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Neuropeptides 51 (2015) 63–73

Neuropeptides 51 (2015) 63–73 Contents lists available at ScienceDirect Neuropeptides journal homepage:

Contents lists available at ScienceDirect


journal homepage:

journal homepage: The secretion patterns and roles of cardiac and circulating

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

Xuanlan Chen, Guihua Lu, Kaiyu Tang, Qinglang Li, Xiuren Gao *

Chen, Guihua Lu, Kaiyu Tang, Qinglang Li , Xiuren Gao * a Department of Cardiology, First

a Department of Cardiology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China



Article history:

Received 19 November 2014 Accepted 6 March 2015 Available online 18 March 2015


Heart failure

Myocardial fibrosis Arginine vasopressin Aldosterone Cardiac microvascular endothelial cells


Objective The aim of this study is to investigate local cardiac and circulating AVP secretion during heart failure and to determine whether AVP mediates ventricular remodeling. Methods We assessed cardiac function and AVP levels of post-myocardial infarction (MI) heart-failure rats 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 hearts and subjected to Ang II to induce AVP expression and secretion. Besides, the effects of AVP stimulation on 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 mRNA and protein expression increased. In addition, AVP promoted CFs proliferation and up-regulated the ex- pression of endothelin-1 and connective tissue growth factor. Conclusion CMECs are the cellular sources of elevated local heart AVP stimulated with Ang II/AT1. An intrinsic 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 is primarily synthesised by the neurosecretory cells of the supraop- tic and paraventricular hypothalamus nuclei and released via the posterior pituitary in response to hyperosmolarity, hypotension, or hypovolemia. 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 water retention 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 Administration approval for the treatment of hyponatremia in heart failure patients.

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 510080, China. E-mail address: (X. Gao).

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

Changes in plasma nerve-endocrine-cytokines provide insights into the development of heart failure and guide the treatment and prognosis of heart failure. B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) are gen- erally considered biomarkers for diagnosing heart failure; however, BNP and NT-pro-BNP have several limitations, particularly grey areas in diagnosis that are greatly affected by renal function, gender, age, obesity, body mass index, heart rate, genetic polymorphism and blood volume (Korenstein et al., 2007). Patients and rats with heart failure present elevated plasma AVP concentration and aggravated water retention (Francis et al., 2001; Goldsmith et al., 1983; Nakamura et al., 2006; Szatalowicz et al., 1981). AVP increases the cardiac preload and therefore promotes the progression of heart failure. In ad- vanced heart failure, changes in neurohumoral factors and the inappropriate administration of diuretics result in renal haemodynamic abnormalities with refractory water retention and progressive renal impairment (Damman et al., 2014), which affect the diagnosis rate of BNP and NT-pro-BNP. AVP is considered the major neurohormone that mediates fluid retention in advanced heart failure. Hence, AVP is a promising biomarker for the diagnosis of advanced heart failure and disease severity evaluation at certain stages. The purpose of the present study is to investigate the plasma


X. Chen et al./Neuropeptides 51 (2015) 63–73

AVP concentration at various time points in the progression of heart failure. AVP is present in plasma of homozygous Bratttelboro, centrally AVP-deficient and hypophysectomised rats (Balment et al., 1986; Kim et al., 1997), which led us to conclude that peripheral organs produce AVP. Earlier studies have shown the distribution of AVP in the aortas of rabbits and rats (Loesch et al., 1991; Simon and Kasson, 1995). Later, Hupf et al. (1999) observed that the presence of AVP was in the isolated perfused rat hearts and the release of AVP in- creased with pressure overload. Previous researches have suggested that cardiovascular tissue is responsible for the AVP autocrine. However, the patterns and regulative mechanisms of local cardiac AVP secretion remain unclear. In the current study, we focused on

serum (NBS, Gibco, USA) and 100 μg/mL ECGS (Sigma, USA). The CMECs were characterised by typical cobblestone morphology and positive staining for CD31 (sc-1506, Santa Cruz Biotechnology, USA) and factor VIII (sc-33584, Santa Cruz Biotechnology, USA), which are surface markers for microvascular endothelial cells. The medium was changed every 2 days, and cells from passages 2–4 were used in all of the experiments. After starvation for 24 hours, the CMECs were exposed to Ang II, losartan, AVP, SR49059, or vehicle for 24 hours. Primary cardiac fibroblasts (CFs) were isolated as previously de- scribed (Gao et al., 2009), and grown in supplemented DMEM media containing 10% FBS. The CFs were treated with AVP, SR49059, or vehicle.

