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The secretion patterns and roles of cardiac and circulating argininevasopressin during the development of heart failure

Xuanlan Chen, Guihua Lu, Kaiyu Tang, Qinglang Li, Xiuren Gao*a Department of Cardiology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China

A R T I C L E I N F O

Article history:

Received 19 November 2014

Accepted 6 March 2015Available online 18 March 2015

Keywords:

Heart failure

Myocardial fibrosis

Arginine vasopressin

Aldosterone

Cardiac microvascular endothelial cells

A B S T R A C T

ObjectiveThe aim of this study is to investigate local cardiac and circulating AVP secretion during heartfailure and to determine whether AVP mediates ventricular remodeling.

MethodsWe assessed cardiac function and AVP levels of post-myocardial infarction (MI) heart-failurerats 3 weeks (n =10), 4 weeks (n =10), 6 weeks (n =10), 9 weeks (n =15) after the proximal left ante-rior descending coronary artery (LAD) ligation. Ten sham-operated rats were used as the control group.In vitro, cardiac microvascular endothelial cells (CMECs) were initiated from isolated Wistar rat heartsand subjected to Ang II to induce AVP expression and secretion. Besides, the effects of AVP stimulationon CMECs and cardiac fibroblasts (CFs) were studied using methylthiazol tetrazolium assay, Western blot-ting and real-time PCR.ResultsWith cardiac dysfunction, plasma and local cardiac AVP, aldosterone levels increased over time,peaking at 9 weeks post-MI. AVP levels were negatively correlated with serum Na + and LVEF but posi-tively correlated with LVEDD and myocardial hydroxyproline. In CMECs treated with Ang II, AVP mRNAand protein expression increased. In addition, AVP promoted CFs proliferation and up-regulated the ex-pression of endothelin-1 and connective tissue growth factor.ConclusionCMECs are the cellular sources of elevated local heart AVP stimulated with Ang II/AT1. Anintrinsic cardiac AVP system exists. Local cardiac and circulating AVP secretion were enhanced by dete-riorating cardiac function. AVP may promote ventricular remodeling. Thus, AVP could be an important

mediator of myocardial fibrosis in late-stage heart failure. 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Arginine vasopressin (AVP) is a neuroendocrine peptide that isprimarily synthesised by the neurosecretory cells of the supraop-tic and paraventricular hypothalamus nuclei and released via theposterior pituitary in response to hyperosmolarity, hypotension, orhypovolemia. AVP is attracting attention for its biological proper-ties such as the regulation of body fluid osmolality, blood volume,and vascular tone(Baylis, 1987; Thibonnier, 2003). Studies have re-ported that AVP is the key factor in the development of chronic waterretention and the main cause of hyponatremia (Adrogue and Madias,2000). AVP receptor antagonists are under development (Finley et al.,2008): for example, conivaptan, the combined V1a/V2-receptor an-tagonist, recently received U.S. Food and Drug Administrationapproval for the treatment of hyponatremia in heart failure patients.

Changes in plasma nerve-endocrine-cytokines provide insightsinto the development of heart failure and guide the treatment andprognosis of heart failure. B-type natriuretic peptide (BNP) andN-terminal pro-B-type natriuretic peptide (NT-pro-BNP) are gen-erally considered biomarkers for diagnosing heart failure; however,BNP and NT-pro-BNP haveseveral limitations,particularly grey areasin diagnosis that are greatly affected by renal function, gender, age,obesity, bodymass index, heart rate, genetic polymorphismand bloodvolume (Korenstein et al., 2007). Patientsand rats with heart failurepresent elevated plasma AVP concentration and aggravated waterretention (Francis et al., 2001;Goldsmith et al., 1983;Nakamura et al.,2006; Szatalowicz et al., 1981). AVP increases the cardiac preloadand therefore promotes the progression of heart failure. In ad-vanced heart failure, changes in neurohumoral factors and theinappropriate administration of diuretics result in renalhaemodynamic abnormalities with refractory water retention andprogressive renal impairment (Damman et al., 2014), which affectthe diagnosis rate of BNP and NT-pro-BNP. AVP is considered themajor neurohormone thatmediates fluidretentionin advanced heartfailure. Hence, AVP is a promising biomarker for the diagnosis ofadvanced heart failure and disease severity evaluation at certainstages. The purpose of thepresentstudy is to investigate theplasma

This work was conducted in the Key Laboratory on Assisted Circulation, Min-

istry of Health, Guangzhou, China.

