logy 559 (2007) 89–97www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Vasopressin promotes cardiomyocyte hypertrophy via thevasopressin V1A receptor in neonatal mice

Masami Hiroyama a,1, Shuyi Wang a,1, Toshinori Aoyagi a,1, Ryo Oikawa b,1, Atsushi Sanbe a,Satoshi Takeo b, Akito Tanoue a,⁎

a Department of Pharmacology, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japanb Department of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received 14 August 2006; received in revised form 7 December 2006; accepted 8 December 2006Available online 29 December 2006

Abstract

[Arg8]-vasopressin (AVP) is an essential hormone for maintaining osmotic homeostasis and is known to be a potent vasoconstrictor thatregulates the cardiovascular system. In the present study, cardiomyocytes were isolated from neonatal mice and used to investigate the effects ofAVP on cardiac hypertrophy. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that vasopressin V1A receptor mRNA,but not V1B or V2 receptor mRNA, was expressed in primary cultured neonatal mouse cardiomyocytes. By exposing the cultured neonatalcardiomyocytes to AVP for 24 h, cell surface areas were significantly increased, suggesting that AVP could induce cardiomyocyte growth. We theninvestigated the expression level of the atrial natriuretic peptide (ANP), which is a marker of cardiac hypertrophy. Stimulation with AVP increasedthe expression of cardiomyocyte ANP mRNA in a dose- and time-dependent manner. Immunocytochemical studies showed that stimulation withAVP significantly increased the expression of the ANP protein as well. Furthermore, AVP administration activated extracellular signal-regulatedkinase (ERK)1/2 in cardiomyocytes. The effects of AVP on these parameters were significantly inhibited by a selective vasopressin V1A receptorantagonist, OPC-21268, and were not observed in cardiomyocytes from mice lacking the vasopressin V1A receptor. In vivo cardiac hypertrophy inresponse to pressure overload was attenuated in vasopressin V1A receptor-deficient (V1AR-KO) mice. Taken together, our data suggest that AVPpromotes cardiomyocyte hypertrophy via the vasopressin V1A receptor, which is in part regulated by the pathway of ERK1/2 signaling.© 2006 Elsevier B.V. All rights reserved.

Keywords: Vasopressin; Hypertrophy; Cardiomyocyte; Atrial natriuretic peptide (ANP)

1. Introduction

[Arg8]-vasopressin (AVP), the neurohypophyseal hormone,is involved in important physiological functions, such asosmotic regulation, vasoconstriction, and adrenocorticotropichormone (ACTH) release. These physiological effects aremediated through the binding of AVP to specific membranereceptors in the target cells. AVP receptors are coupled with theG protein and have been divided into at least three types (V1A,V2, and V1B, also termed V3). All of these receptors have beencloned (de Keyzer et al., 1994; Lolait et al., 1992; Morel et al.,1992) and belong to the family of seven membrane spanning

⁎ Corresponding author. Tel.: +81 3 5494 7058; fax: +81 3 5494 7057.E-mail address: atanoue@nch.go.jp (A. Tanoue).

1 Authors that equally contributed to this work.

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.12.010

receptors, which signal through G proteins (Thibonnier et al.,2002). The vasopressin V1A (vascular/hepatic) and V1B

(anterior pituitary) receptors act through phosphatidylinositolhydrolysis to mobilize intracellular Ca2+ (Jard et al., 1986). Thevasopressin V1A receptor mediates physiological effects, suchas cell contraction and proliferation, platelet aggregation,coagulation factor release, and glycogenolysis. The vasopressinV1B receptor exists in the anterior pituitary, where it stimulatescorticotrophin release (Tanoue et al., 2004). The vasopressin V2

receptors are found primarily in the kidney. They are linked toadenylate cyclase and the production of cAMP and areassociated with antidiuresis (Thibonnier, 1988).