AVP expression in rat myocardial tissue with heart failure and ex-



Echocardiography measurements

plored the cellular origin of AVP. Moreover, AVP plays an important role in cardiovascular ho- meostasis.Whereas the vascular effects of AVP are well characterised, AVP’s direct cardiac actions are less clear. AVP has been observed to enhance the synthesis of protein, creating cell hypertrophy in cardiomyocytes and smooth vascular muscle cells (Geisterfer and Owens, 1989; Tahara et al., 1998). Nevertheless, the role of AVP in cardiovascular remodelling, especially myocardial fibrosis, has yet to be illustrated. Connective tissue growth factor (CTGF) mediates the development of myofibroblasts by enhancing transforming growth factor (TGF) β1’s ability to induce fibroblasts to differentiate into myofibroblasts, 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-aldosterone

Echocardiography was performed 3 weeks post-surgery and 1 day before the sacrifice to evaluate the changes in cardiac mor- phology and blood flow. The echocardiography was performed by an experienced operator using ESAOTE ultrasound Doppler equip- ment. M-mode tracings of the long-axis view of the left ventricle were captured, and the following indexes were collected: the left ventricular systolic diameter (LVESD), the left ventricular diastolic diameter (LVEDD), the left ventricular ejection fraction (LVEF), and the left ventricular fractional shortening (LVFS). The echocardiograph operator was blinded to the group allocation at all times. All of the echocardiograms were recorded for off-line analysis. The enumer- ated data were presented as the average of three cardiac cycles.

system (RAAS), the AVP system, the natriuretic peptide system, and


Tissue preparation and immunohistochemistry

the endothelial system. AVP shares several properties with RAAS and the endothelial system such as the regulation of hydromineral balance and vasoconstriction. Nonetheless, the exact relations among these systems have not been clarified. This study explores these neu- rohormonal systems activated in heart failure, with a focus on the role of AVP.

The heart was arrested in diastole with an intraventricular in- jection of KCl (10%). The atria and the right ventricular free wall were excised; the ventricles were rinsed with isotonic saline and then dissected and weighed. The weights of the ventricles were normalised


to the body weight and used as an index of ventricular hypertro-


Materials and methods

phy. To estimate collagen production, the hydroxyproline level in


Heart failure rat model in vivo

the left ventricle was determined using the hydroxyproline assay

Normal male Wistar rats (N = 75) weighing 200 g to 250 g were obtained from the Experimental Animal Centre of Sun Yat-sen Uni- versity (Guangzhou, China). All procedures were approved by the

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

Experimental Animal Ethics Committee of Sun Yat-sen University.



Enzyme-linked immunosorbent assay

Experimental myocardial infarction-induced heart failure was pro- duced by ligating the left anterior descending coronary artery, as previously described (Klocke et al., 2007). Ten sham-operated rats were used as the control group. Echocardiography was performed

Blood from the abdominal aortas was collected in sodium citrate anticoagulant tubes before sacrifice. The blood was centrifuged at

