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Clinical Science (2015) 128, 307319 (Printed in Great Britain) doi: 10.1042/CS20140215

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Angiotensin-(17) decreases skeletal muscle


atrophy induced by angiotensin II through a Mas
receptor-dependent mechanism

Laboratorio de Biologa y Fisiopatologa Molecular, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas & Facultad de Medicina,
Universidad Andres Bello, Santiago, Chile
Laboratorio de Fisiopatologa Integrativa, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas & Facultad de Medicina,
Universidad Andres Bello, Santiago, Chile
Millennium Institute on Immunology and Immunotherapy, Santiago, Chile
Centro de Regulacion Celular y Patologa (CRCP), Centro de Regeneracion y Envejecimiento (CARE), Laboratorio de Diferenciacion Celular y
Patologa, Departamento de Biologa Celular y Molecular, MIFAB, Pontificia Universidad Catolica de Chile, Santiago, Chile

Abstract

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Skeletal muscle atrophy is a pathological condition characterized by the loss of strength and muscle mass, an
increase in myosin heavy chain (MHC) degradation and increase in the expression of two muscle-specific ubiquitin
ligases: atrogin-1 and MuRF-1. Angiotensin II (AngII) induces muscle atrophy. Angiotensin-(17) [Ang-(17)], through
its receptor Mas, produces the opposite effects than AngII. We assessed the effects of Ang-(17) on the skeletal
muscle atrophy induced by AngII. Our results show that Ang-(17), through Mas, prevents the effects induced by
AngII in muscle gastrocnemius: the decrease in the fibre diameter, muscle strength and MHC levels and the
increase in atrogin-1 and MuRF-1. Ang-(17) also induces AKT phosphorylation. In addition, our analysis in vitro
using C2 C12 myotubes shows that Ang-(17), through a mechanism dependent on Mas, prevents the decrease in
the levels of MHC and the increase in the expression of the atrogin-1 and MuRF-1, both induced by AngII. Ang-(17)
induces AKT phosphorylation in myotubes; additionally, we demonstrated that the inhibition of AKT with MK-2206
decreases the anti-atrophic effects of Ang-(17). Thus, we demonstrate for the first time that Ang-(17) counteracts
the skeletal muscle atrophy induced by AngII through a mechanism dependent on the Mas receptor, which involves
AKT activity. Our study indicates that Ang-(17) is novel molecule with a potential therapeutical use to improve
muscle wasting associated, at least, with pathologies that present high levels of AngII.

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Clinical Science

www.clinsci.org

Franco Cisternas1 , Mara Gabriela Morales1 , Carla Meneses , Felipe Simon, Enrique Brandan,
Johanna Abrigo , Yaneisi Vazquez and Claudio Cabello-Verrugio

Key words: angiotensin-(17), atrophy, Mas, muscle wasting, skeletal muscle

INTRODUCTION

Skeletal muscle atrophy is the loss of muscle mass concomitantly with a decrease in strength generated by muscle wasting.
Several characteristic features are observed in skeletal muscle atrophy, such as the decrease in the sarcomeric proteins as the
heavy and light chains of myosin, which is translated into a
decrease in the generation of net force [1,2]. The causes of
skeletal muscle atrophy are diverse and include chronic diseases
such as cardiac, renal and pulmonary failure [37]. One of the

common aspects of these diseases is the high level of circulating angiotensin II (AngII), the main peptide of the classical
axis of the reninangiotensin system (RAS). Recently, AngII
has been demonstrated to be a key peptide in the regulation of
skeletal muscle function by modulating tissue structure, damage and contractile activity in muscle diseases, such as in muscular dystrophy or insulin resistance [810]. In addition, several studies have described the effects of AngII as an atrophic
factor in skeletal muscle [11,12]. One of the main mechanisms
involved in the skeletal muscle atrophy induced by AngII is the

Abbreviations: AngII, angiotensin II; Ang-(17), angiotensin-(17); ACE, angiotensin-converting enzyme; AT1 R, angiotensin type 1 receptor; AT2 R, angiotensin type 2 receptor; FoxO,
forkhead box O; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF-1, insulin-like growth factor-I; MHC, myosin heavy chain; RAS, reninangiotensin system; UPS,
ubiquitinproteasome system; WGA, wheat germ agglutinin.
1 These authors contributed equally to the present work
Correspondence: Dr Claudio Cabello-Verrugio (email claudio.cabello@unab.cl).