AVP is a potent antidiuretic, vasoconstricting, and growth-stimulating neuropeptide that contributes to the pathogenesis ofhypertension and heart failure (Schrier and Abraham, 1999).Patients with heart failure have chronically elevated plasma

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vasopressin concentrations, which may contribute to theirclinical syndrome of fluid retention (Sanghi et al., 2005).Furthermore, AVP has adverse effects in heart failure andincreasing peripheral resistance via constrictor actions at thevasopressin V1A receptor (Sanghi et al., 2005; Yatsu et al.,1999). Thus, blockade of the vasopressin V1A receptor could bea useful adjunct to standard therapy in congestive heart failure.

In addition to their involvement in the pathogenesis of heartfailure, AVP as well as other neurohormonal factors, such ascatecholamines, angiotensin II, and endothelin-1 (ET-1), mayplay important roles in the development of cardiac hypertrophyin symptomatic congestive heart failure by increasing peripheralvascular resistance and cardiac afterload (Cowley et al., 1981;Kawano et al., 1997; Riegger and Liebau, 1982). Catechola-mines, angiotensin II, and ET-1 can induce cardiac hypertrophyin the cardiomyocytes and directly affect cardiac remodeling(Aceto and Baker, 1990; Ito et al., 1991; Simpson, 1985). AVPalso could induce cardiac hypertrophy (Nakamura et al., 2000).However, it has not been clarified whether AVP promoted thecardiomyocyte hypertrophy by increasing vascular resistance ordirectly stimulated cardiomyocytes.

Several studies have shown that AVP increases the rate ofprotein synthesis in cardiomyocytes and heart in rats and humans,suggesting that AVP might be a promoter of ventricularhypertrophy. However, little is known about AVP-inducedventricular hypertrophy in mice. An insight into the mechanismsregulating the hypertrophy of cardiomyocytes may provide cluesfor the treatment of patients with heart failure. Recently, wedeveloped a mutant mouse lacking the vasopressin V1A receptor,which is considered to be a good model for analyzing themechanism of cardiomyocyte hypertrophy by AVP stimulation.The current study was conducted to determine whether AVPinduces the hypertrophy of cardiac myocytes, and, if so, todetermine whether the vasopressin V1A receptor is involved inthese effects.

2. Materials and methods

2.1. Materials and animals

[Arg8]-vasopressin (AVP) and endothelin-1 (ET-1) werepurchased from the Peptide Institute, Inc. (Osaka, Japan). Thevasopressin V1A receptor antagonist, OPC-21268 (1-{1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-quinolinone), was a gift from the Otsuka Pharmaceutical Co.(Tokyo, Japan). U0126, an ERK inhibitor, was from PromegaCo. (Madison, USA). The anti-α-actinin antibody was pur-chased from Sigma (St. Louis, USA). Phospho-ERK1/2 andERK1/2 antibodies were purchased from Cell SignalingTechnology (Beverly, USA). The atrial natriuretic peptide(ANP) was from Santa Cruz Biotechnology (Santa Cruz, USA).Vasopressin V1A receptor-deficient (V1AR-KO) mice weregenerated by homologous recombination, as described pre-viously (Koshimizu et al., 2006; Egashira et al., 2004). Non-vasopressin V1A receptor-deficient littermates were used as wildtype control subjects for V1AR-KO mice. Cardiomyocytes wereisolated from one-day-old wild type or V1AR-KO mice and

cultured for experiments. Mice were maintained undercontrolled conditions at 23 °C with food and water availablead libitum. The experiments were approved by the committeeon the ethical aspects of research involving animals of theNational Research Institute for Child Health and Development.