* Corresponding author. Department of Cardiology, First Affiliated Hospital, Sun

Yat-sen University, Guangzhou 510 080, China.

E-mail address:[email protected](X. Gao).

http://dx.doi.org/10.1016/j.npep.2015.03.003

0143-4179/ 2015 Elsevier Ltd. All rights reserved.

Neuropeptides 51 (2015) 6373

Contents lists available atScienceDirect

Neuropeptides

j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / n p e p
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AVP concentrationat various time pointsin theprogression of heartfailure.

AVP is present in plasma of homozygous Bratttelboro, centrallyAVP-deficient and hypophysectomised rats(Balment et al., 1986;Kim et al., 1997), which led us to conclude that peripheral organsproduce AVP. Earlier studies have shown the distribution of AVP inthe aortas of rabbits and rats (Loesch et al., 1991; Simon and Kasson,1995). Later,Hupf et al. (1999)observed that the presence of AVPwas in the isolated perfused rat hearts and the release of AVP in-creased with pressure overload. Previous researches have suggestedthat cardiovascular tissue is responsible for the AVP autocrine.However, the patterns and regulative mechanisms of local cardiacAVP secretion remain unclear. In the current study, we focused onAVP expression in rat myocardial tissue with heart failure and ex-plored the cellular origin of AVP.

Moreover, AVP plays an important role in cardiovascular ho-meostasis.Whereas the vascular effectsof AVP are wellcharacterised,AVPs direct cardiac actions are less clear. AVP has been observed toenhance the synthesis of protein, creating cell hypertrophy incardiomyocytes and smooth vascular muscle cells(Geisterfer andOwens, 1989; Tahara et al., 1998). Nevertheless, the role of AVP incardiovascular remodelling, especially myocardial fibrosis, has yetto be illustrated. Connective tissue growth factor (CTGF) mediates

the developmentof myofibroblastsbyenhancingtransforminggrowthfactor (TGF) 1s ability to induce fibroblasts to differentiate intomyofibroblasts, a critical process in fibrosis(Zeisberg et al., 2000).

Multiple neuroendocrine systems are involved in the develop-ment of heart failure, including the renin-angiotensin-aldosteronesystem (RAAS), the AVP system, the natriuretic peptide system, andthe endothelial system. AVP shares several properties with RAASand the endothelial system such as the regulation of hydromineralbalance and vasoconstriction. Nonetheless, the exact relations amongthese systems have not beenclarified. Thisstudy explores these neu-rohormonal systems activated in heart failure, with a focus on therole of AVP.

2. Materials and methods

2.1. Heart failure rat model in vivo

Normal male Wistar rats (N = 75) weighing 200 g to 250 g wereobtained from the Experimental Animal Centre of Sun Yat-sen Uni-versity (Guangzhou, China). All procedures were approved by theExperimental Animal Ethics Committee of Sun Yat-sen University.Experimental myocardial infarction-induced heart failure was pro-duced by ligating the left anterior descending coronary artery, aspreviously described (Klocke et al., 2007). Ten sham-operated ratswere used as the control group. Echocardiography was performed3 weeks post-surgery. The rats with a left ventricular ejection frac-tion (LVEF) no higher than 45% and a weak cardiac impulse in theleft ventricular anterior wall were randomly divided into five ex-

perimental heart failure groups: (1) the sham-operated group, whichwas used as a control group (sham, n = 10); (2) the 3 weeks post-infarction group (3w-HF, n =10); (3) the 4 weeks post-infarctiongroup (4w-HF, n = 10); (4) the 6 weeks post-infarction group (6w-HF, n = 10); and (5) the 9 weeks post-infarction group (9w-HF, n = 15).Twenty rats with LVEF >45 % or death were excluded.