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agonists were osmotically infused through micropumps (AlzetDurect) implanted under ketamine/xylazine anaesthesia into the
dorsal area of the animal [35]. At the end of the experiment,
the animals were killed under anaesthesia and the gastrocnemius
muscles were dissected, removed and rapidly frozen and stored at
80 C until processing. All protocols were conducted in strict
accordance and with the formal approval of the Animal Ethics
Committee at the Universidad Andres Bello.

Cell cultures

The skeletal muscle cell line C2 C12 (American Type Culture Collection) was grown and differentiated until day 5 as described
previously [42]. The myotubes were incubated with the following peptides or antagonists: AngII (Sigma), Ang-(17) (Phoenix
Pharmaceuticals), A779 (10 M; CPC Scientific), MK-2206
(10 M; Selleckchem), losartan (5 m; Tocris) and PD123319
(5 m; Tocris).

increase in protein breakdown, although an inhibitory component


on protein synthesis has also been reported [1216]. The enhanced protein degradation induced by AngII is dependent
on the activation of the ubiquitinproteasome system (UPS)
[13,16]. The muscle-specific E3 ubiquitin ligases atrogin-1 and
MuRF-1 belong to the UPS and are involved at the beginning of
skeletal muscle atrophy [1720]. Thus, they are up-regulated in
muscle atrophy caused by different situations, including AngII
treatment [18,21,22].
Among the key pathways that modulate skeletal muscle atrophy is AKT signalling. In atrophic conditions, AKT phosphorylation has been described as being decreased [23]. It has
been demonstrated that the activation of the AKT pathway can
up-regulate protein synthesis and decrease proteolysis, whereas
inactivation of this pathway is involved in protein degradation
through UPS activation [2427].
There is an alternative axis to AngII in which the main
peptide is angiotensin-(17) [Ang-(17)]. This peptide exerts
a number of opposite actions to AngII, including the inhibition of cell proliferation, vasodilation and antihypertensive effects [2832]. Ang-(17) produces its effects through
the G-protein-coupled transmembrane receptor Mas [33,34].
Among the effects of Ang-(17) through the Mas receptor
in skeletal muscle are the prevention of fibrosis and autonomic dysfunction associated with Duchenne muscular dystrophy, as well as the decrease in AngII-induced insulin resistance
and transforming growth factor (TGF)- signalling induced by
AngII [3541].
The purpose of the present study was to assess the effects of
Ang-(17) on skeletal muscle atrophy induced by AngII in vitro
and in vivo. Our results have demonstrated that Ang-(17) prevents the atrophic effects mediated by AngII in the gastrocnemius
muscle and C2 C12 myotubes, which are dependent on the Mas
receptor. We also demonstrated that Ang-(17), through Mas, induces AKT phosphorylation in the gastrocnemius muscle, which
is dependent on Mas. In addition, we have demonstrated that
the inhibition of AKT with the inhibitor MK-2206 decreases the
anti-atrophic effect of Ang-(17) in the myotubes.
This is the first report that demonstrates the anti-atrophic
effects of Ang-(17) in skeletal muscle, counteracting the atrophy
induced by AngII, through a mechanism dependent on the Mas
receptor and AKT activity.

RNA isolation, reverse transcription and


quantitative real-time PCR

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The total RNA was isolated from the diaphragm muscles


using TRIzol (Invitrogen) according to the manufacturers instructions. The total RNA (1 g) was reverse-transcribed to
cDNA using random hexamers and the Superscript II reverse
transcriptase (Invitrogen). TaqMan quantitative real-time PCRs
were performed in triplicate, using an Eco Real-Time PCR System (Illumina), with pre-designed primer sets for mouse atrogin1, MuRF-1, angiotensin type 1 receptor (AT1 R), angiotensin
type 2 receptor (AT2 R), the housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) and -actin (TaqMan
Assays-on-Demand; Applied Biosystems). The mRNA expression was quantified using the comparative C t method (2CT ),
with GAPDH as the reference gene. The mRNA levels are expressed relative to the mean expression in the normal mice [43].