2.2. Cell culture

Primary ventricular myocytes were prepared as describedpreviously (Gray et al., 1998). Ventricles from one-day-old wildtype and V1AR-KO mice were minced, and cells were isolatedby multiple rounds of 8-min-long tissue dissociation with0.01% trypsin. After incubation with trypsin, the supernatantwas added to an equal volume of Dulbecco's modified Eagle'smedium containing 10% fetal bovine serum, and all of thesupernatants were combined. The cardiomyocytes were col-lected by different adhesiveness. Cardiomyocyte-enrichedsuspensions were removed from the culture dishes and platedat a density of 150–200 cells/mm2 after overnight incubation.Cultures were maintained in Dulbecco's Modified Eagle'smedium supplemented with 10% fetal bovine serum and0.1 mM bromodeoxyuridine to prevent fibroblast proliferation.

2.3. Protein, DNA, and RNA content in cardiomyocytes

Cardiomyocytes were plated on six-well plates and cultured ina serum-free condition for 24 h before experiments. Cardiomyo-cytes were treated with 0.1 μMAVP and further cultured for 24 h.After stimulation with AVP, each dish was rinsed three times withice-cold phosphate-buffered saline. The cell layer was scrapedfrom each dish with 1 ml standard sodium citrate (200 mM, pH4.8) containing 0.25% (wt/vol) sodium dodecyl sulfate and frozenat −80 °C until the assay. The extracts were thawed and vortexedextensively. The 100 μl extract was used for the protein assayaccording to the instructions for use of a bicinchoninic acidprotein assay kit (PIERCE, Rockford, USA). DNA was isolatedfrom 200 μl extracts by the phenol–chloroform method andmeasured using a spectrometer (BioSpec Mini analyzer, Shi-madzu Co., Kyoto, Japan). RNAwas isolated from 100 μl extractsaccording to the instructions for the use of a mini-RNA kit(Qiagen, Valencia, USA) and measured using the spectrometer.

2.4. RT-PCR analysis

For the RT-PCR analysis, total RNA was extracted fromcultured neonatal myocytes. The samples were treated withDNase and then subjected to first-strand synthesis using an oligo(dT) primer and reverse transcriptase (Superscript II, Invitrogen,Carlsbad, USA). The following mouse V1A, V1B, and V2 receptorprimers were used: V1A, 5′-CCGTGCTGGGTAATAGCAGT-3′and 5′-TCTTCACTGTGCGGATCTTG-3′ ; V1B, 5′-TACAGGTGCTCAGCATGTTT-3′ and 5′-ATTCTCGTCCCA-CACAGACC-3′; and V2, 5′-TGTGGCTCTGTTTCAAGTGC-3′ and 5′-CACAGCTGCACAAGGAAGAA-3′. Mouse ANPand GAPDH mRNAwere amplified by PCR using the followingprimers: mouse ANP, 5′-GGGGGTAGGATTGACAGGAT-3′and 5′-CAGAGTGGGAGA GGCAAGAC-3′; and mouse

Fig. 2. Effects of AVP on the protein, DNA, and RNA contents were determined.Cardiomyocytes were plated at a density of 150–200 cells/mm2 and incubated ina serum-free medium, and AVP was added to a final concentration of 100 nM for24 h. The ratios of total protein to DNA (A) and RNA to DNA (B) weremeasured. Values are the means±S.E.M. from 3 to 4 dishes in four differentpreparations. ⁎, significant differences (P<0.05) from the value of the wild typecontrol.

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GAPDH, 5′-CAATCACCATCTTCCAGGAG-3′ and 5′-TTCAGCTCTGGGATGACCTT-3′. Thermal cycling was per-formed for 1 min at 95 °C, 30 s at 60 °C, and 30 s at 72 °C for25 cycles with an initial denaturation at 95 °C for 5 min and a finalextension step at 72 °C for 7min.After amplification, the productswere analyzed by electrophoresis on a 1.5% agarose gel.

2.5. Immunocytochemistry

Cardiomyocytes were prepared for immunocytochemicalstaining as described previously (Taigen et al., 2000). For α-actinin and ANP staining, cardiomyocytes were cultured ongelatin-precoated glass covers for 24 h and then free serum for24 h and fixed by 4% paraformaldehyde for 5 min aftertreatment with 0.1 μM AVP and diluted water. The glass coverswere incubated with antibodies to α-actinin (1:500) and ANP(1:100) overnight at 4 °C followed by Alexor Fluor 594 anti-mouse antibody and anti-rabbit antibody at a dilution of 1:500.The quantification of ANP expression was performed bycounting the ANP expression in 100 cardiomyocytes.