2.2. Cell culture in vitro

Wistar rats aged 57 days were obtained from the Experimen-tal Animal Center at Sun Yat-sen University. Primary rat cardiacmicrovascular endothelial cells (CMECs) were isolated as previ-ously described(Nishida et al., 1993). The CMECs were grown inDulbeccos modified Eagles medium (DMEM, HyClone, USA) con-

taining 10% foetal bovine serum (FBS, Gibco, USA), 10% newborn calf

serum (NBS, Gibco, USA) and 100 g/mL ECGS (Sigma, USA). TheCMECs were characterised by typical cobblestone morphology andpositive staining for CD31 (sc-1506, Santa Cruz Biotechnology, USA)and factor VIII (sc-33584, Santa Cruz Biotechnology, USA), whichare surface markers for microvascular endothelial cells. The mediumwas changed every 2 days, and cells from passages 24 were usedin all of the experiments. After starvation for 24 hours, the CMECswere exposed to Ang II, losartan, AVP, SR49059, or vehicle for 24hours.

Primary cardiac fibroblasts (CFs) were isolated as previously de-scribed (Gao et al., 2009), and grown in supplemented DMEM mediacontaining 10% FBS. The CFs were treated with AVP, SR49059, orvehicle.

2.3. Echocardiography measurements

Echocardiography was performed 3 weeks post-surgery and 1day before the sacrifice to evaluate the changes in cardiac mor-phology and blood flow. The echocardiography was performed byan experienced operator using ESAOTE ultrasound Doppler equip-ment. M-mode tracings of the long-axis view of the left ventriclewere captured, and the following indexes were collected: the leftventricular systolic diameter (LVESD), the left ventricular diastolic

diameter (LVEDD), the left ventricular ejection fraction (LVEF), andthe left ventricular fractional shortening (LVFS). The echocardiographoperator was blinded to the group allocation at all times. All of theechocardiograms were recorded for off-line analysis. The enumer-ated data were presented as the average of three cardiac cycles.

2.4. Tissue preparation and immunohistochemistry

The heart was arrested in diastole with an intraventricular in-jection of KCl (10%). The atria and the right ventricular free wall wereexcised; the ventricles were rinsed with isotonic saline and thendissectedand weighed.The weightsof the ventricles were normalisedto the body weight and used as an index of ventricular hypertro-phy. To estimate collagen production, the hydroxyproline level in

the left ventricle was determined using the hydroxyproline assayaccording to the manufacturers instructions (BioVision, USA).

Left ventricle tissue specimens were cross-sectioned at the levelof the papillary muscle, fixed and dehydrated in 10% formalde-hyde, and embedded in paraffin for immunohistochemistry of AVP(1:5000; Millipore, USA).

2.5. Enzyme-linked immunosorbent assay

Blood from the abdominal aortas was collected in sodium citrateanticoagulant tubes before sacrifice. The blood was centrifuged at3000 r/min at 4 C, and the supernatant was collected and kept at80 C. The myocardial tissue samples were grounded thoroughlywith a glass homogeniser in phosphate-buffered saline solution

(0.01 M, pH 7.4) and centrifuged at 3000 r/min for 20 minutes. Thesupernatant was collected for the detection of AVP and aldoste-rone. The cell culture medium of the CMECs was collected todetermine AVP levels. The AVP and aldosterone levels weredetermined using a commercially obtained enzyme-linkedimmunosorbent assay (ELISA) kit (ADI-900-017,Enzo; 10004377,Cayman) according to the manufacturers instructions.

2.6. Quantitative real-time PCR

Total RNA was extracted using Trizol (Sigma, USA) according tothemanufacturersinstructionsand quantified using NanoDropsspec-trophotometer. Then 1 g of the isolated total RNA was reversetranscribed using an Omniscript RT Kit (Qiagen, Australia) accord-

ing to the manufacturers protocol. The single-strand cDNA was

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amplified using real-time polymerase chain reaction (real-time PCR)with the TaqMan system(ABI-7500fast PCRSystem) andSYBR Greendye. The mRNA expression was normalised to an endogenous 18 srRNA.

