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The Novel Mas agonist, CGEN-856S, Attenuates
Isoproterenol-Induced Cardiac Remodeling and
Myocardial Infarction Injury in Rats
Sı
´lvia Q. Savergnini
1
, Danielle Ianzer
1
, Mariana B. L. Carvalho
1
, Anderson J. Ferreira
2
, Gerluza A. B. Silva
2
,
Fu
´lvia D. Marques
1
, Anto
ˆnio Augusto B. Peluso
1
, Merav Beiman
3
, Gady Cojocaru
3
, Yossi Cohen
3
,
Alvair P. Almeida
1
, Galit Rotman
3
, Robson A. S. Santos
1
*
1Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil, 2Department of Morphology, Federal University of Minas
Gerais, Belo Horizonte, MG, Brazil, 3Compugen Ltd., Tel Aviv, Israel
Abstract
CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac
remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying
mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg?kg
21
?day
21
)in
Wistar rats. After a 7-day treatment period with CGEN-856S (90 mg?kg
21
?day
21
) or vehicle, the cardiomyocyte diameter was
evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and
quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left
ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 mg?kg
21
?day
21
) or saline was administered for 14
days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with
Masson’s trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we
evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S
reduced the degree of ISO-induced hypertrophy (13.9160.17 mm vs. 12.4160.16 mm in the ISO+CGEN-856S group). In
addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-
856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-
856S administration significantly decreased the infarct area (23.6862.78% vs. 13.9564.37% in the MI+CGEN-856S group).
These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and
p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and
MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.
Citation: Savergnini SQ, Ianzer D, Carvalho MBL, Ferreira AJ, Silva GAB, et al. (2013) The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac
Remodeling and Myocardial Infarction Injury in Rats. PLoS ONE 8(3): e57757. doi:10.1371/journal.pone.0057757
Editor: Michael Bader, Max-Delbru
¨ck Center for Molecular Medicine (MDC), Germany
Received October 29, 2012; Accepted January 24, 2013; Published March 1, 2013
Copyright: ß2013 Savergnini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: S.Q.S. was supported by a fellowship from Conselho Nacional de Desenvolvimento Cientı
´fico e Tecnolo
´gico (CNPq) and F.D.M. was supported by
a fellowship from Coordenac¸a
˜o de Aperfeic¸oamento de Pessoal de Nı
´vel Superior (CAPES). This study was supported by a Compugen Grant and by Ministe
´rio de
Cie
ˆncia e Tecnologia/Fundac¸a
˜o de Amparo a
`Pesquisa do Estado de Minas Gerais (Fapemig)/Instituto Nacional de Cie
ˆncia e Tecnologia-INCT-NanoBiofar. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors would like to declare that Merav Beiman, Gady Cojocaru, Yossi Cohen and Galit Rotman are affiliated to the commercial
funders of this research (Compugen Ltd.) and this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: robsonsant@gmail.com
Introduction
Cardiac remodeling is an adaptive response to the pathogenesis
of several heart diseases that interferes with the function and
structure of the myocardium [1–3]. This structural remodeling
process predisposes patients to an increased risk of adverse cardiac
events, including myocardial ischemia, myocardial infarction (MI),
arrhythmias, and sudden cardiac death [3]. A growing body of
evidence indicates that the renin-angiotensin system (RAS) plays
an important role in the development and progression of cardiac
remodeling. Angiotensin (Ang) II, the main end product of the
RAS cascade, stimulates the biosynthesis of cardiac extracellular
matrix (ECM) proteins, leading to interstitial and perivascular
fibrosis [4,5], and cardiomyocyte hypertrophy [6]. On the other
hand, numerous studies have proposed that the cardioprotective
axis of the RAS composed by Ang-converting enzyme (ACE) 2,
Ang-(1–7), and the Mas receptor counterregulates these Ang II
actions in the heart [7–11]. Indeed, it has been reported that Ang-
(1–7) reduces the growth of cardiac myocytes through activation of
the G protein-coupled receptor (GPCR) Mas [12] and inhibits
cardiac fibroblast-mediated collagen deposition [13]. Additionally,
the hearts of Mas-deficient mice exhibit marked changes in ECM
protein expression leading to a profibrotic profile accompanied by
cardiac dysfunction [14,15]. Grobe et al. [8,16] demonstrated that
Ang-(1–7) prevented cardiac fibrosis elicited by either deoxycorti-
costerone acetate (DOCA)-salt treatment or Ang II infusion,
independent of blood pressure changes. Moreover, AVE 0991,
a nonpeptide Ang-(1–7) analog, prevents the development of
isoproterenol (ISO)-induced hypertrophy and collagen deposition
[17]. Recently, He et al. [18] reported that AVE 0991 prevents
Ang II-induced myocardial hypertrophy in a dose-dependent
manner. Taken together, these findings indicate that the ACE2/
PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e57757
Ang-(1–7)/Mas axis is involved in the prevention and attenuation
of cardiac remodeling.