2.6. Cardiomyocyte surface area

The cardiomyocyte surface area was measured on α-actinin-stained cardiomyocytes by the method of Simpson (Simpson,1985). Cell images, which were viewed using a video camera(Nikon Co., Tokyo, Japan) fixed to a microscope (Nikon Co.,Tokyo, Japan), were projected onto a monitor and traced. NIHimage analysis software directed the computation of thecardiomyocyte area. The area was then doubled to accountfor the surface portion in contact with the dish. All cells fromrandomly selected fields in 2 or 3 wells were examined foreach condition. We measured 100 cells in each condition. Thecardiomyocyte area was determined after 24 h of treatmentwith AVP (1 μM) and ET-1 (1 μM), in contrast to the controlcells, which were treated with their diluents (diluted water).

2.7. Western blot analysis

Protein was extracted from the cultured cardiomyocytes afterstimulation with AVP and ET-1 for 30 min. The cells were lysed

Fig. 1. Expression of the AVP receptor mRNAwas determined by RT-PCR usingspecific primers in primary cultured cardiomyocyte isolated from wild type orV1AR-KO mice. The amplified PCR products for V1A, V1B, V2, and GAPDHwere 696-, 581-, 618-, and 475-bp in size, respectively. The expression ofGAPDH was measured as an internal control to evaluate sample-to-sampledifferences in RNA concentration.

in a lysis buffer (100 mM NaCl, 50 mM Tris-HCl (pH 7.6),10 mM β2-mercaptoethanol, 1 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride (PMSF), and 0.25% TritonX-100). Theconcentration of whole protein was measured using thebicinchoninic acid protein assay kit. Protein lysates (20 μg)were analyzed by 12.5% sodium dodecyl sulfate polyacryla-mide gel electrophoresis (SDS-PAGE) gels, electrotransferredto a polyvinylidene difluoride (PVDF) membrane (Bio-RadLaboratories, Hercules, USA), and probed with antibodies tophospho-ERK1/2 (1:1000) and ERK1/2 (1:1000), followed byappropriate horseradish peroxidase (HRP)-conjugated second-ary antibodies. The peroxidase reaction products were visua-lized using a LumiGLO chemiluminescent substrate (NewEngland Lab, Woburn, USA). The same membrane was thenstripped and re-probed with the anti-ERK1/2 antibody todetermine the total protein abundance using a similar procedure.

2.8. In vivo hypertrophic response

In order to determine the hypertrophic response in vivo,V1AR-KO mice were subjected to a thoracic aortic constriction(TAC) surgery as described previously (Rockman et al., 1991).Eight-to 12-week-old male mice from either genotype were

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anesthetized with a ketamine/xylazine cocktail (100 mg/kg and10 mg/kg, respectively, i.p.) and connected to a rodent ventilatorafter endotracheal intubation. The aorta was partially ligatedwith 7-0 silk around a 27-gauge blunted needle, which wassubsequently removed to generate a calibrated constriction. Todetermine the hemodynamic overload imposed on the leftventricle, the systolic pressure gradient between right carotidand systemic arterial pressures was measured under pentobar-bital anesthesia (40 mg/kg, i.p.) one week after the TAC surgery.After measurement of the pressure gradient, the heart wasisolated, and the heart weight was measured.

2.9. Statistical analysis

All values were expressed as the means±S.E.M. Statisticalsignificance was determined using the unpaired Student's t-test

Fig. 3. Effects of AVP on primary cultured cardiomyocytes. Cardiomyocytes were pla1 μM AVP and endothelin-1 (ET-1) stimulation. (A) Cardiomyocyte immunostamorphological hypertrophy. (B) Surface areas of cardiomyocytes were quantified froareas were calculated using NIH image software (n=100 cells each). Data were precontrols. ⁎⁎⁎, significant differences (P<0.001) from the value of the wild type con

or one-way analysis of variance. The results were considered tobe significant when P<0.05.

