Neuropeptides 51 (2015) 6373

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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

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 *
a

Department of Cardiology, First Aliated 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 2015
Available online 18 March 2015
Keywords:
Heart failure
Myocardial brosis
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 heart
failure and to determine whether AVP mediates ventricular remodeling.
MethodsWe 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 anterior 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 broblasts (CFs) were studied using methylthiazol tetrazolium assay, Western blotting 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 positively 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 expression of endothelin-1 and connective tissue growth factor.
ConclusionCMECs 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 deteriorating cardiac function. AVP may promote ventricular remodeling. Thus, AVP could be an important
mediator of myocardial brosis 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 supraoptic and paraventricular hypothalamus nuclei and released via the
posterior pituitary in response to hyperosmolarity, hypotension, or
hypovolemia. AVP is attracting attention for its biological properties such as the regulation of body uid osmolality, blood volume,
and vascular tone (Baylis, 1987; Thibonnier, 2003). Studies have reported 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 antagonist, 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, Ministry of Health, Guangzhou, China.
* Corresponding author. Department of Cardiology, First Aliated Hospital, Sun
Yat-sen University, Guangzhou 510080, China.
E-mail address: xiurengao@163.com (X. Gao).
http://dx.doi.org/10.1016/j.npep.2015.03.003
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 generally 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 advanced 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 uid 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

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X. Chen et al./Neuropeptides 51 (2015) 6373

AVP concentration at various time points in the progression of heart

failure.
AVP is present in plasma of homozygous Bratttelboro, centrally
AVP-decient 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 increased 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
AVP expression in rat myocardial tissue with heart failure and explored the cellular origin of AVP.
Moreover, AVP plays an important role in cardiovascular homeostasis. Whereas the vascular effects of AVP are well characterised,
AVPs 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 brosis, has yet
to be illustrated. Connective tissue growth factor (CTGF) mediates
the development of myobroblasts by enhancing transforming growth
factor (TGF) 1s ability to induce broblasts to differentiate into
myobroblasts, a critical process in brosis (Zeisberg et al., 2000).
Multiple neuroendocrine systems are involved in the development of heart failure, including the renin-angiotensin-aldosterone
system (RAAS), the AVP system, the natriuretic peptide system, and
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 claried. This study explores these neurohormonal systems activated in heart failure, with a focus on the
role 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 were
obtained from the Experimental Animal Centre of Sun Yat-sen University (Guangzhou, China). All procedures were approved by the
Experimental Animal Ethics Committee of Sun Yat-sen University.
Experimental myocardial infarction-induced heart failure was produced 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
3 weeks post-surgery. The rats with a left ventricular ejection fraction (LVEF) no higher than 45% and a weak cardiac impulse in the
left ventricular anterior wall were randomly divided into ve experimental heart failure groups: (1) the sham-operated group, which
was used as a control group (sham, n = 10); (2) the 3 weeks postinfarction group (3w-HF, n = 10); (3) the 4 weeks post-infarction
group (4w-HF, n = 10); (4) the 6 weeks post-infarction group (6wHF, n = 10); and (5) the 9 weeks post-infarction group (9w-HF, n = 15).
Twenty rats with LVEF >45 % or death were excluded.

serum (NBS, Gibco, USA) and 100 g/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 24 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 broblasts (CFs) were isolated as previously described (Gao et al., 2009), and grown in supplemented DMEM media
containing 10% FBS. The CFs were treated with AVP, SR49059, or
vehicle.
2.3. Echocardiography measurements
Echocardiography was performed 3 weeks post-surgery and 1
day before the sacrice to evaluate the changes in cardiac morphology and blood ow. The echocardiography was performed by
an experienced operator using ESAOTE ultrasound Doppler equipment. 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 enumerated 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 injection 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 hypertrophy. To estimate collagen production, the hydroxyproline level in
the left ventricle was determined using the hydroxyproline assay
according to the manufacturers instructions (BioVision, USA).
Left ventricle tissue specimens were cross-sectioned at the level
of the papillary muscle, xed and dehydrated in 10% formaldehyde, and embedded in paran for immunohistochemistry of AVP
(1:5000; Millipore, USA).
2.5. Enzyme-linked immunosorbent assay
Blood from the abdominal aortas was collected in sodium citrate
anticoagulant tubes before sacrice. The blood was centrifuged at
3000 r/min at 4 C, and the supernatant was collected and kept at
80 C. The myocardial tissue samples were grounded thoroughly
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 aldosterone. 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
immunosorbent assay (ELISA) kit (ADI-900-017,Enzo; 10004377,
Cayman) according to the manufacturers instructions.