2.7. Immunofluorescence staining

CMECs in 24-well plateswere pre-treatedwith losartan (10 M)or vehicle for1 hour andthenstimulatedwithAng II (1 M)or vehicle.After 24 hours, the cells were fixed in 4% paraformaldehyde (PFA),blocked with 5% bovineserum albumin, and stainedwith rabbitanti-vasopressin antibody (1:1000; Millipore, USA) at 4 C overnight. Onthe second day, after 3 washes, the cells were incubated with FITC-conjugated goat anti-rabbit antibody (Proteintech, USA) at roomtemperature for 1 hour. After 3 washes, the samples were stainedwith DAPI (Sigma, USA). Images were taken using a fluorescencemicroscope (Olympus BX51) and analysed with ImageJ software.

2.8. MTT assay

CF proliferation was performed using the MTT reagent (Sigma,USA) according to the suppliers instructions. Briefly, the CFs wereseeded onto 96-well plates at a final density of 5 103 cells/mL. After

exposure to AVP, SR49059, or vehicle, 20 L/mL of 5% MTT solu-tion (Sigma, USA) was added to each well. The plates were thenincubated for 4 hours at 37 C. The supernatant was aspirated, and150 L of dimethyl sulfoxide (Sigma, USA) was added to each well.After 10 minutes of shaking, absorbance was measured with amicroplate reader (Tecan Sunrise, Japan) at a wavelength of 490 nm,which represents a viable cell number.

2.9. Western blotting

The cells were lysed in RIPA buffer (Millipore, USA) containingprotease inhibitor cocktail (Roche, Switzerland) at 4 C. The proteinconcentration was measured using a BCA assay (Pierce, Rockford,Illinois, USA). Equal amounts of protein (25 g) were loaded onto

12% SDS electrophoresis plates and transferred onto PVDF mem-branes (Millipore, USA). The blots were incubated with theappropriate primary antibodies (goat anti-CTGF polyclonal anti-body, Santa Cruz; mouse anti--tubulin monoclonal antibody, ProteinTech), followed by the corresponding HRP-conjugated secondary an-tibodies, and the proteins were revealed using the ECL system(Millipore). -Tubulin was used as the loading control. The devel-oped films were scanned, and quantitative analysis was performedusing ImageJ software.

2.10. Statistical analysis

All analyses were performed with SPSS software (Version 13.0).Data were expressed as the mean SEM. For two-group compari-sons, Students t-test was performed; for multiple-groupcomparisons, ANOVA was used after the homogeneity of vari-ances test was applied. Correlations between two groups wereanalysed using Pearsons chi-square test. Heteroscedastic data wereanalysed using the KruskalWallis test, and the correlation betweenthe two groups was analysed using Spearmans rank correlation test.P < 0.05 was considered statistically significant.

3. Results

3.1. Heart weight index and cardiac function in rats with heart

failure

The left ventricular mass index (LVW/BW), the heart weight index(HW/BW), and the lung wet/dry (W/D) weight ratio of the heartfailure groups were higher than those of the sham group (P < 0.05).The LVW/BW and HW/BW increased with the progression of heartfailure, and the data for each group were significantly different. The

W/D also increased with the progression of heart failure; however,there was no significant difference between the 3-week-HF and the4-week-HF groups (P >0.05). The data of the other groups differedsignificantly. Compared with the sham group, the LVESD and LVEDDof the heart failure groups increased, whereas the LVEF and LVFSsignificantly decreased (P 0.05).

3.2. Aldosterone and AVP concentrations in plasma and heart tissue

during the development of heart failure in rats

The aldosterone concentrations in the plasma and heart tissuewere significantly elevated compared with the sham group (P < 0.05,Fig.1A and B). The aldosterone concentration in the plasma and hearttissue increased with the progression of heart failure, with the

Table 1

Echocardiography data and serum electrolyte levels in rats with heart failure.