3. Results

3.1. Expression of vasopressin receptor mRNA in culturedcardiomyocytes from neonatal mice

To determine whether vasopressin receptors are involved inthe response of cardiomyocytes to AVP, we first examined theexpression of three kinds of vasopressin receptor subtypes inprimary cardiomyocytes isolated from neonatal mice. We foundthat the vasopressin V1A receptor mRNA was expressed in theprimary cardiomyocytes and did not detect the expression ofV1B and V2 receptor mRNA (Fig. 1). Therefore, V1AR-KO micewere useful to understand the AVP effects on cardiomyocytes.

ted on glass covers and immunostained with the α-actinin antibody after 24 h ofined with α-actinin antibody (red). The cells stimulated with AVP revealedm different treatment cultures. α-actinin-stained cells were imaged, and surfacesented as a percentage change of experimental groups compared with untreatedtrol. OPC: OPC-21268.

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mRNAs from the kidney and pituitary were used as positivecontrol for V2 and V1B, respectively.

3.2. Changes of total protein, DNA, and RNA components onprimary cardiomyocytes after treatment with AVP

To determine the effects of AVP on the total protein, DNA,and RNA content, we used cultured cardiomyocytes fromneonatal wild type and V1AR-KO mice. These cardiomyo-cytes were cultured under a serum-free condition for 24 hbefore the experiments, treated with AVP, and further culturedfor 24 h. After stimulation with AVP, the protein-to-DNAratio and RNA-to-DNA ratio were measured. The protein-to-DNA ratios in AVP-treated cardiomyocytes were significantlyhigher than those of the vehicle-treated controls (Fig. 2A).The RNA-to-DNA ratio was significantly increased afterexposure to AVP (Fig. 2B). The total DNA concentration wasnot altered in each treated cell. These findings indicated thatAVP increased the protein synthesis that led to theacceleration of cardiomyocyte growth. In order to determinewhether AVP increased protein synthesis via the vasopressinV1A receptor, we next treated the cardiomyocytes from wildtype neonatal mice with OPC-21268, a vasopressin V1A

receptor-selective antagonist. The protein-to-DNA ratios andRNA-to-DNA ratios were not increased in the cardiomyocytesfrom the wild type neonatal mice by treatment with OPC-21268 for 15 min before AVP administration (Fig. 2A and B).Similarly, the protein-to-DNA and RNA-to-DNA ratios werenot increased in the cardiomyocytes from the V1AR-KOneonatal mice by exposure to AVP (Fig. 2A and B), suggestingthat AVP could promote cardiomyocyte growth via thevasopressin V1A receptor.

3.3. Increase in cardiomyocyte areas by exposure to AVP

Because cardiac hypertrophy is characterized by theenlargement of cardiomyocytes, we examined the influenceof exposure to AVP on cardiomyocyte surface areas. Thecardiomyocyte surface areas were significantly increased in thecultured cardiomyocytes exposed to AVP (1 μM) for 24 h butnot in the cardiomyocytes from the V1AR-KO neonatal mice(Fig. 3). In addition, the increase in cardiomyocyte surfaceareas was inhibited by pre-administration of the vasopressinV1A receptor antagonist, OPC-21268, in the cultured cardio-myocytes from the wild type neonatal mice (Fig. 3). Theseresults suggest that AVP can promote cardiomyocyte hyper-