2.2. Cell culture in vitro

2.6. Quantitative real-time PCR
Wistar rats aged 57 days were obtained from the Experimental Animal Center at Sun Yat-sen University. Primary rat cardiac
microvascular endothelial cells (CMECs) were isolated as previously described (Nishida et al., 1993). The CMECs were grown in
Dulbeccos modied Eagles medium (DMEM, HyClone, USA) containing 10% foetal bovine serum (FBS, Gibco, USA), 10% newborn calf

Total RNA was extracted using Trizol (Sigma, USA) according to

the manufacturers instructions and quantied using NanoDrops spectrophotometer. Then 1 g of the isolated total RNA was reverse
transcribed using an Omniscript RT Kit (Qiagen, Australia) according to the manufacturers protocol. The single-strand cDNA was

X. Chen et al./Neuropeptides 51 (2015) 6373

amplied 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. Immunouorescence staining
CMECs in 24-well plates were pre-treated with losartan (10 M)
or vehicle for 1 hour and then stimulated with Ang II (1 M) or vehicle.
After 24 hours, the cells were xed in 4% paraformaldehyde (PFA),
blocked with 5% bovine serum albumin, and stained with rabbit antivasopressin antibody (1:1000; Millipore, USA) at 4 C overnight. On
the second day, after 3 washes, the cells were incubated with FITCconjugated 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 uorescence
microscope (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. Briey, the CFs were
seeded onto 96-well plates at a nal density of 5 103 cells/mL. After
exposure to AVP, SR49059, or vehicle, 20 L/mL of 5% MTT solution (Sigma, USA) was added to each well. The plates were then
incubated for 4 hours at 37 C. The supernatant was aspirated, and
150 L 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 g) were loaded onto
12% SDS electrophoresis plates and transferred onto PVDF membranes (Millipore, USA). The blots were incubated with the
appropriate primary antibodies (goat anti-CTGF polyclonal antibody, Santa Cruz; mouse anti--tubulin monoclonal antibody, Protein
Tech), followed by the corresponding HRP-conjugated secondary antibodies, and the proteins were revealed using the ECL system
(Millipore). -Tubulin was used as the loading control. The developed lms were scanned, and quantitative analysis was performed
using ImageJ software.

65

2.10. Statistical analysis

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

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 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 signicantly different. The
W/D also increased with the progression of heart failure; however,
there was no signicant difference between the 3-week-HF and the
4-week-HF groups (P > 0.05). The data of the other groups differed
signicantly. Compared with the sham group, the LVESD and LVEDD
of the heart failure groups increased, whereas the LVEF and LVFS
signicantly decreased (P < 0.05, Table 1). Among the heart failure
groups, the LVESD and LVEDD of the 6-week-HF and the 9-weekHF groups were signicantly higher than the LVESD and LVEDD of
the 3-week-HF and the 4-week-HF groups, whereas the LVEF and
LVFS were signicantly lower (P < 0.05). The serum Na+ levels of the
heart failure groups were signicantly 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 signicant difference 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 signicantly 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.
Parameters

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)

Sham

3 weeks-HF

4 weeks-HF

6 weeks-HF

9 weeks-HF

(n = 10)

(n = 10)

(n = 10)

(n = 10)

(n = 15)

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

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

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

3.72 0.15abcd
2.59 0.12abcd
5.13 0.43abcd
19.57 1.01abcd
9.34 1.14abcd
12.21 0.61abcd
11.00 0.62abcd
110.76 13.49abcd
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 weeksHF, 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.

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X. Chen et al./Neuropeptides 51 (2015) 6373

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 correlated with plasma aldosterone (correlation coecient 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 coefcient 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 expression 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 signicantly different (P < 0.05).
3.5. AVP expression and secretion in CMECs upregulated by Ang
II/AT1
Primary CMECs showed the antigenic characteristics of endothelial cell-specic marker CD31 and coagulation factors VIII (Fig. 4A).

X. Chen et al./Neuropeptides 51 (2015) 6373

67

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 uorescent intensity compared with the
control group. Although the losartan-incubated group showed weakened uorescent intensity, Ang II induced AVP mRNA expression in
a concentration-dependent manner (Fig. 5A). AVP protein expression 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-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 signicantly 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. 6CE).

4. Discussion
In our study, we demonstrated that the elevation of plasma AVP
levels is related to the severity of cardiac dysfunction. Another important discovery was the presence of local cardiac AVP autocrine
in rats with heart failure. We rst identied that CMECs were the
cellular sources of increased cardiac AVP levels. Moreover, we observed 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 myocardial remodelling. It causes cardiac dysfunction associated with
the activation of various interrelated neurohormonal systems, including the sympathetic nervous system, RAAS, endothelin-1 and
AVP (Chatterjee, 2005). In our study, neurohormonal activation manifested as increases in the plasma levels of AVP and aldosterone.
Increased plasma AVP levels are associated with impaired heart function. Notably, there were positive correlations between AVP and