Parameters Sham 3 weeks-HF 4 weeks-HF 6 weeks-HF 9 weeks-HF

(n =10) (n = 10) (n = 10) (n = 10) (n = 15)

HW/BW (mg/g) 2.66 0.18 2.92 0.08a 3.13 0.03ab 3.28 0.06abc 3.72 0.15abcd

LVW/BW (mg/g) 1.88 0.10 2.10 0.03a 2.23 0.03ab 2.35 0.07abc 2.59 0.12abcd

Lung W/D (mg/mg) 2.17 0.19 2.58 0.12a 2.83 0.03a 3.40 0.34abc 5.13 0.43abcd

LVEF (%) 59.24 1.55 42.33 3.54a 36.23 2.97a 31.03 1.86abc 19.57 1.01abcd

LVFS (%) 32.65 1.94 21.30 3.52a 18.68 3.39a 15.47 1.94abc 9.34 1.14abcd

LVEDD (mm) 8.30 0.26 9.50 0.48a 9.82 0.54a 10.89 0.64abc 12.21 0.61abcd

LVESD (mm) 6.3 0.14 7.88 0.49a 8.35 0.56a 9.40 0.51abc 11.00 0.62abcd

Na+ (mmol/l) 144.16 3.81 135.10 1.59a 132.54 1.42a 124.48 3.62abc 110.76 13.49abcd

K+ (mmol/l) 4.90 0.57 4.74 0.38 4.82 0.24 4.57 0.43 4.38 0.39

BW, body weight; HW, heart weight; LVW, left ventricular weight; Lung W/D, the ratio of lung wet weight to dry weight; LVEF, left ventricular ejection fraction; LVFS, left

ventricular fractional shortening; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension. n =10, 10, 10, 10, 15 for sham, 3 weeks-

HF, 4 weeks-HF 6 weeks-HF, 9 weeks-HF, respectively. Data are means SE.a P


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highest level in the 9-week-HF group. The plasma AVP levels of theheart failure groups were higher than the levels of the sham groupand increased with the development of heart failure, peaking in the9-week-HF group (P < 0.05,Fig. 1C). Plasma AVP was positively cor-related with plasma aldosterone (correlation coefficient r =0.907,Fig. 1D).

3.3. Correlation between plasma AVP and cardiac function index and

serum electrolyte levels in heart failure rats

Statistical analysis showed that plasma AVP concentration wasnegatively correlated with LVEF, serum Na+ level (correlation coef-ficient r= 0.856,Fig. 2A; r= 0.904,Fig. 2C, respectively) andpositively correlated with LVEDD and myocardial hydroxyproline

level (r=

0.900,Fig. 2B;r=

0.904,Fig. 2D,respectively).

3.4. Local cardiac secretion of AVP

The immunohistochemistry results suggested marked AVP ex-pression in the vascular tissue of heart failure rats (P < 0.05, Fig. 3A).Enhanced AVP expression in the myocardial tissues of heart failurerats was observed in both the mRNA (Fig. 3B) and protein (Fig. 3C)levels compared with the sham group (P < 0.05). AVP protein andmRNA expression increased with the development of heart failure,peaking at 9 weeks after MI. The AVP mRNA levels of each groupwere statistically significantly different (P


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AVP was mainly distributed in the cytoplasm(Fig. 4B). The Ang IIgroup showed elevated fluorescent intensity compared with thecontrol group. Although the losartan-incubated group showed weak-

ened fluorescent intensity, Ang II induced AVP mRNA expression ina concentration-dependent manner(Fig. 5A). AVP protein expres-sion increased with Ang II concentration, peaking at 107 mol/L(Fig. 5B). Losartan inhibited AVP expression in both the mRNA(Fig. 5C) and protein(Fig. 5D).

3.6. The direct effect of AVP on cardiac effector cells

AVP stimulated CFs proliferation in a concentration-dependentand time-dependent manner (Fig. 6A and 6B). The ET-1 mRNA levelin rat CMECs also increased after AVP treatment (P


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aldosterone (r= 0.907). The plasma levels of AVP in the rats dete-riorating cardiac function increased over time, peaking at 9 weekspost-MI. Further, plasma AVP levels were negatively correlated withserum Na+ and LVEF, but positively correlated with LVEDD and myo-cardial hydroxyproline contents. Besides, it is well documented thataldosterone concentration is proportional to the severity of heartfailure (Pitt, 2012). Therefore, the plasma AVP levels in the rats withheart failure inversely related to the cardiac function. Clinically,