The evidence supporting the cardioprotective effects of the
ACE2/Ang-(1–7)/Mas axis prompted us to search for novel Mas
ligands. We used a computational biology discovery platform that
utilizes machine learning algorithms designed to predict novel
GPCR ligands cleaved from secreted proteins by convertase
proteolysis, which are extracted from the Swiss-Prot protein
database [19,20]. The predicted ligands were synthesized and
screened for activation of 152 GPCRs using calcium flux and
cyclic adenosine monophosphate (cAMP) assays [20]. Two novel
peptide ligands, P61S and P33V, displayed high specificity for
Mas, eliciting calcium influx in Mas-overexpressing Chinese
hamster ovary (CHO) cells [20]. Furthermore, P61S, designated
CGEN-856S, does not activate either Ang II type 1 (AT
1
) or type
2 (AT
2
) receptors [20,21].
We recently reported that CGEN-856S elicits nitric oxide (NO)-
dependent vasodilation mediated by Mas in rat and mice aorta
rings [21]. Additionally, picomolar concentrations of this peptide
(40 pmol/L) induced an antiarrhythmogenic effect, as demon-
strated by reduced incidence and duration of reperfusion
arrhythmias in isolated rat hearts [21]. In addition, acute
CGEN-856S administration produced a dose-dependent decrease
in the blood pressure of spontaneously hypertensive rats (SHR)
[21]. Importantly, the actions of CGEN-856S are inhibited by the
Mas antagonist A-779 or by genetic deletion of this receptor [21].
In general, the data obtained following the use of this peptide
resemble to those reported previously for Ang-(1–7) and AVE
0991. Thus, in the present study, we hypothesized that CGEN-
856S might mimic the antihypertrophic and antifibrotic effects
induced by Ang-(1–7) in rat hearts. To test this hypothesis, we
evaluated the cardiac structure of ISO-treated and of infarcted
rats. In addition, the effects of CGEN-856S administration on
AKT and endothelial NO synthase (e-NOS) activation were
investigated.
Materials and Methods
Ethics Statement and Animals
Male Wistar rats weighing 2402300 g were obtained from the
animal facility of the Biological Sciences Institute of the Federal
University of Minas Gerais. The experimental protocols were
approved by the Ethics Committee in Animal Experimentation of
the Federal University of Minas Gerais, Brazil (CETEA-UFMG),
in accordance with the National Institutes of Health (NIH)
Guidelines for the Care and Use of Laboratory Animals (protocol
#149/10).
Isoproterenol Induction of Hypertrophy
Osmotic mini-pumps (AlzetH, model 2001) containing CGEN-
856S (90 mg?kg
21
?day
21
, 7 days) or saline were implanted
subcutaneously (sc) under anesthesia (10% ketamine/2% xylazine,
0.1 mL/100 g, intraperitoneally [ip]). After recovery from anes-
thesia, the animals were divided into 4 groups: i) saline +vehicle
(olive oil, 1 mL?g
21
?day
21
, sc, 7 days), ii) saline +ISO
(2 mg?kg
21
?day
21
, sc, 7 days), iii) CGEN-856S +vehicle; and
(iv) CGEN-856S +ISO. Moreover, as a positive control, an
additional group of rats were treated with losartan
(1 mg?kg
21
?day
21
, gavage, 7 days) +vehicle (olive oil,
1mL?kg
21
?day
21
, sc, 7 days) or losartan (1 mg?kg
21
?day
21
,
gavage, 7 days) +ISO (2 mg?kg
21
?day
21
, sc, 7 days). The final
gavage and sc injection volumes were approximately 0.5 and
0.2 mL, respectively.