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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 < 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 deteriorating 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 myocardial 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 signicantly higher in patients with heart failure than in healthy agematched 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 watersodium retention and myocardial brosis, thus accelerating the
development of heart failure (Pitt, 2012). AVP is crucial for uid homeostasis 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 primarily 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 cortisolproducing adrenocortical tumours or hyperplasias (Hinson et al.,
1987; Perraudin et al., 2006). The increased AVP levels and the exaggerated release of aldosterone may participate in water-sodium
retention, resulting in volume expansion that exacerbates diastolic wall stress and heart function in heart failure. Furthermore,
increased V1a receptor expression in failing hearts has been reported (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 overload that causes cardiac hypertrophy and cardiac function decrease
in heart failure. Additionally, in our study, plasma AVP levels were
consistent with HW/BW. This nding indicates that AVP may mediate
cardiac hypertrophy. The above effects, if sustained, may exacerbate 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 hypothalamus is considered the principal locus of AVP synthesis (Vincent and

X. Chen et al./Neuropeptides 51 (2015) 6373

69

Fig. 4. Immunouorescence staining of CMECs (200). (A) Identication of CMECs with CD31 and factors VIII (green) using immunouorescence. 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).

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. Signicantly, our ndings 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 nding that is consistent with previous reports of immunoreactive 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 Laplaces 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 signicantly enlarged and thinner,
resulting in signicant ventricular wall stress. The ndings in the
present study indicate that local cardiac AVP expression during the

early and middle stages of heart failure is mainly regulated by ventricular 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 vasoconstriction, 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 systemic 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
myobroblasts and promotes the secretion of pro-brosis 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 intracardiac pressure and leads to water retention in heart failure (Chatterjee,
2005). With the progression of heart failure, the increasing ventricle 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

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X. Chen et al./Neuropeptides 51 (2015) 6373

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, 109, 108, 107, 106, 105 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 (105 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 losartan105 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 thinning. Nevertheless, we do not know which neurohormonal systems
activation plays leading roles in eccentric hypertrophy in advanced heart failure.
In our study, increased plasma AVP was involved in dilutional
hyponatremia and regulated the cardiovascular system via V1a receptors. 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, indicating 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 hyponatremia is an independent predictor of the mortality and
readmission rates of heart failure patients (Klein et al., 2005). Therefore, identifying an effective method for correcting hyponatremia
is an absolute necessity. AVP plays a central role in water retention 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 antagonists 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 brosis is a particular
characteristic of this remodelling. CF proliferation and excessive accumulation of extracellular matrix collagen are believed to be the
major pathologic causes of myocardial brosis. ET-1, a mitogenic
factor and pro-brotic cytokine, induces the synthesis of type I and
type III collagen bres and inhibits matrix metalloproteinases-1 expression in the CFs; consequently, extracellular matrix (ECM)
accumulates, promoting cardiac brosis (Shi-Wen et al., 2001). In
patients with heart failure, ET-1 is signicantly increased (Ohmae,
2011). CTGF, an important brogenic cytokine and downstream molecule of Ang II and TGF-1, can stimulate CFs to excrete ECM and
synthesise collagen, thereby causing cardiac brosis (Mori et al.,
1999). As other studies show, CTGF, a new marker of cardiac dysfunction (Koitabashi et al., 2008), is involved in the generation and
persistence of cardiac brosis (Dean et al., 2005). We demonstrated 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 observed that AVP is positively correlated with the cardiac
hydroxyproline content. Therefore, AVP participated in myocardial brosis. Furthermore, the circulating AVP levels continuously

X. Chen et al./Neuropeptides 51 (2015) 6373

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 with107 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 106 M SR49059 followed by 107 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, 107 M AVP, and 107 M AVP+106 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.

71

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X. Chen et al./Neuropeptides 51 (2015) 6373

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

increased in advanced heart failure. These ndings suggest that AVP

may be an important cytokine of cardiac brosis 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 coronary blood ow and cardiac contractility and promote peripheral
vasoconstriction, causing an increase in cardiac afterload and deteriorating cardiac function (Large, 2002). Ventricle remodeling is
shown as changes in myocardial brosis (molecular, cellular, interstitial), decreased capillary density, and function of the heart (Cohn
et al., 2000). Accordingly, based on the above discussion, AVP mediates ventricular remodelling in heart failure. The specic
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 contribution to cardiomyocytes hypertrophy by increasing protein synthesis
(Tahara et al., 1998), promotion proliferation of cardiac broblasts
and upregulation of pro-brogenic 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 antagonist treatment of heart failure show benets (OConnor et al.,
2003). Therefore, AVP receptor antagonists may provide a new therapeutic 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 brosis, which impairs ventricular wall compliance 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 important 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 brillar collagen degradation, 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 secretion, whereas AVP induces aldosterone and ET-1 expression. In
late-stage heart failure, the dilutional hyponatremia caused by increased AVP secretion may activate RAAS. Notably, AVPs 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 AVPs role in heart failure induced by myocardial infarction (Fig. 7). Furthermore, this study provides an
experimental basis for AVP receptor antagonists in the therapeutic window for effective heart failure treatment.

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

X. Chen et al./Neuropeptides 51 (2015) 6373

Funding
This work was supported by a grant from the Science Fund Committee 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
doi:10.1016/j.npep.2015.03.003.
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