Nakamura et al. reported that plasma AVP activity was signifi-cantly higher in patients with heart failure than in healthy age-matched controls, and AVP levels were highest in patients with overtsymptoms of heart failure (Nakamura et al., 2006). The present studymight support the notion of AVP as a potential biomarker for thediagnosis and severity of heart failure. Aaldosterone promotes water-sodium retention and myocardial fibrosis, thus accelerating thedevelopment of heart failure(Pitt, 2012). AVP is crucial for fluid ho-meostasis but also serves as cardiovascular control (Bao et al., 2014),acting via three different receptor subtypes (V1a, V2, and V1b). TheV1a receptors mediate vasoconstriction and are localized primar-ily on vascular smooth muscle cells. V2 receptors mediate antidiureticeffects and are highly expressed in the kidneys, which is crucial forwater homeostasis. V1b receptors found in the anterior pituitary

brain are involved in central nervous system effects (Vincent and

Su, 2008). AVP has been shown to stimulate aldosterone secretionfrom the normal human or rat adrenal gland and some cortisol-producing adrenocortical tumours or hyperplasias (Hinson et al.,1987; Perraudin et al., 2006). The increased AVP levels and the ex-aggerated release of aldosterone may participate in water-sodiumretention, resulting in volume expansion that exacerbates dia-stolic wall stress and heart function in heart failure. Furthermore,increased V1a receptor expression in failing hearts has been re-

ported (Zhu et al., 2014), as well as in the brain and kidney aftermyocardial infarction(Milik et al., 2014). The consequence of V1areceptor activation is vasoconstriction, leading to pressure over-load that causes cardiac hypertrophy and cardiac function decreasein heart failure. Additionally, in our study, plasma AVP levels wereconsistent with HW/BW. This finding indicates that AVP may mediatecardiac hypertrophy. The above effects, if sustained, may exacer-bate cardiac dysfunction to a greater extent in the failing heart, thuscreating a vicious cycle. That is, excessive AVP activation can leadto heart failure deterioration if it is not corrected promptly.

Vasopressin content in the hypothalamus increased in the ratswith heart failure (Muders et al., 2002). AVP can be released intothe plasma from the pituitary glands of rats with heart failure vianon-osmotic regulation(Szatalowicz et al., 1981). The hypothala-

mus is considered the principal locus of AVP synthesis (Vincent and

Fig. 3. Local heart AVP expression in rats with heart failure. (A) Immunohistochemistry of heart failure rats (100). Representative images of the left ventricle at week 4 in

the sham and HF groups. Scale bar: 10 m.

(B) AVP mRNA expression in myocardial tissue, determined using RT-PCR. 3w-HF, 4w-HF, 6w-HF, and 9w-HF refer to heart-failure rats 3, 4, 6, and 9 weeks after myocardial

infarction surgery, respectively. n =8 for all groups.

*P


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Su, 2008). Indeed, experimental heart failure in rats augments AVPin the hypothalamus and raises plasma AVP levels (Ciosek andDrobnik, 2012). Moreover, with the enhanced activation of RAASin heart-failure rats, elevated levels of Ang II pass through the bloodbrain barrier, activate vasopressin neurons (Grobe et al., 2010), andexaggerate AVP expression. Significantly, our findings revealed thatcardiac tissue is another source of circulating AVP. The present study

showed that cardiac AVP in rats was localised near the blood vessels,a finding that is consistent with previous reports of immunoreac-tive AVP in the vascular beds(Gutkowska et al., 2007; Loesch et al.,1991). Furthermore, in this study, AVP mRNA and protein levels wereincreased in Ang II/AT1 receptor-mediated CMECs. In the failing heart,AVP levels increased with the cardiac diameter, indicating that AVPsecretion was related to ventricular volume overload. Data suggestthat AVP expression increased in isolated, perfused rat hearts stressedby pressure overload(Hupf et al., 1999). According to Laplaces law,the thickening of the ventricular wall in the early-middle stages ofheart failure could maintain the wall stress at an appropriate levelthat avoids stress from too much change. However, in the late stagesof heart failure, the ventricle is significantly enlarged and thinner,resulting in significant ventricular wall stress. The findings in the

present study indicate that local cardiac AVP expression during the

early and middle stages of heart failure is mainly regulated by ven-tricular dilation, whereas in the late stage, AVP expression isregulated mainly by the increased ventricular wall stress.