Fig. 4. The effects of AVP on ANP mRNA expression on neonatal mousecardiomyocytes were determined by semi-quantitative RT-PCR analysis. (A)ANP mRNA increased in a dose-dependent manner in cardiomyocytes derivedfrom wild type mice after stimulation with 1, 10, and 100 nM AVP for 24 h. (B)ANP mRNA increased in a time-dependent manner in the cardiomyocytesderived from wild type mice after stimulation with 100 nM AVP for 12, 24, and48 h. (C) ANP mRNA expression in the cardiomyocytes derived from wild typeor V1AR-KO mice after stimulation with 100 nM AVP for 24 h. The results wereaveraged from three independent experiments. ⁎ and ⁎⁎, significant differences(P<0.05 and P<0.01, respectively) from the value of the wild type control.

trophy through the vasopressin V1A receptor. Another hyper-trophic agonist, ET-1, was used as a positive control. ET-1significantly increased the cell surface areas from wild typeand V1AR-KO mice, but OPC-21268 did not affect the cellsurface areas (Fig. 3).

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3.4. Expression of ANP mRNA and protein translation afterAVP stimulation

We further characterized AVP-induced cardiomyocytehypertrophy by examining mRNA and protein expression ofANP, a marker for cardiac hypertrophy. The expression of ANPmRNAwas increased in a dose- and time-dependent manner inthe cardiomyocytes stimulated with AVP (Fig. 4A and B).Immunocytochemical results revealed that AVP also inducedthe expression of the ANP protein in the perinuclear regions ofthe cell, as demonstrated by the appearance of a circular band atthe nuclear membrane (Fig. 5). Approximately 70–85% of theAVP-stimulated cells under 20-times magnification exhibitedthe expression of ANP around the perinuclear regions.Pretreatment with the vasopressin V1A receptor antagonist,OPC-21268, inhibited the expression of ANP mRNA andprotein (Figs. 4C and 5). Similarly, no ANP mRNA or proteininduction was observed in the cardiomyocytes from the V1AR-KO mice (Figs. 4 and 5).

3.5. AVP induced ERK1/2 phosphorylation

Previous results have suggested that the Ras-Raf-1-Erk1/2signaling pathway plays an important role in cardiomyocytehypertrophy (Molkentin and Dorn, 2001; Wang and Proud,

Fig. 5. Effects of AVP on ANP protein expression. Cardiomyocytes were plated on100 nM AVP for 24 h. (A) The ANP (red)-immunostained cardiomyocytes revealed hphotography. (B) Percentage of cardiomyocytes expressing ANP protein (n=100independent experiments from A–B. ⁎⁎, significant differences (P<0.01) from the

2002). To analyze the signaling pathway after vasopressin V1A

receptor activation, we examined changes in ERK1/2 phos-phorylation by Western blot analysis. Our results showed thatAVP induced ERK1/2 phosphorylation, which was inhibited bypretreatment with OPC-21268 (Fig. 6A). Similarly, AVP couldnot induce ERK1/2 phosphorylation in the cardiomyocytesfrom V1AR-KO neonatal mice (Fig. 6A). ET-1 also enhancedERK1/2 phosphorylation, but OPC-21268 had no effect on thephosphorylation by ET-1 and the basal level (Fig. 6B).Furthermore, ERK inhibitor, U0126, was used to ensure therole of ERK in AVP-induced hypertrophy. U0126 inhibitedAVP- and ET-1-induced cardiomyocyte hypertrophy (Fig. 6Cand D). These results suggest that AVP induces cardiomyocytehypertrophy via ERK phosphorylation.

3.6. In vivo hypertrophic response

All our results using isolated cardiomyocytes indicate thatthe AVP can induce cardiac hypertrophy via the vasopressinV1A receptor. To extend these findings to in vivo studies, weperformed TAC surgery. The heart/body weight ratio in V1AR-KO mice after the TAC surgery was lower than that in wild typemice (Fig. 7), whereas the pressure gradient was similarbetween V1AR-KO and wild type mice (wild type mice; 106±8 mmHg, V1AR-KO mice; 132±7 mmHg). The results indicate

glass covers and immunostained with the ANP antibody after stimulation withypertrophical ANP expression. Perinuclear ANP expression is shown in enlargedcells, 25 fields at 200× magnification). The results were averaged from threevalue of the wild type control.