3 weeks post-surgery. The rats with a left ventricular ejection frac-

3000 r/min at 4 °C, and the supernatant was collected and kept at

80 °C. The myocardial tissue samples were grounded thoroughly

tion (LVEF) no higher than 45% and a weak cardiac impulse in the left ventricular anterior wall were randomly divided into five ex- perimental heart failure groups: (1) the sham-operated group, which was used as a control group (sham, n = 10); (2) the 3 weeks post- infarction group (3w-HF, n = 10); (3) the 4 weeks post-infarction group (4w-HF, n = 10); (4) the 6 weeks post-infarction group (6w-

with a glass homogeniser in phosphate-buffered saline solution (0.01 M, pH 7.4) and centrifuged at 3000 r/min for 20 minutes. The supernatant was collected for the detection of AVP and aldoste- rone. The cell culture medium of the CMECs was collected to determine AVP levels. The AVP and aldosterone levels were

determined using a commercially obtained enzyme-linked

HF, n = 10); and (5) the 9 weeks post-infarction group (9w-HF, n = 15). Twenty rats with LVEF > 45 % or death were excluded.

immunosorbent assay (ELISA) kit (ADI-900-017,Enzo; 10004377, Cayman) according to the manufacturer’s instructions.

2.2. Cell culture in vitro

Wistar rats aged 5–7 days were obtained from the Experimen- tal Animal Center at Sun Yat-sen University. Primary rat cardiac microvascular endothelial cells (CMECs) were isolated as previ- ously described (Nishida et al., 1993). The CMECs were grown in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, USA) con- taining 10% foetal bovine serum (FBS, Gibco, USA), 10% newborn calf

2.6. Quantitative real-time PCR

Total RNA was extracted using Trizol (Sigma, USA) according to themanufacturer’s instructions and quantified using NanoDrops spec- trophotometer. Then 1 μg of the isolated total RNA was reverse transcribed using an Omniscript RT Kit (Qiagen, Australia) accord- ing to the manufacturer’s protocol. The single-strand cDNA was

X. Chen et al./Neuropeptides 51 (2015) 63–73


amplified using real-time polymerase chain reaction (real-time PCR) with the TaqMan system (ABI-7500 fast PCR System) and SYBR Green dye. The mRNA expression was normalised to an endogenous 18 s rRNA.

2.7. Immunofluorescence staining

CMECs in 24-well plates were pre-treated with losartan (10 μM) or vehicle for 1 hour and then stimulated with Ang II (1 μM) or vehicle. After 24 hours, the cells were fixed in 4% paraformaldehyde (PFA), blocked with 5% bovine serum albumin, and stained with rabbit anti- vasopressin antibody (1:1000; Millipore, USA) at 4 °C overnight. On the second day, after 3 washes, the cells were incubated with FITC- conjugated goat anti-rabbit antibody (Proteintech, USA) at room temperature for 1 hour. After 3 washes, the samples were stained with DAPI (Sigma, USA). Images were taken using a fluorescence microscope (Olympus BX51) and analysed with 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, Student’s t-test was performed; for multiple-group comparisons, ANOVA was used after the homogeneity of vari- ances test was applied. Correlations between two groups were analysed using Pearson’s chi-square test. Heteroscedastic data were analysed using the Kruskal–Wallis test, and the correlation between the two groups was analysed using Spearman’s rank correlation test. P < 0.05 was considered statistically significant.

3. Results

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


2.8. MTT assay

CF proliferation was performed using the MTT reagent (Sigma, USA) according to the supplier’s instructions. Briefly, the CFs were seeded onto 96-well plates at a final density of 5 × 10 3 cells/mL. After exposure to AVP, SR49059, or vehicle, 20 μL/mL of 5% MTT solu- tion (Sigma, USA) was added to each well. The plates were then incubated for 4 hours at 37 °C. The supernatant was aspirated, and 150 μL of dimethyl sulfoxide (Sigma, USA) was added to each well. After 10 minutes of shaking, absorbance was measured with a microplate 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) containing protease inhibitor cocktail (Roche, Switzerland) at 4 °C. The protein concentration was measured using a BCA assay (Pierce, Rockford, Illinois, USA). Equal amounts of protein (25 μg) were loaded onto 12% SDS electrophoresis plates and transferred onto PVDF mem- branes (Millipore, USA). The blots were incubated with the appropriate primary antibodies (goat anti-CTGF polyclonal anti- body, Santa Cruz; mouse anti-α-tubulin monoclonal antibody, Protein Tech), followed by the corresponding HRP-conjugated secondary an- tibodies, and the proteins were revealed using the ECL system (Millipore). α-Tubulin was used as the loading control. The devel- oped films were scanned, and quantitative analysis was performed using ImageJ software.