Histological Analysis
At the end of the 7-day ISO treatment period, the rats were
sacrificed by decapitation and the hearts were immediately
removed. The left ventricles were fixed in 4% paraformaldehyde
for 48 h at room temperature. The tissues were dehydrated by
sequential washes with 70%, 80%, 90%, and 100% ethanol and
embedded in ParaplastHX-tra Tissue Embedding Medium
(McCormick Scientific). Transversal sections (6 mm) were cut
starting from the base area of the left ventricle at 40-mm intervals
and stained with hematoxylin and eosin for cell morphometry
(n = 4 for each group). The cardiomyocyte diameter was evaluated
in the tissue sections (324 for each animal) using an ocular
micrometer calibrated with a stage micrometer adapted to a light
microscope (BX 60, Olympus) at 1006magnification and analyzed
using Image Pro Express software. Only cardiomyocytes cut
longitudinally with the nuclei and cellular limits visible were used
for analysis (an average of 15 cardiomyocytes for each slice). The
diameter of each myocyte was measured across the region
corresponding to the nucleus. Approximately 50 cardiomyocytes
were analyzed for each animal.
Immunofluorescence Analysis
Immunofluorescence labeling and quantitative confocal micros-
copy were used to investigate the distribution and quantity of type
I and III collagen and fibronectin present in the left ventricles of
the animals included in the ISO protocol (n = 425 rats/group).
The hearts were enclosed in Tissue Tek OCT compound (Miles
Scientific, Chicago, IL, USA), immediately frozen in liquid
nitrogen, and stored at -80uC. Ventricular sections (7 mm) were
obtained using a cryostat at -20uC, mounted on slides, fixed with
ethanol for 10 min, and dried at room temperature. The slides
were rehydrated with phosphate-buffered saline (PBS) for 10 min
and incubated in blocking solution (1% bovine serum albumin
[BSA] and 0.1% Tween 20 in PBS) at room temperature for
30 min. The sections were incubated overnight at 4uC with one of
the following primary antibodies: rabbit anti-human type I
collagen (1:100, Rockland), rabbit anti-human type III collagen
(1:100, Rockland), or rabbit anti-human fibronectin (1:200,
Rockland). All antibodies were diluted with a 1:10 dilution of
blocking solution. After 425 PBS rinses, donkey anti-rabbit
immunoglobulin G (IgG) conjugated with Alexa Fluor 488
Figure 1. Effects of CGEN-856S and losartan administration on
the cardiomyocyte diameters of isoproterenol-treated rats.
Animals were treated with isoproterenol (ISO) for 7 days to induce heart
hypertrophy or with olive oil as a control. The effects of CGEN-856S
were compared to those of saline as a negative control (Veh) or losartan
(LOS) as a positive control. Values are expressed as mean 6standard
error of the mean (SEM), n = 425 animals. *P,0.05 vs. oil +Veh;
#
P,0.05 vs. ISO+Veh;
a
P,0.05 vs. ISO+LOS.
doi:10.1371/journal.pone.0057757.g001
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Figure 2. Effects of CGEN-856S and losartan administration on the deposition of type I collagen (CO I), type III collagen (CO III), and
fibronectin (FN) in the left ventricles of isoproterenol (ISO)-treated rats. (A) Representative confocal photomicrographs and (B)
quantification of CO I, CO III, and FN in the left ventricles of animals treated with CGEN-856S. (C) Representative confocal photomicrographs and (D)
quantification of CO I, CO III, and FN in the left ventricles of animals treated with losartan. Values are expressed as arbitrary units (mean gray value 6
SEM, n = 425 animals). *P,0.05 vs. ISO+vehicle.
doi:10.1371/journal.pone.0057757.g002
Cardiac Effects of a Novel Mas Agonist
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(1:200, Molecular Probes) and DRAQ5 (1:1000, Biostatus) were
added for 1 h in the dark at room temperature. Following PBS
washes, the sections were mounted in 25% glycerol/75% PBS and
viewed with a laser scanning confocal microscope (Zeiss 510
Meta). Optimal confocal settings (aperture, gain, and laser power)
were determined at the beginning of each imaging session and
Figure 3. Effects of CGEN-856S and captopril administration on (A) systolic tension, (B) diastolic tension, (C) +dT/dt, (D) -dT/dt, (E)
coronary flow, and (F) heart rate of rat hearts with myocardial infarction (MI). Values are expressed as mean 6SEM, n = 728 animals.
*P,0.05 vs. sham;
#
P,0.05 vs. MI+vehicle;
a
P,0.05 vs. MI+captopril.
doi:10.1371/journal.pone.0057757.g003
Cardiac Effects of a Novel Mas Agonist
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then held constant during the analysis of all samples. For
quantitative analysis of collagen I and III and fibronectin, we
used ImageTool 2.0 image analysis software to measure the
fluorescence intensity of the randomly selected images. The 12-bit
images were captured and analyzed in the gray scale range of
02255. The fluorescence intensity was calculated as an average of
the area (i.e., the sum of the gray values of all pixels divided by the
number of pixels in the area) and the values were recorded as
arbitrary units.