In recent years, numerous studies have focused on the effectsof RAAS on the heart. RAAS activation induces systemic vasocon-striction, adjusts the water and electrolyte balance and activates othersystems (e.g., AVP and aldosterone). However, chronic activation of

these systems in heart failure can impair cardiac function andpromote heart failure. Ang II increases cardiac after load via sys-temic vasoconstriction and can also induce myocyte hypertrophyand alter the myocardial matrix structure (Morgan and Baker, 1991).Aldosterone stimulates CFs proliferation, induces the activation ofmyofibroblasts and promotes the secretion of pro-fibrosis factors,leading to collagen matrix deposition (Johar et al., 2006). Inthe longterm, these effects result in cardiac concentric hypertrophy(Chatterjee, 2005). AVP regulates blood volume and vascular tone(Thibonnier, 2003). Excessive activation of AVP increases intracar-diac pressure and leads to water retention in heart failure (Chatterjee,2005). With the progression of heart failure, the increasing ventri-cle volume load and wall stress result in an imbalance betweenmatrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs)

(Spinale et al., 2000), which transition to a process of eccentric

Fig. 4. Immunofluorescence staining of CMECs (200). (A) Identification of CMECs with CD31 and factors VIII (green) using immunofluorescence. PBS(negative control without

primary antibody) scale bar: 5 m. The nuclei were dyed with DAPI (blue).

(B) AVP expression (green) in CMECs. The nuclei were dyed with DAPI (blue) .

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hypertrophy with progressive ventricular dilation and wall thin-ning. Nevertheless, we do not know which neurohormonal systemsactivation plays leading roles in eccentric hypertrophy in ad-vanced heart failure.

In our study, increased plasma AVP was involved in dilutionalhyponatremia and regulated the cardiovascular system via V1a re-ceptors. We observed that serum Na+ concentration decreased along

with cardiac function in the rats with heart failure and that AVP wasnegatively correlated with the serum Na+ concentration, indicat-ing that AVP was the leading mechanism of hyponatremia in heartfailure. Hyponatremia is the most common electrolyte disorderamong patients with heart failure; it occurs in 18% to 27% of heartfailure patients (Klein et al., 2005). It is well documented that hy-ponatremia is an independent predictor of the mortality andreadmission rates of heart failure patients (Klein et al., 2005). There-fore, identifying an effective method for correcting hyponatremiais an absolute necessity. AVP plays a central role in water reten-tion in chronic heart failure patients (Nielsen et al., 1999) and AVPdysregulation is the most common cause of hypotonic-hypervolemichyponatremia(Anderson et al., 1985). Thus, AVP receptor antago-nists are a promising approach for treating hyponatremia in patients

with heart failure(Finley et al., 2008).

Ventricular remodelling is the pathophysiological basis for theprogression of heart failure, and myocardial fibrosis is a particularcharacteristic of this remodelling. CF proliferation and excessive ac-cumulation of extracellular matrix collagen are believed to be themajor pathologic causes of myocardial fibrosis. ET-1, a mitogenicfactor and pro-fibrotic cytokine, induces the synthesis of type I andtype III collagen fibres and inhibits matrix metalloproteinases-1 ex-

pression in the CFs; consequently, extracellular matrix (ECM)accumulates, promoting cardiac fibrosis (Shi-Wen et al., 2001). Inpatients with heart failure, ET-1 is significantly increased(Ohmae,2011). CTGF, an important fibrogenic cytokine and downstream mol-ecule of Ang II and TGF-1, can stimulate CFs to excrete ECM andsynthesise collagen, thereby causing cardiac fibrosis (Mori et al.,1999). As other studies show, CTGF, a new marker of cardiac dys-function(Koitabashi et al., 2008), is involved in the generation andpersistence of cardiac fibrosis (Dean et al., 2005). We demon-strated that AVP up-regulated CTGF and ET-1 expression andpromoted CF proliferation. Additionally, AVP acts on cardiomyocytesto stimulate protein synthesis(Tahara et al., 1998). In vivo, we ob-served that AVP is positively correlated with the cardiachydroxyproline content. Therefore, AVP participated in myocar-

dial fibrosis. Furthermore, the circulating AVP levels continuously

Fig. 5. The mRNA and protein expression of AVP in CMECs, determined using RT-PCR and ELISA.