The left ventricular mass index (LVW/BW), the heart weight index (HW/BW), and the lung wet/dry (W/D) weight ratio of the heart failure groups were higher than those of the sham group (P < 0.05). The LVW/BW and HW/BW increased with the progression of heart failure, 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 the 4-week-HF groups (P > 0.05). The data of the other groups differed significantly. Compared with the sham group, the LVESD and LVEDD of the heart failure groups increased, whereas the LVEF and LVFS significantly decreased (P < 0.05, Table 1). Among the heart failure groups, the LVESD and LVEDD of the 6-week-HF and the 9-week- HF groups were significantly higher than the LVESD and LVEDD of the 3-week-HF and the 4-week-HF groups, whereas the LVEF and LVFS were significantly lower (P < 0.05). The serum Na + levels of the heart failure groups were significantly lower than the levels of the sham group (P < 0.05; Table 1). The serum Na + levels continued to decrease with the development of heart failure, and the lowest level was observed in the 9-week-HF group. There was no significant dif- ference in the serum K + levels for each group (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 tissue were significantly elevated compared with the sham group (P < 0.05, Fig. 1A and B). The aldosterone concentration in the plasma and heart tissue increased with the progression of heart failure, with the

Table 1 Echocardiography data and serum electrolyte levels in rats with heart failure.



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) LVW/BW (mg/g) Lung W/D (mg/mg) LVEF (%) LVFS (%) LVEDD (mm) LVESD (mm) Na + (mmol/l) K + (mmol/l)

2.66 ± 0.18 1.88 ± 0.10 2.17 ± 0.19 59.24 ± 1.55 32.65 ± 1.94 8.30 ± 0.26 6.3 ± 0.14 144.16 ± 3.81 4.90 ± 0.57

2.92 ± 0.08 a 2.10 ± 0.03 a 2.58 ± 0.12 a 42.33 ± 3.54 a 21.30 ± 3.52 a 9.50 ± 0.48 a 7.88 ± 0.49 a 135.10 ± 1.59 a 4.74 ± 0.38

3.13 ± 0.03 ab 2.23 ± 0.03 ab 2.83 ± 0.03 a 36.23 ± 2.97 a 18.68 ± 3.39 a 9.82 ± 0.54 a 8.35 ± 0.56 a 132.54 ± 1.42 a 4.82 ± 0.24

3.28 ± 0.06 abc 2.35 ± 0.07 abc 3.40 ± 0.34 abc 31.03 ± 1.86 abc 15.47 ± 1.94 abc 10.89 ± 0.64 abc 9.40 ± 0.51 abc 124.48 ± 3.62 abc 4.57 ± 0.43

3.72 ± 0.15 abcd 2.59 ± 0.12 abcd 5.13 ± 0.43 abcd 19.57 ± 1.01 abcd 9.34 ± 1.14 abcd 12.21 ± 0.61 abcd 11.00 ± 0.62 abcd 110.76 ± 13.49 abcd 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 < 0.05 vs. sham.

b P < 0.05 vs. 3 weeks-HF.

c P < 0.05 vs. 4 weeks-HF.

d P < 0.05 vs. 6 weeks-HF.


X. Chen et al./Neuropeptides 51 (2015) 63–73

66 X. Chen et al./Neuropeptides 51 (2015) 63–73 Fig. 1. AVP and aldosterone levels in the

Fig. 1. AVP and aldosterone levels in the plasma and heart tissue of rats with heart failure.

(A) Plasma aldosterone levels. n = 5, 6, 5, 8, 5 for the sham, 3w-HF, 4w-HF, 6w-HF and 9w-HF groups, respectively.

*P < 0.05 vs. sham.