Myocardial Infarction Procedure
Under anesthesia with 10% ketamine/2% xylazine (0.1 mL/
100 g, ip), the rats were placed in the supine position on a surgical
table, tracheotomized, intubated, and ventilated with room air
using a respirator for small rodents. Subdermal electrodes were
placed for electrocardiography (ECG). The chest was opened by
left thoracotomy at the fourth or fifth intercostal space. To expose
the heart, a small retractor was used to maintain rib separation.
After pericardial incision, the heart was quickly removed from the
Figure 4. Effects of CGEN-856S and captopril administration on left ventricular infarct area. (A) Representative photomicrographs and (B)
quantification of the infarct area of animals treated with CGEN-856S or captopril. Values are expressed as mean 6SEM, n = 728 animals. MI:
myocardial infarction. *P,0.05 vs. sham;
#
P,0.05 vs. MI+vehicle.
doi:10.1371/journal.pone.0057757.g004
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Figure 5. Effects of CGEN-856S and Ang-(1–7) administration on AKT phosphorylation and on the quantity of p-AKT. (A)
Representative immunoblots demonstrating the presence of Mas in Mas-transfected CHO cells (CHO-Mas) and the absence of Mas in untransfected
cells (CHO-K1). (B) Effects of CGEN-856S and Ang-(1–7) administration (10
29
and 10
27
mol/L) for 10 min on p-AKT levels in CHO-Mas cells. (C) The
absence of effects of CGEN-856S and Ang-(1–7) administration (10
27
mol/L) for 10 min on p-AKT levels in CHO-K1 cells. (D) Effects of CGEN-856S and
Ang-(1–7) administration (10
29
mol/L) for 5 min on AKT phosphorylation in CHO-Mas cells. Ang-(1–7) (10
29
and 10
27
mol/L) was used as a positive
control and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and total AKT were used as loading controls. *P,0.05 vs. control. Results are
expressed as the mean 6SEM of 426 experiments.
doi:10.1371/journal.pone.0057757.g005
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Figure 6. Effects of CGEN-856S and Ang-(1–7) administration on p-eNOS levels. CGEN-856S and Ang-(1–7) administration (10
27
mol/L)
increased the quantity of (A) p-eNOS Ser1177, but not (B) p-eNOS Thr495 in CHO-Mas cells. (C) The absence of the effects of CGEN-856S and Ang-(1–
7) administration (10
27
mol/L) on p-eNOS Ser1177 levels in CHO-K1 cells. Cells were exposed to the agonist for 10 min. Ang-(1–7) was used as
a positive control and GAPDH was used as a loading control. *P,0.05 vs. control. Results are expressed as the mean 6SEM of 426 experiments.
doi:10.1371/journal.pone.0057757.g006
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thoracic cavity and turned to the left to allow access to the
proximal left anterior descending (LAD) coronary artery. A 4-0 silk
suture was snared around the LAD and tightly ligated to occlude
the vessel. To increase the survival rate of the animals, coronary
ligation was performed on a more distal portion of the LAD. The
heart was then replaced and the chest was closed with 4-0 silk
sutures. Sham-operated rats were treated in the same manner,
although the coronary artery was not ligated. After the surgical
procedures, ECG tracings were obtained to confirm myocardial
ischemia, i.e., ST-segment elevation and increased R-wave
amplitude. Infarcted rats received CGEN-856S
(90 mmg?kg
21
?day
21
) or vehicle (saline) administered through
osmotic mini-pumps (AlzetH, model 2002) for 14 days. An
additional group of infarcted rats received captopril
(1 mg?kg
21
?day
21
, 14 days) through daily gavage. Fourteen days
after infarction induction, the rats were sacrificed and cardiac
function was evaluated.
Isolated Heart Preparation
The animals (n = 728 rats/group) were decapitated 10215 min
after ip injection of 400 IU of heparin. The thorax was opened,
then the heart was carefully dissected and perfused with Krebs-
Ringer solution (KRS) containing (in mmol/L): 118.4 NaCl,
4.7 KCl, 1.2 KH
2
PO
4
, 1.2 MgSO
4
,7H
2
O, 2.5 CaCl
2
,2H
2
O,
11.7 glucose, and 26.5 NaHCO
3
. The perfusion fluid was
maintained at 3761uC with a pressure of 65275 mmHg and
constant oxygenation (5% CO
2
/95% O
2
). A force transducer was
attached through a heart clip to the apex of the ventricles to record
the contractile force (tension, g) on a computer by a data-
acquisition system (Biopac System). A diastolic tension of
1.060.2 g was applied to the hearts. Electrical activity was
recorded on ECG (Nihon Kohden) with the aid of 2 cotton wicks
placed directly on the surface of the right atrium and left ventricle.