Fig. 6. AVP induces cardiomyocyte hypertrophy via ERK phosphorylation. (A) The cultured cardiomyocyte cells were pretreated with or without 1 μM OPC-21268for 30 min and stimulated with 0.1 μM AVP for 30 min. (B) The cells were pretreated with or without 1 μM OPC-21268 for 30 min and stimulated with 0.1 μMendothelin-1 for 30 min. Whole-cell lysates were immunoblotted with anti-phospho-ERK. Membranes were stripped and re-probed using the anti-ERK antibody.Three independent experiments produced similar results. (C) The cells were treated with 1 μMAVP or 1 μM endothelin-1 (ET-1) with or without the pretreatment with10 μMU0126. The cells were then stained with anti-actinin antibody. (D) Surface areas of cardiomyocytes were quantified (n=100 cells each). Data were presented asa percentage change of experimental groups compared with untreated controls. ⁎⁎⁎, significant differences (P<0.001) from the value of the wild type control.

Fig. 7. Heart/body weight ratio 1 week after thoracic aortic constriction (TAC)surgery. The heart weight in V1AR-KO mice was lower than that of wild typemice after the TAC surgery. ⁎, significant differences (P<0.05) from the value ofwild type mice.

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that the AVP-V1A receptor signaling pathway plays a significantrole in cardiac hypertrophy in response to pressure overload.

4. Discussion

AVP is a peptide hormone that modulates a number ofprocesses implicated in the pathogenesis of heart failure(Krismer et al., 2004). Through the activation of vasopressinV1A and V2 receptors, AVP regulates various physiologicalprocesses, including bodily fluid regulation, vascular toneregulation, and cardiovascular contractility (Krismer et al.,2004). Vasopressin V1A receptors are expressed on bothvascular smooth muscle cells and cardiomyocytes and havebeen shown to modulate blood vessel vasoconstriction andmyocardial function in rats and humans (Penit et al., 1983;Thibonnier et al., 1994). In the present study, we found thatvasopressin V1A receptors, but not V1B and V2 receptors, wereexpressed in the neonatal mouse heart and investigated AVP-induced cardiomyocyte hypertrophy with cultured cardiomyo-cytes from the V1AR-KO and wild type neonatal mice.

AVP is implicated in the stimulation of myocardial cellhypertrophy by enhancing protein synthesis and cellular growthwithout affecting cell division in neonatal rat cardiomyocytes(Nakamura et al., 2000; Tahara et al., 1998). Similar to theobservation in rats, our results also showed that AVP increasedthe protein synthesis and cell surface areas of cardiomyocytes inneonatal mice. Since there was no cell division, as indicated bythe stable DNA content, the AVP-induced growth response was

probably cardiomyocyte hypertrophy. At the same time, wefound that AVP stimulation increased the expression of ANP.ANP is mainly synthesized in the atria of the normal adult heart,but a small amount of ANP mRNA has also been found in thenormal adult ventricular myocytes, where the expression level isapproximately 0.5 to 3% of that in the atria (Ruskoaho, 1992).The expression of atrial ANP is initially low and increasesduring development. In contrast to the low expression of ANPin the neonatal atria, ventricular myocytes from neonatal heartscontain substantial amounts of ANP mRNA, which declinerapidly after birth (Ruskoaho, 1992). The ventricular expressionand secretion of ANP are substantially elevated in cardiac

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hypertrophy, suggesting the involvement of ANP in developingcardiac hypertrophy. ANP secretion is induced by mechanicalstimulation, such as the stretching of myocytes (Lang et al.,1985), as well as by a number of hormones and vasoactivepeptides, such as AVP (Ruskoaho, 1992). In addition, AVP isknown to stimulate prostacyclin and ANP release from culturedrat cardiomyocytes (Van der Bent et al., 1994). Therefore, AVPcould induce cardiomyocyte hypertrophy by mediating totalprotein increase and ANP release.