(B) Heart aldosterone levels. n = 5, 6, 5, 8, 5 for the sham, 3w-HF, 4w-HF, 6w-HF and 9w-HF groups, respectively.

*P < 0.05 vs. sham.

(C) Plasma AVP levels. n = 5 for all groups.

*P < 0.05 vs. sham.

(D) Correlation analysis for AVP and aldosterone.

highest level in the 9-week-HF group. The plasma AVP levels of the heart failure groups were higher than the levels of the sham group and increased with the development of heart failure, peaking in the 9-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 was negatively correlated with LVEF, serum Na + level (correlation coef- ficient r = − 0.856, Fig. 2A; r = − 0.904, Fig. 2C, respectively) and positively 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 failure rats was observed in both the mRNA (Fig. 3B) and protein (Fig. 3C) levels compared with the sham group (P < 0.05). AVP protein and mRNA expression increased with the development of heart failure, peaking at 9 weeks after MI. The AVP mRNA levels of each group were statistically significantly different (P < 0.05).

3.5. AVP expression and secretion in CMECs upregulated by Ang


Primary CMECs showed the antigenic characteristics of endo- thelial cell-specific marker CD31 and coagulation factors VIII (Fig. 4A).

X. Chen et al./Neuropeptides 51 (2015) 63–73


X. Chen et al./Neuropeptides 51 (2015) 63–73 67 Fig. 2. Correlation analysis between AVP and cardiac

Fig. 2. Correlation analysis between AVP and cardiac function index. Correlation between AVP concentration and LVEF (A), LVEDD (B), serum Na + (C), and hydroxyproline (D).

AVP was mainly distributed in the cytoplasm (Fig. 4B). The Ang II group showed elevated fluorescent intensity compared with the control group. Although the losartan-incubated group showed weak- ened fluorescent intensity, Ang II induced AVP mRNA expression in a concentration-dependent manner (Fig. 5A). AVP protein expres- sion increased with Ang II concentration, peaking at 10 7 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-dependent and time-dependent manner (Fig. 6A and 6B). The ET-1 mRNA level in rat CMECs also increased after AVP treatment (P<0.05; Fig. 6D). The CTGF expression in rat CFs significantly increased after 24 hours of AVP treatment compared with the control group (Fig. 6E). SR49059 inhibited the proliferation of CFs and the expression of CTGF and ET-1 (Fig. 6C–E).

4. Discussion

In our study, we demonstrated that the elevation of plasma AVP levels is related to the severity of cardiac dysfunction. Another im- portant discovery was the presence of local cardiac AVP autocrine in rats with heart failure. We first identified that CMECs were the cellular sources of increased cardiac AVP levels. Moreover, we ob- served that AVP participates in hyponatremia, promotes the proliferation of CFs and up-regulates the expression of ET-1 and CTGF, thus contributing to ventricular remodelling. Heart failure is a complex clinical disorder with progressive myo- cardial remodelling. It causes cardiac dysfunction associated with the activation of various interrelated neurohormonal systems, in- cluding the sympathetic nervous system, RAAS, endothelin-1 and AVP (Chatterjee, 2005). In our study, neurohormonal activation mani- fested as increases in the plasma levels of AVP and aldosterone. Increased plasma AVP levels are associated with impaired heart func- tion. Notably, there were positive correlations between AVP and


X. Chen et al./Neuropeptides 51 (2015) 63–73

68 X. Chen et al./Neuropeptides 51 (2015) 63–73 Fig. 3. Local heart AVP expression in rats

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 μm.

(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 < 0.05 vs. sham; # P < 0.05 vs. 3w-HF; **P <0.05 vs. 4w-HF; ***P < 0.05 vs. 6w-HF.

(C) AVP protein expression in myocardial tissue, detected using ELISA. n = 10, 8, 10, 8, 8 for the sham, 3w-HF, 4w-HF, 6w-HF and 9w-HF groups, respectively.