Coronary flow was measured every 5 min by collecting and
determining the volume of heart effluent during a 1-min interval.
After 15220 min of stabilization, the functional parameters
(systolic tension, diastolic tension, 6dT/dt, heart rate, and
coronary flow) were recorded for an additional 30-min period.
Quantification of the Myocardial Infarct Area
At the end of the perfusion, the left ventricles (n =628 for each
group) were fixed in 4% Bouin’s fixative for 24 h at room
temperature. The tissues were dehydrated by sequential washes
with 70%, 80%, and 90% ethanol, 3 washes with 100% ethanol,
and 3 washes with xylene, and then imbedded in paraffin.
Transverse sections (6 mm) of the left ventricles were cut starting
from the median area immediately below the left coronary artery
ligation at 40-mm intervals and stained with Masson’s trichrome to
quantify the infarct area. The infarct area was measured in 2 tissue
sections (both at the median area, one proximal and the other
distal to the coronary ligation of the left ventricle) of each animal.
Images (406magnification) were obtained using a JVC TK-1270/
RGB microcamera. The built-in KS300 software built of
a Kontron Elektronick/Carl Zeiss image analyzer was used for
infarct area quantification using the image segmentation function.
The data were expressed as mm
2
.
Cell Culture and Western Blot Analysis
CHO cells (American Type Culture Collection) stably trans-
fected with Mas and selected by neomycin (CHO-Mas) were
serum-starved 3 h before all experiments. Cells were stimulated for
10 min in serum-free Dulbecco’s modified Eagle’s medium
(DMEM) F-12 medium (Sigma-Aldrich, St. Louis, MO, USA)
with Ang-(1–7) (10
27
mol/L and 10
29
mol/L) or CGEN-856S
(10
27
mol/L and 10
29
mol/L) (Compugen Ltd., Israel). Optimal
conditions, such as the duration of the incubation and peptide
concentration, were chosen based on our initial concentration-
response studies [22]. Untransfected CHO cells were used as
controls and submitted to similar experimental conditions.
After the incubation period, the cells were washed with PBS to
remove metabolic residues and most of the floating cells. The
remaining cells were scraped into 180 mL of lysis buffer (50 mM
Na
4
P
2
O
7
, 50 mM NaF, 5 mM Na
2
EDTA, 5 mM NaCl, 5 mM
EGTA, 10 mM HEPES, 1% Triton X-100, and a specific EDTA-
free inhibitor cocktail) for each cell culture flask (75 cm
2
). The
lysate was transferred to a 1.5 mL tube, homogenized, centrifuged
at 14000 rpm for 20 min at 4uC, and the supernatant was
transferred for another tube. The protein concentration was
assayed using the Bradford protein method. Sixty micrograms of
protein were loaded on a 10% sodium dodecyl sulfate (SDS)
polyacrylamide gel, electrophoresed, and transferred to a nitrocel-
lulose membrane (Bio-Rad, Hercules, CA, USA). The membranes
were blocked in 5% dry milk for 1 h and incubated overnight with
one of the following primary antibodies at 4uC: total AKT (1:1000,
Cell Signaling Technology, Danvers, MA, USA), p-AKT Ser473
(1:500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS
Ser1177 (1:500, Cell Signaling Technology, Danvers, MA, USA),
p-eNOS Thr495 (1:500, Cell Signaling Technology, Danvers,
MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
(1:1000, Cell Signaling Technology, Danvers, MA, USA), and
Mas (1:500) [22]. The secondary antibody was added for 1 h at
room temperature. Protein band detection was performed using
the Odyssey scanning system (Li-Cor, USA) using Odyssey
software. The results were quantified by densitometry (Odyssey
software), normalized for the GAPDH or total AKT levels, and
then the ratio of the experimental values to the control values was
calculated.
Statistical Analysis
Data were expressed as mean 6standard error of the mean
(SEM). Histological data from each animal were obtained by
averaging all values acquired in each tissue section. Statistical
analysis was performed using one-way analysis of variance
(ANOVA) followed by the Bonferroni post hoc test. Confocal
microscopy data were expressed as the percentage of the mean
gray value in relation to the maximum value acquired in the ISO-
treated group of each imaging session and the statistical analysis
was performed using an unpaired Student’s t-test followed by the
Mann-Whitney Utest. One-way ANOVA followed by the
Newman-Keuls post hoc test was used to evaluate the cardiac
function. P,0.05 was considered statistically significant.