OPC-21268 was reported as an orally effective, non-peptidevasopressin V1A receptor antagonist that specifically antago-nized the responses to AVP in vitro and in vivo (Yamamura et al.,1991). Furthermore, among vasopressin receptors, only thevasopressin V1A receptors were found in the neonatal mousecardiomyocytes by RT-PCR analysis. Therefore, we investigatedthe effects of OPC-21268 on AVP-induced protein synthesis andcell growth in neonatal mouse cardiomyocytes. We found thatOPC-21268 could inhibit AVP-induced total protein synthesisand AVP-induced growth of cell surface areas and that AVPinduced the increase of ANPmRNA and protein, which suggeststhat stimulation with AVP could cause neonatal cardiomyocytehypertrophy via the vasopressin V1A receptor. To confirm theseresults, we used cardiomyocytes from V1AR-KO and wild typemice to examine these effects and found that AVP could notincrease the protein synthesis, growth of cell surface areas, andexpression of ANP mRNA and protein in the cardiomyocytesfrom V1AR-KO neonatal mice. Thus, analysis with the selectiveantagonist and gene-mutated mice for the vasopressin V1A

receptor demonstrated that AVP could induce cardiomyocytehypertrophy via the vasopressin V1A receptor. In addition tothese results, cardiac hypertrophy in response to pressureoverload was attenuated in V1AR-KO mice, suggesting thatAVP-V1A receptor signaling contributes to cardiac hypertrophyin response to pressure overload. This finding is consistent withanother finding, namely, that the activation of vasopressin V1A

receptors was strongly associated with left ventricular hyper-trophy and collagen deposition in rats, which was attenuated byspecific vasopressin V1A receptor antagonists (Nakamura et al.,2000). Thus, AVP stimulation may play a causal role inmyocardial hypertrophy under certain circumstances.

Several factors, including phenylephrine and ET-1, areknown to stimulate cardiomyocytes. While phenylephrine andET-1 have been shown to induce cardiomyocyte hypertrophyvia the mitogen-activated protein kinase (MAPK) pathway(Bogoyevitch et al., 1993), it remains unclear which pathwaysare involved in AVP-induced cardiomyocyte hypertrophy. Sincethe ERK family of MAPKs (ERK1/2), known as p42/44 MAPkinase, was the first of the MAPK pathways proposed toparticipate in hypertrophy in cardiomyocytes (Bogoyevitch etal., 1993; Sugden, 2001), we investigated the possibleinvolvement of ERK1/2 in the cardiomyocyte hypertrophyinduced by AVP stimulation. Our results revealed that AVPcould induce ERK1/2 phosphorylation and that pretreatmentwith the V1A antagonist OPC-21268 inhibited this effect. Inaddition, AVP stimulation could not induce ERK1/2 phosphor-ylation in cardiomyocytes from V1AR-KO neonatal mice.Furthermore, the ERK inhibitor, U0126, specifically inhibited

AVP-induced hypertrophy. Thus, our results show that AVPstimulation induces neonatal cardiomyocyte hypertrophy partlyvia the ERK pathway, which is mediated by the vasopressin V1A

receptor.In summary, we demonstrated that AVP increased the total

protein synthesis and expression of ANP mRNA and protein inmouse neonatal cardiomyocytes, in part by stimulating thephosphorylation of ERK1/2. Our results from this study with thevasopressin V1A receptor antagonist, OPC-21268, and V1AR-KO mice demonstrated that neonatal cardiomyocyte hypertro-phy by exposure to AVP was mediated by the vasopressin V1A

receptor and ERK signaling and that this AVP-V1A receptorsignaling pathway plays a significant role in cardiac hypertrophyin response to pressure overload.

Acknowledgements

We thank Dr. T. Mori for providing OPC-21268. This workwas supported by research grants from The Japan HealthSciences, the NOVARATIS Foundation, and the TakedaScience Foundation.

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