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Immunoblot analysis

MATERIALS AND METHODS

For skeletal muscle extracts, the diaphragm muscles were homogenized in Tris/EDTA buffer with a cocktail of protease inhibitors and 1 mM PMSF. Proteins were subjected to SDS/PAGE,
transferred on to PDVF membranes (Millipore) and probed
with mouse anti-MHC (1:3000 dilution) (MF-20; Developmental
Studies, Hybridoma Bank, University of Iowa), rabbit antiphospho-AKT (1:1000 dilution), rabbit anti-AKT (1:1000 dilution) (Cell Signaling Technology) and mouse anti-tubulin (1:5000
dilution) (Santa Cruz Biotechnology). All immunoreactions were
visualized by enhanced chemiluminescence (Thermo Scientific).

Animals

Fibre diameter determination and quantification

The C57BL/10J (12 weeks old) strain of mice was used, and the
animals were kept at room temperature with a 24 h nightday
cycle, water available ad libitum, and paired feeding with pellets. The male mice were randomized and separated into groups.
Three independent experiments were performed using 46 animals/group. Six experimental groups were designed: those treated
with the vehicle (water), AngII (1 g/kg per min), Ang-(17)
(100 ng/kg per min), AngII plus Ang-(17), A779 (100 ng/kg per
min) and AngII plus Ang-(17) plus A779. The peptides or ant-

Fibre diameter was detected by using wheat germ agglutinin


(WGA) staining, which labels glycoproteins at the sarcolemma
[44,45]. Briefly, freshly frozen gastrocnemius muscles were sectioned and cryosections (7 m) were placed on glass slides,
fixed in 4 % paraformaldehyde followed by incubation with
AlexaFluor -594-conjugated WGA (Molecular Probes). Three
washes with PBS were performed between each step. After rinsing, the sections were mounted with a fluorescence mounting medium (DAKO) under glass cover slips, viewed and photographed


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Ang-(17) prevents skeletal muscle atrophy induced by AngII

Skeletal muscle histology


Freshly frozen gastrocnemius muscles were sectioned and cryosections (7 m) were placed on glass slides. Haematoxylin
and eosin staining was performed according to standard
procedures.

Contractile properties

RESULTS
Ang-(17) prevents the decrease in the muscle
strength and fibre diameter induced by AngII in
skeletal muscle in vivo
We evaluated the effects of Ang-(17) and the Mas receptor on
the AngII-induced atrophy in skeletal muscle. For this, the mice
were infused with AngII, Ang-(17), or their mixture, in the absence or presence of antagonist A779. The diameter of the gastrocnemius fibres was evaluated by staining with WGA around
the fibres (Figure 1A). Figure 1(B) shows the quantification of
the diameters measured in Figure 1(A), showing that AngII decreases the diameter, which is prevented by the co-treatment with
Ang-(17), but maintained when Ang-(17) and A779 were coadministrated together with AngII. AngII displaces the diameter
observed in control muscles towards a small size (Figure 1B,
panel a). Thus, AngII increases the percentage of small fibres
relative to the control (<20 m, 11 % compared with 5 %; 20
30 m, 55 % compared with 21 %). Concomitantly, AngII produces a decrease in the proportion of bigger fibres than control
(3040 m, 32 % compared with 61 %; >40 m, 2 % compared
with 13 %). Figure 1(B, panel b) shows that the administration of
Ang-(17) does not change the diameter relative to control. Interestingly, Figure 1(B, panel c) shows that the co-administration of
AngII plus Ang-(17) prevents the displacement of the fibre size
induced by AngII reaching a diameter similar to Ang-(17) alone
(<20 m, 4 %; 2030 m, 28 %; 3040 m, 57 %; and >40 m,
11 %). Figure 1(B panel c) shows that the co-administration of
A779 together AngII plus Ang-(17) abolishes the effects of
Ang-(17), showing an effect similar to AngII and suggesting
the participation of Mas receptor. The same Figure shows that
A779 alone presents a distribution of fibre size similar to control.
Similar results were obtained when histological analysis was performed in muscle section stained with haematoxylin and eosin
(Supplementary Figure S1) corroborating the effect of Ang-(17)
on the fibre size.
Next, we evaluated whether the effect of Ang-(17) on the
diameter is translated to an effect on muscle strength, and
we decided to measure the maximal isometric tetanic force in
the gastrocnemius of mice treated with AngII, Ang-(17), or
their mixture, in the absence or presence of A779. Figure 2(A)
shows that AngII decreases the strength by 25 % when compared
with the mice treated with the vehicle. The same Figure shows
that the administration of Ang-(17) prevents this decrease induced by AngII, restoring the strength to 97 % of the maximum
force of the mice treated with the vehicle. Figure 2(B) shows
that the administration of A779, together with AngII and Ang(17), abolished the prevention mediated by Ang-(17), reaching
a decrease in the strength similar to the AngII alone, confirming the participation of the receptor Mas. Other tests to measure
muscle strength in live mice (weightlifting test) showed that animals infused with AngII have less capacity to generate strength