(A) AVP mRNA levels at 24 hours after the CMECs were treated with Ang II. n = 6, 4, 4, 4, 4, 4 for Ang II; 0, 10 9, 10 8, 1 07, 10 6, 1 05 mol/L, respectively. *P


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Fig. 6. Effect of AVP on CFs and CMECs.

AVP-induced CF proliferation in a dose-dependent (A) and time-dependent (B) manner via MTT. n =6 for all groups.

(C) SR49059 can inhibit CFs proliferation induced with10 7 M AVP. n =4 for all groups.

*P


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increased in advanced heart failure. These findings suggest that AVPmay be an important cytokine of cardiac fibrosis during late-stageheart failure. Moreover, ET-1 is the most potent vasoconstrictor. Inthe development of heart failure, elevated AVP may participate inthe exaggerated release of ET-1. Both AVP and ET-1 can reduce cor-onary blood flow and cardiac contractility and promote peripheralvasoconstriction, causing an increase in cardiac afterload and de-teriorating cardiac function (Large, 2002). Ventricle remodeling isshown as changes in myocardial fibrosis (molecular, cellular, inter-stitial), decreased capillary density, and function of the heart(Cohnet al., 2000). Accordingly, based on the above discussion, AVP me-diates ventricular remodelling in heart failure. The specificmechanisms were as follows: pressure overload is associated with

peripheral vasoconstrictor, AVP itself or elevation ET-1 by AVP;volume overload results from water reabsorption by stimulation ofthe V2 receptors; AVP directly acts on cardiac including contribu-tion to cardiomyocytes hypertrophy by increasing protein synthesis(Tahara et al., 1998), promotion proliferation of cardiac fibroblastsand upregulation of pro-fibrogenic cytokine, aldosterone (Perraudinet al., 2006), CTGF and ET-1. At present, there are no therapies aimedat reducing CTGF production, nor did the endothelium receptor an-tagonist treatment of heart failure show benefits (OConnor et al.,2003). Therefore, AVP receptor antagonists may provide a new ther-apeutic strategy for heart failure.

During the heart failure process, excessive AVP secretion causeshyponatremia and an increase in cardiac preload, and excessive AVPmediates myocardial fibrosis, which impairs ventricular wall com-

pliance and cardiac function. Furthermore, AVP, as a vasoconstrictor,

causes myocardial ischaemia and increases the cardiac afterload, thusnegatively affecting cardiac function. Hence, AVP plays an impor-tant role in the development and progression of heart failure.Sustained increases in preload that aggravates diastolic wall stressin heart failure promote activation of matrix metalloproteinases(Spinale et al., 2000) and thereby myocardial fibrillar collagen deg-radation, myocardial remodelling and subsequent chamber dilation,resulting in eccentric hypertrophy. During end-stage heart failure,AVP-mediated reabsorption of free water by the renal tubules mayplay a role in the transition to eccentric hypertrophy, thus causingheart function to deteriorate. In addition, Ang II potentiates AVP se-cretion, whereas AVP induces aldosterone and ET-1 expression. Inlate-stage heart failure, the dilutional hyponatremia caused by in-

creased AVP secretion may activate RAAS. Notably, AVPs synergisticinteractions with Ang II, aldosterone, and endothelin-1 cannot beneglected when examining impaired cardiac function.

This study demonstrates the dynamic changes in local cardiacand circulating AVP and AVPs role in heart failure induced by myo-cardial infarction (Fig. 7). Furthermore, this study provides anexperimental basis for AVP receptor antagonists in the therapeu-tic window for effective heart failure treatment.

Acknowledgments

We would like to thank the Key Laboratory on Assisted Circu-lation, Ministry of Health, Guangzhou, China, for the excellent

technical assistance.

Fig. 7. Schematic of the dynamic changes in AVP and AVPs role in heart failure.

72 X. Chen et al./Neuropeptides 51 (2015) 6373
http://-/?-http://-/?-


11/11

Funding

This work was supported by a grant from the Science Fund Com-mittee of Guang Zhou City, Guang Dong Province, China(No.2011J4100111) and the Junhong Company (No.078231),Dongguan, China.

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.npep.2015.03.003 .

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