*P < 0.05 vs. sham; # P < 0.05 vs. 3w-HF; **P <0.05 vs. 4w-HF; ***P < 0.05 vs. 6w-HF.

aldosterone (r = 0.907). The plasma levels of AVP in the rats dete- riorating cardiac function increased over time, peaking at 9 weeks post-MI. Further, plasma AVP levels were negatively correlated with serum Na + and LVEF, but positively correlated with LVEDD and myo- cardial hydroxyproline contents. Besides, it is well documented that aldosterone concentration is proportional to the severity of heart failure (Pitt, 2012). Therefore, the plasma AVP levels in the rats with heart 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 overt symptoms of heart failure (Nakamura et al., 2006). The present study might support the notion of AVP as a potential biomarker for the diagnosis and severity of heart failure. Aaldosterone promotes water- sodium retention and myocardial fibrosis, thus accelerating the development 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). The V1a receptors mediate vasoconstriction and are localized primar- ily on vascular smooth muscle cells. V2 receptors mediate antidiuretic effects and are highly expressed in the kidneys, which is crucial for water 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 secretion from 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-sodium retention, 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 after myocardial infarction (Milik et al., 2014). The consequence of V1a receptor activation is vasoconstriction, leading to pressure over- load that causes cardiac hypertrophy and cardiac function decrease in heart failure. Additionally, in our study, plasma AVP levels were consistent with HW/BW. This finding indicates that AVP may mediate cardiac hypertrophy. The above effects, if sustained, may exacer- bate cardiac dysfunction to a greater extent in the failing heart, thus creating a vicious cycle. That is, excessive AVP activation can lead to heart failure deterioration if it is not corrected promptly. Vasopressin content in the hypothalamus increased in the rats with heart failure (Muders et al., 2002). AVP can be released into the plasma from the pituitary glands of rats with heart failure via non-osmotic regulation (Szatalowicz et al., 1981). The hypothala- mus is considered the principal locus of AVP synthesis (Vincent and

X. Chen et al./Neuropeptides 51 (2015) 63–73


X. Chen et al./Neuropeptides 51 (2015) 63–73 69 Fig. 4. Immunofluorescence staining of CMECs ( ×

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

Su, 2008). Indeed, experimental heart failure in rats augments AVP in the hypothalamus and raises plasma AVP levels (Ciosek and Drobnik, 2012). Moreover, with the enhanced activation of RAAS in heart-failure rats, elevated levels of Ang II pass through the blood– brain barrier, activate vasopressin neurons (Grobe et al., 2010), and exaggerate AVP expression. Significantly, our findings revealed that cardiac 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 were increased in Ang II/AT1 receptor-mediated CMECs. In the failing heart, AVP levels increased with the cardiac diameter, indicating that AVP secretion was related to ventricular volume overload. Data suggest that AVP expression increased in isolated, perfused rat hearts stressed by pressure overload (Hupf et al., 1999). According to Laplace’s law, the thickening of the ventricular wall in the early-middle stages of heart failure could maintain the wall stress at an appropriate level that avoids stress from too much change. However, in the late stages of 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 is regulated mainly by the increased ventricular wall stress. In recent years, numerous studies have focused on the effects of RAAS on the heart. RAAS activation induces systemic vasocon- striction, adjusts the water and electrolyte balance and activates other systems (e.g., AVP and aldosterone). However, chronic activation of these systems in heart failure can impair cardiac function and promote heart failure. Ang II increases cardiac after load via sys- temic vasoconstriction and can also induce myocyte hypertrophy and alter the myocardial matrix structure (Morgan and Baker, 1991). Aldosterone stimulates CFs proliferation, induces the activation of myofibroblasts and promotes the secretion of pro-fibrosis factors, leading to collagen matrix deposition (Johar et al., 2006). In the long term, 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 between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) (Spinale et al., 2000), which transition to a process of eccentric


X. Chen et al./Neuropeptides 51 (2015) 63–73

70 X. Chen et al./Neuropeptides 51 (2015) 63–73 Fig. 5. The mRNA and protein expression of

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 , 10 7 , 10 6 , 10 5 mol/L, respectively. *P < 0.05 vs. Ang II

0 mol/L.