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Strength test

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After treatment, the mice were anaesthetized, the gastrocnemius


muscles were removed and the muscle contractile properties were
measured as previously described. Briefly, the maximum isometric tetanic force (Po) was determined from the plateau of
the frequencyforce relationship with stimuli ranging between
1 and 100 Hz, measuring three repeated tetanic stimulations.
After functional testing, the muscles were removed from the bath,
trimmed of their tendons and any adhering non-muscle tissue,
blotted once on filter paper, and weighed. The muscle mass and
optimum muscle length (Lo) were used to calculate the specific
net force [force normalized per total muscle fibre cross-sectional
area (CSA) in mN/mm2 ] [8,47,48].

test (Sigma Stat). A difference was considered statistically significant at a P value < 0.05.

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on the Motic BA310 epifluorescence microscope (Motic). Fibre


sizes were determined using the ImageJ software (NIH) on five
randomly captured images of gastrocnemius from each experimental condition (in a blinded fashion). Fibres were manually
selected and the minimal Ferets diameter of each fibre was quantified by the ImageJ software [46].

At the end of the treatment, the mice were subjected to a


measurement of muscle strength by a weightlifting test as described previously [49]. Briefly, the apparatus consisted of a
series of chain links of increasing length attached to a ball of
tangled fine wire. The number of links ranged from one to seven
with total weights between 20 and 98 g. Before performing the
test, the mice were subject to previous training (once for day,
during 2 weeks) before the treatment with peptides. To perform
the test, the mouse must grasp (with its forepaws) the first weight
(20 g) which is lying on the laboratory bench. When it grasps the
wire, the mouse is raised until the link is elevated from the bench.
At that moment, the time is registered. The criterion for success
is that the mouse maintains the links for 3 s, in which case the
mouse was tested on the next heavier weight (after testing all
of its cage mates on the first weight). If the mouse drops the
weight in less than 3 s, the time it held the weight was registered,
it was allowed to rest for approximately 10 s and the weight was
tried once again. If it failed three times, that terminated the trial,
and the mouse was assigned the maximum time/weight achieved.
If it held the weight for 3 s, then the next heaviest weight was
tried. The final score was calculated as the summation of the
product between links weight and the timed hold. The average
of three measures by each mouse was normalized by the body
weight.

