPR-A receptor in the suprarenal aortabanded mouse heart results in increased cardiac hypertrophy and fibrosis [23]. The abdominal aorta-banded rat heart is also characterized by hypertrophy and fibrosis [8]. Although increased gene expression does not necessarily convey a higher concentration of the protein, it is conceivable that the observed increase in NPR-A mRNA expression reflects increased production of the NPR-A protein and that it participates in modulation of the myocardial response to pressure overload. We also observed an increase in NPR-C mRNA expression after abdominal aortic banding. Cardiac expression of the NPR-C gene has been assigned to the same cells as the NPR-A gene, i.e., myocytes, fibroblasts, and endothelial cells [19]. NPR-C has been proposed to regulate tissue levels of the NPs [24], and changes in NPR-C gene expression may thus have important physiological consequences in the pressureoverloaded left ventricle. The finding of upregulation of NPR-C mRNA expression by pressure overload is in accordance with previous studies [16]. Increased NPR-C gene expression may thus dampen the increased production of ANP and BNP in the pressure-overloaded left ventricle. It may seem contradictory that both NPR-A and NPR-C are upregulated in aorta-banded rats, when they may have opposing effects. However, NPR-C may confer signalling in addition to its proposed role as a clearance receptor [2]. There now exists a wealth of experimental evidence that indicates NPR-C is coupled to an inhibitory heteromeric G protein, Gi , and cause inhibition of adenylyl cyclase and activation of phospholipase-C. Both the ␣ and ␤␥ subunits of this Gi protein mediate a number of important physiological effects in the heart and vessels such as cardiac protection against ischemia and hypertrophy by increasing myocardial perfusion and optimize heart metabolism [25]. It is expected that new roles and functional implications for NPR-C signalling will continue to be elucidated. Gene expression of NPR-B was similar to the sham group after aortic stenosis, as a response to pressure overload. Time dependent changes in gene expression may reflect ongoing

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myocardial remodelling involving NPR-B gene expression during the cardiac response to pressure overload. In conclusion, in our left ventricular hypertrophy model, the NPR-A and NPR-C receptors were increased in association to the increased BNP level. Increased number of NPR-A receptors may amplify beneficial paracrine/autocrine effects of BNP on the heart, whereas increasing expression of NPR-C may counteract this by enhancing the removal of the NPs. This relationship may be important in cardiac remodelling in response to hemodynamic overload. Acknowledgments This work was supported by grants from the University of Saint Joseph, Beirut, Lebanon. We thank Nadine Jalkh for excellent technical assistance and the staff at the Department for genetics and histopathology at the University of Saint Joseph and National Institute of Pathology respectively. References [1] Nishikimi T, Maeda N, Matsuoka H. The role of natriuretic peptides in cardioprotection. Cardiovasc Res 2006;69:318–28. [2] Barry SP, Davidson M, Townsend PA. Molecular regulation of cardiac hypertrophy. Int J Biochem Cell Biol 2008;40:2023–39. [3] Gardner DG, Chen S, Glenn DJ, Grigsby CL. Molecular biology of the natriuretic peptide system: implications for physiology and hypertension. Hypertension 2007;49:419–26. [4] Gardner DG. Natriuretic peptides: markers or modulators of cardiac hypertrophy? Trends Endocrinol Metab 2003;4:411–6. [5] Richards AM. Natriuretic peptides: update on peptide release, bioactivity, and clinical use. Hypertension 2007;50:25–30. [6] Kailash NP. Biology of natriuretic peptides and their receptors. Peptides 2004;26:901–32. [7] Gu J, D’Andrea M, Seethapathy M, McDonnell C, Cichon R. Physical overdistension converts ventricular cardiomyocytes to acquire endocrine property and regulate ventricular atrial natriuretic peptide production. Angiol 1991;42:173–86. [8] Nader L, Smayra V, Jebara V, Bois P, Potreau D, Farès N. Brain natriuretic peptide secretion in adult rat heart muscle cells: the role of calcium channels. Arch Cardiovasc Dis 2008;101:459–63. [9] Christoffersen C, Goetze JP, Bartels ED, Larsen MO, Ribel U, Rehfeld JF, et al. Chamber-dependent expression of brain natriuretic peptide and its mRNA in normal and diabetic pig heart. Hypertension 2002;40:54–60. [10] Goetze JP, Christoffersen C, Perko M, Arendrup H, Rehfeld JF, Kastrup J, et al. Increased cardiac BNP expression associated with myocardial ischemia. FASEB J 2003;17:1105–7.

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