Results
The cardiomyocyte cross-sectional area, a measure of cardiac
hypertrophy, was significantly increased in ISO +vehicle-treated
animals compared to oil +vehicle-treated rats (11.1760.09 mm vs.
13.9160.17 mm in ISO +vehicle-treated rats, Figure 1). CGEN-
856S treatment reduced the degree of cardiac hypertrophy, as
evidenced by a significant decrease in the cardiomyocyte diameter
(13.9160.17 mm vs. 12.4160.16 mm in ISO +CGEN-856S-
treated rats, Figure 1). Losartan administration also attenuated the
ISO-induced increase in the cardiomyocyte diameter
(13.9160.17 mm vs. 11.6360.08 mm in ISO +losartan-treated
rats, Figure 1). CGEN-856S or losartan +oil treatment did not
significantly affect the cardiomyocyte diameter.
To investigate the effects of CGEN-856S on cardiac fibrosis, we
evaluated the deposition of cardiac type I and III collagen and
Cardiac Effects of a Novel Mas Agonist
PLOS ONE | www.plosone.org 8 March 2013 | Volume 8 | Issue 3 | e57757
fibronectin using immunofluorescence labeling and quantitative
confocal microscopy. ISO administration resulted in a significant
increase in the deposition of type I and III collagen and fibronectin
(Figure 2). CGEN-856S treatment significantly reduced the
deposition of type I collagen (60.5765.52% vs. 30.6363.00% in
ISO+CGEN-856S-treated rats, Figures 2A and 2B), type III
collagen (63.5464.84% vs. 45.3562.96% in ISO+CGEN-856S-
treated rats, Figures 2A and 2B), and fibronectin (55.2865.84%
vs. 29.9364.48% in ISO+CGEN-856S-treated rats, Figures 2A
and 2B). Similar effects on cardiac fibrosis were obtained when
ISO-treated rats were administered losartan (Figures 2C and 2D).
The cardioprotective actions of CGEN-856S in the ISO model
prompted us to study its effects on MI. As shown in Figure 3, MI
resulted in reduced systolic tension and decreased velocities of
contraction and relaxation (+dT/dt and -dT/dt, respectively)
when compared to the sham-operated group. CGEN-856S
treatment normalized the systolic tension and attenuated the
decrease in the 6dT/dt induced by MI (Figures 3A, 3C, and 3D).
In addition, CGEN-856S administration produced a slight but
significant increase in the coronary flow when compared with
infarcted rats (Figure 3E). No significant changes were observed in
the diastolic function or heart rate among the groups (Figures 3B
and 3F). The actions of CGEN-856S on the cardiac function of
infarcted rats were similar to those effects observed in infarcted
rats treated with captopril, an ACE inhibitor used as a positive
control (Figure 3). Importantly, CGEN-856S treatment reduced
the infarct area when compared to the vehicle-treated group
(23.6861.94% vs. 15.6863.15% in MI+CGEN-856S-treated
rats). In contrast, captopril did not induce any significant effect
on the size of the infarct area (Figure 4).
In order to ascertain whether CGEN-856S stimulates similar
intracellular pathways as Ang-(1–7), we assessed the effects of
CGEN-856S administration on AKT phosphorylation and the
level of p-AKT and p-eNOS in CHO cells transfected with Mas.
As observed in Figure 5A, transfected CHO cells expressed Mas
while untransfected CHO cells (CHO-K1) did not. After 10 min
of stimulation, Ang-(1–7) and CGEN-856S (10
27
mol/L) signif-
icantly increased the level of p-AKT in CHO-Mas cells (Figure 5B).
On the other hand, administration of 10
27
mol/L of CGEN-856S
or Ang-(1–7) did not affect the level of p-AKT in untransfected
CHO cells (Figure 5C). Of note, 10
29
mol/L of Ang-(1–7) and
CGEN-856S also augmented AKT phosphorylation in CHO-Mas
cells (Figure 5D). Furthermore, we observed that both Ang-(1–7)
and CGEN-856S (10
27
mol/L) significantly increased the level of
p-eNOS Ser1177 (Figure 6A), but not p-eNOS Thr495 (Figure 6B)
in CHO-Mas cells. No significant changes in the level of p-eNOS
Ser1177 were observed in untransfected CHO cells (Figure 6C).