(B) AVP protein level in the supernatant after 24 hours of treatment with Ang II. n = 4 for all groups.

*P < 0.05 vs. Ang II 0 mol/L.

(C) AVP mRNA level in CMECs after losartan (10 5 mol/L) intervention. n = 5 for all groups.

*P <0.05 vs. control.

# P < 0.05 vs. Ang II.

(D) AVP protein levels in the supernatant CMECs pre-treated with losartan10 5 mol/L. n = 4, 4, 5 for control, Ang II, and Ang II + losartan, respectively.

*P <0.05 vs. control. # P < 0.05 vs. Ang II.

hypertrophy with progressive ventricular dilation and wall thin- ning. Nevertheless, we do not know which neurohormonal systems’ activation plays leading roles in eccentric hypertrophy in ad- vanced heart failure. In our study, increased plasma AVP was involved in dilutional hyponatremia 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 was negatively correlated with the serum Na + concentration, indicat- ing that AVP was the leading mechanism of hyponatremia in heart failure. Hyponatremia is the most common electrolyte disorder among patients with heart failure; it occurs in 18% to 27% of heart failure patients (Klein et al., 2005). It is well documented that hy- ponatremia is an independent predictor of the mortality and readmission rates of heart failure patients (Klein et al., 2005). There- fore, identifying an effective method for correcting hyponatremia is an absolute necessity. AVP plays a central role in water reten- tion in chronic heart failure patients (Nielsen et al., 1999) and AVP dysregulation is the most common cause of hypotonic-hypervolemic hyponatremia (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 the progression of heart failure, and myocardial fibrosis is a particular characteristic of this remodelling. CF proliferation and excessive ac- cumulation of extracellular matrix collagen are believed to be the major pathologic causes of myocardial fibrosis. ET-1, a mitogenic factor and pro-fibrotic cytokine, induces the synthesis of type I and type 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). In patients with heart failure, ET-1 is significantly increased (Ohmae, 2011). CTGF, an important fibrogenic cytokine and downstream mol- ecule of Ang II and TGF-ß1, can stimulate CFs to excrete ECM and synthesise 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 and persistence of cardiac fibrosis (Dean et al., 2005). We demon- strated that AVP up-regulated CTGF and ET-1 expression and promoted CF proliferation. Additionally, AVP acts on cardiomyocytes to stimulate protein synthesis (Tahara et al., 1998). In vivo, we ob- served that AVP is positively correlated with the cardiac hydroxyproline content. Therefore, AVP participated in myocar- dial fibrosis. Furthermore, the circulating AVP levels continuously

X. Chen et al./Neuropeptides 51 (2015) 63–73


X. Chen et al./Neuropeptides 51 (2015) 63–73 71 Fig. 6. Effect of AVP on CFs and

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< 0.05 vs. control. # P <0.05 vs. AVP.

(D) The ET-1 mRNA expressed in CMECs pre-treated with 10 6 M SR49059 followed by 10 7 M AVP, as measured using RT-PCR. n = 5 for all groups.

*P <0.05 vs. control. #P < 0.05 vs. AVP.

(E) CTGF protein in CFs exposed to vehicle, 10 7 M AVP, and 10 7 M AVP + 10 6 M SR49059 for 24 hours, determined by Western blotting.

n = 5 for each group. The upper panel shows representative Western blot images, and the lower panel shows the pooled results. *P <0.05 vs. control. #P <0.05 vs. AVP.