Discussion
The most important findings of the present study were that
CGEN-856S, a novel Mas agonist, attenuated the cardiac damage
induced by ISO treatment and MI. Specifically, CGEN-856S
treatment prevented ISO-induced myocardial hypertrophy and
fibrosis, improved heart function, and reduced myocardial injury
in infarcted rats. CGEN-856S has been described as a specific Mas
agonist with a .1000-fold lower affinity for AT
2
receptors than
Ang II. In addition, no evidence of CGEN-856S binding to AT
1
receptors was observed [21]. Thus, we presented strong evidence
that Mas activation using a specific agonist induces cardioprotec-
tive effects, as demonstrated in 2 distinct models of cardiac failure.
Importantly, we were able to exclude any contribution of AT
2
receptors to these effects, as the affinity of CGEN-856S for AT
2
receptors is quite low.
CGEN-856S was first described as a Mas agonist by Shemesh
et al. in 2008 [20]. The authors showed that this peptide elicits
calcium influx in Mas-transfected CHO cells [20]. Additionally,
we observed that CGEN-856S induces vasorelaxation through an
NO- and Mas-dependent mechanism in rat and mouse aorta rings
[21]. Further evidence of CGEN-856S binding to Mas was
obtained by the observation that the Ang-(1–7) analogue D-Ala
7
-
Ang-(1–7) (A-779) abolishes the vasorelaxant effects of CGEN-
856S and by the effective CGEN-856S-induced displacement of
fluorescent Ang-(1–7) [FAM-Ang-(1–7)] binding in Mas-trans-
fected CHO cells [21]. Additionally, this vasodilative effect of
CGEN-356S was absent in the aortic rings of Mas-knockout mice
[21].
The antihypertrophic effect of Ang-(1–7) has been extensively
reported. Transgenic rats [TGR(A1–7)3292] that possess a 2.5-
fold increase in plasma Ang-(1–7) levels showed attenuated ISO-
induced heart hypertrophy [11]. Tallant et al. [12] observed that
Ang-(1–7) directly inhibits the growth of cultured cardiomyocytes
through Mas activation. The antihypertrophic effects of Ang-(1–7)
on Ang II-treated cardiomyocytes were prevented by N(G)-nitro-l-
arginine methyl ester and 1H-1,2,4oxadiazolo4,2-aquinoxalin-1-
one, suggesting that these effects are mediated by the NO/cyclic
guanosine monophosphate (cGMP) pathway [23]. Furthermore,
the nonpeptide analog of Ang-(1–7), AVE 0991, prevented Ang II-
induced myocardial hypertrophy by putatively inhibiting the
transforming growth factor (TGF)-b1/Smad2 signaling pathway
[18]. In line with these findings, the novel Mas agonist, CGEN-
856S, also reduced cardiomyocyte hypertrophy in rats challenged
with ISO, a b-adrenergic agonist capable of inducing cardiac
remodeling.
Another well-characterized action elicited by Ang-(1–7)/Mas is
its antifibrotic effect. Grobe et al. [16] demonstrated that Ang-(1–
7) selectively prevents collagen deposition in DOCA-salt rats. In
an in vitro study, Ang-(1–7) inhibited collagen formation as
measured by [
3
H]proline incorporation in cardiac fibroblasts of
adult rats and decreased the mRNA expression of various growth
factors, including TGF-1, endothelin-1, and leukemia inhibitory
factor [13]. Nadu et al. [9] reported that the ISO-induced increase
of type I and III collagen and fibronectin deposition observed in
normal rats was attenuated in transgenic rats expressing an Ang-
(1–7)-producing fusion protein [TGR(A1–7)3292]. In addition, it
was reported that these transgenic rats are protected against
cardiac dysfunction and fibrosis and show an attenuated increase
in blood pressure after DOCA-salt treatment [24]. DOCA-salt
[TGR(A1–7)3292] rats showed an important local increase in left
ventricular Ang-(1–7) levels, which might have contributed to the
reduced cardiac dysfunction and fibrotic lesions observed in these
animals [24]. The compound AVE 0991 also decreased ISO-
induced ECM protein deposition [17]. Thus, our current data are
in agreement with previous studies using 2 well-known Mas
agonists, Ang-(1–7) and AVE 0991 [25,26]. These data corrob-
orate the concept that Mas stimulation regulates cardiac
remodeling and indicate that the cardioprotective effects of
CGEN-856S are mediated by Mas activation.