X. Chen et al./Neuropeptides 51 (2015) 63–73

72 X. Chen et al./Neuropeptides 51 (2015) 63–73 Fig. 7. Schematic of the dynamic changes in

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

increased in advanced heart failure. These findings suggest that AVP may be an important cytokine of cardiac fibrosis during late-stage heart failure. Moreover, ET-1 is the most potent vasoconstrictor. In the development of heart failure, elevated AVP may participate in the exaggerated release of ET-1. Both AVP and ET-1 can reduce cor- onary blood flow and cardiac contractility and promote peripheral vasoconstriction, causing an increase in cardiac afterload and de- teriorating cardiac function (Large, 2002). Ventricle remodeling is shown as changes in myocardial fibrosis (molecular, cellular, inter- stitial), decreased capillary density, and function of the heart (Cohn et al., 2000). Accordingly, based on the above discussion, AVP me- diates ventricular remodelling in heart failure. The specific mechanisms 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 of the 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 fibroblasts and upregulation of pro-fibrogenic cytokine, aldosterone (Perraudin et al., 2006), CTGF and ET-1. At present, there are no therapies aimed at reducing CTGF production, nor did the endothelium receptor an- tagonist treatment of heart failure show benefits (O’Connor 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 causes hyponatremia and an increase in cardiac preload, and excessive AVP mediates myocardial fibrosis, which impairs ventricular wall com- pliance and cardiac function. Furthermore, AVP, as a vasoconstrictor,

causes myocardial ischaemia and increases the cardiac afterload, thus negatively 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 stress in 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 may play a role in the transition to eccentric hypertrophy, thus causing heart function to deteriorate. In addition, Ang II potentiates AVP se- cretion, whereas AVP induces aldosterone and ET-1 expression. In late-stage heart failure, the dilutional hyponatremia caused by in- creased AVP secretion may activate RAAS. Notably, AVP’s synergistic interactions with Ang II, aldosterone, and endothelin-1 cannot be neglected when examining impaired cardiac function. This study demonstrates the dynamic changes in local cardiac and circulating AVP and AVP’s role in heart failure induced by myo- cardial infarction (Fig. 7). Furthermore, this study provides an experimental basis for AVP receptor antagonists in the therapeu- tic window for effective heart failure treatment.


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

X. Chen et al./Neuropeptides 51 (2015) 63–73



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 at



failure after myocardial infarction in the rat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1734–R1745. Gao, X., He, X., Luo, B., Peng, L., Lin, J., Zuo, Z., 2009. Angiotensin II increases collagen I expression via transforming growth factor-beta1 and extracellular signal- regulated kinase in cardiac fibroblasts. Eur. J. Pharmacol. 606, 115–120. Geisterfer, A.A., Owens, G.K., 1989. Arginine vasopressin-induced hypertrophy of cultured rat aortic smooth muscle cells. Hypertension 14, 413–420. Goldsmith, S.R., Francis, G.S., Cowley, A.J., Levine, T.B., Cohn, J.N., 1983. Increased plasma arginine vasopressin levels in patients with congestive heart failure. J. Am. Coll. Cardiol. 1, 1385–1390. Grobe, J.L., Grobe, C.L., Beltz, T.G., Westphal, S.G., Morgan, D.A., Xu, D., et al., 2010. The brain renin-angiotensin system controls divergent efferent mechanisms to regulate fluid and energy balance. Cell Metab. 12, 431–442. Gutkowska, J., Miszkurka, M., Danalache, B., Gassanov, N., Wang, D., Jankowski, M., 2007. Functional arginine vasopressin system in early heart maturation. Am. J. Physiol. Heart Circ. Physiol. 293, H2262–H2270. Hinson, J.P., Vinson, G.P., Porter, I.D., Whitehouse, B.J., 1987. Oxytocin and arginine vasopressin stimulate steroid secretion by the isolated perfused rat adrenal gland. Neuropeptides 10, 1–7. Hupf, H., Grimm, D., Riegger, G.A., Schunkert, H., 1999. Evidence for a vasopressin system in the rat heart. Circ. Res. 84, 365–370. Johar, S., Cave, A.C., Narayanapanicker, A., Grieve, D.J., Shah, A.M., 2006. Aldosterone mediates angiotensin II-induced interstitial cardiac fibrosis via a Nox2-containing NADPH oxidase. FASEB J. 20, 1546–1548. Kim, J.K., Summer, S.N., Wood, W.M., Brown, J.L., Schrier, R.W., 1997. Arginine vasopressin secretion with mutants of wild-type and Brattleboro rats AVP gene.