Also, according to previous studies using Ang-(1–7) and the Mas
agonist AVE 0991 [25,26], activation of this receptor by CGEN-
856S elicited significant improvements in cardiac function of
infarcted hearts. CGEN-856S treatment restored the systolic
tension, attenuated the MI-induced decrease in 6dT/dt, and
increased the coronary flow as compared to control infarcted rats.
The beneficial effects of Ang-(1–7)/Mas on cardiac function are
one of the most important actions of ACE2/Ang-(1–7)/Mas axis
activation. This includes both vascular and muscular effects
[7,11,14,15,17]. Thus, the availability of compounds such as
Cardiac Effects of a Novel Mas Agonist
PLOS ONE | www.plosone.org 9 March 2013 | Volume 8 | Issue 3 | e57757
CGEN-856S that specifically activate Mas represents an important
step toward translating these findings into clinical practice.
It is important to note that we previously demonstrated that
CGEN-856S treatment produced merely a small decrease in the
blood pressure of normotensive Wistar rats. This effect was
observed only after infusion of higher doses of CGEN-856S (30
and 300 ng?kg
21
?min
21
). No significant changes in the heart rate
were noted [21]. Thus, we believe that the functional and
structural cardiac effects induced by CGEN-856S observed in our
current study were not due to decreases in blood pressure.
We found that CGEN-856S stimulates AKT phosphorylation
and increases the level of p-AKT and p-eNOS. These data suggest
that, similarly to Ang-(1–7), CGEN-856S induces its beneficial
effects through Mas activation via an eNOS/AKT-dependent
pathway. Evidence for the involvement of Mas in the actions of
CGEN-856S has been reported previously [21]. This new peptide
induced NO-dependent vasodilation mediated by Mas in rat and
mouse aorta rings [21]. Therefore, we hypothesized that CGEN-
856S could trigger underlying mechanisms related to Mas
stimulation. Consistent with our hypothesis, we observed that
AKT and e-NOS were activated by CGEN-856S. Indeed, it has
been reported that Ang-(1–7) elicits AKT phosphorylation in
endothelial cells and cardiomyocytes [22,27,28]. It is important to
note that high levels of NO/NOS might induce pro-oxidant
effects. When NO reacts with superoxide, it generates the oxidant
anion peroxynitrite (ONOO
2
), which provokes lipid peroxidation,
nitrosation of amino acid residues, and disruption of cell
membranes, cell signaling, and cell survival. Peroxynitrite also
exerts proinflammatory actions [29]. Thus, a complete future
study evaluating the effects of CGEN-856S on the oxidative
balance of cardiomyocytes is warranted.
One may argue that 10
29
mol/L of CGEN-856S significantly
increased AKT phosphorylation (ratio between p-AKT and total
AKT, Figure 5D) but not the level of p-AKT (ratio between p-
AKT and GAPDH, Figure 5B) in Mas-transfected CHO cells.
However, this assertion is not true, as a careful analysis of the p-
AKT level data (Figure 5B) reveals that CGEN-856S treatment
almost doubled the quantity of p-AKT in CHO cells. Thus, we
believe that these data did not achieve statistical significance
merely due to the manner in which the data were organized, i.e.,
both concentrations of CGEN-856S (10
29
mol/L and 10
27
mol/
L) were placed in the same graph (Figure 5B).
Of note, in contrast to Ang-(1–7), CGEN-856S presented no
evidence of ACE inhibitory activity and showed low affinity to
AT
1
and AT
2
receptors [21]. This suggests that the cardioprotec-
tive effects induced by CGEN-856S in these cardiac remodeling
models might be independent of any action on ACE activity or
other angiotensin receptors. Also, it is interesting to note that the
CGEN-856S compound is more stable than Ang-(1–7) [21].
However, all of these possibilities warrant confirmation in cardiac
tissues.
In summary, our current findings demonstrated that CGEN-
856S treatment attenuates ISO and MI-induced heart damage
likely through a mechanism involving the Mas/eNOS/AKT
pathway. These data further support the protective role of Ang-(1–
7) in the cardiovascular system and provide evidence that
stimulation of this GPCR might be a potential therapeutic
approach for cardiovascular diseases.
Author Contributions
Conceived and designed the experiments: SQS MB GC YC APA GR AJF
RASS. Performed the experiments: SQS DI MBLC GABS FDM AABP.
Analyzed the data: SQS DI MBLC GABS FDM AABP MB GC YC APA
GR AJF RASS. Contributed reagents/materials/analysis tools: RASS.
Wrote the paper: SQS AJF RASS.
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