Statistics
The statistical analysis was evaluated using the one-way or twoway ANOVA with a post-hoc Bonferroni multiple-comparison


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F. Cisternas and others

Figure 1

The decrease in the fibre diameter induced by AngII is prevented by Ang-(17) via the receptor Mas in skeletal
muscle
(A) C57BL10 male mice were systemically treated with the vehicle, AngII, Ang-(17), AngII + Ang-(17), A779 or AngII +
Ang-(17) + A779 for 7 days as described in the Materials and methods section. Cryosections of the gastrocnemius were
incubated with WGA used to delineate the fibre and further determine its diameter. The bar corresponds to 150 m. (B)
Quantitative analysis of the fibre diameters from experiments showed in (A), which was separated in four graphics (a, b,
c and d) to facilitate the analysis. A dotted line is shown as reference of the main pick of the fibre size in the control
muscle. The values are expressed as the percentage of the total fibres quantified (n = 3; P < 0.05).

than the control mice, which was prevented in the mice infused
with AngII plus Ang-(17) (Figure 2C). The same Figure shows
that the administration of A779 inhibits the preventive effects of
Ang-(17).
Together, these results strongly suggest that Ang-(17) improves the diameter of the fibres and the muscle strength of
mice treated with AngII through a mechanism that involves Mas
receptor.

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Systemic administration of Ang-(17) prevents the


decrease in the MHC and the increase in atrogin-1
and MuRF-1 induced by AngII in skeletal muscle
We evaluated the effects of Ang-(17) and A779 on the myosin
heavy chain (MHC) levels in the gastrocnemius muscle upon
the administration of AngII. Figure 3(A) shows that Ang-(17)
prevents the decrease in the levels of MHC induced by AngII. This
effect was abolished by the co-treatment with A779, suggesting

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Ang-(17) prevents skeletal muscle atrophy induced by AngII

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Figure 3 Ang-(17) prevents the decrease in the myosin heavy


chain induced by AngII in skeletal muscle
(A) C57BL10 male mice were systemically treated with the vehicle,
AngII, Ang-(17), AngII + Ang-(17), A779 or AngII + Ang-(17) +A779
for 7 days as described in the Materials and methods section. The
levels of the MHC were detected by Western blot analysis. The levels
of tubulin are shown as the loading control. (B) Quantitative analysis
of the experiments showed in (A). The levels of MHC normalized to
tubulin are expressed as percentages relative to vehicle, and corres
ponding to the means +
S.D. [n = 3; P < 0.05 compared with vehicle;
#
P < 0.05 compared with AngII; P < 0.05 compared with AngII +
Ang-(17)].

Figure 2 Angiotensin-(17), through the Mas receptor, prevents


the decrease in the muscle strength induced by AngII in skeletal
muscle
(A) The tetanic-specific force of the gastrocnemius from C57BL10
male mice with different treatments: vehicle, AngII, Ang-(17) or
AngII+Ang-(17). The values are represented as percentages of the
specific isometric force (mN/mm2 ) shown by muscles of mice treated
with vehicle. (B) Decrease in the tetanic force in the gastrocnemius from
C57BL10 male mice treated with AngII, Ang-(17), AngII+Ang-(17),
A779 or AngII+Ang-(17)+A779. The values are represented as percentages of the diminution relative to the vehicle group. (C) Limb
muscle strength measured by the weightlifting test in C57BL10 male
mice treated with the vehicle, AngII, Ang-(17), AngII+Ang-(17), A779
or AngII + Ang-(17) + A779. The value represents the score normalized by body weight as described in the Materials and methods section [n = 3; P < 0.05 compared with control; # P < 0.05 compared with
AngII; P < 0.05 compared with AngII + Ang-(17)].

the participation of Mas receptor. The quantification of these


experiments is shown in Figure 3(B).
To evaluate the effects of Ang-(17) on the UPS activated by
AngII in the gastrocnemius muscle, we evaluated the expression
of the E3 ligases atrogin-1 and MuRF-1. Figure 4 shows that the

increase in the expression of atrogin-1 and MuRF-1 induced by


AngII is prevented by co-treatment with Ang-(17). The same
Figures show that this effect mediated by Ang-(17) is decreased
by treatment with A779, suggesting the participation of Mas
receptor.
These results suggest that Ang-(17), through Mas, decreases typical molecular markers of atrophy induced by AngII
such as a decrease in MHC and increase in atrogin-1 and
MuRF-1.

Ang-(17) induces AKT phosphorylation through


the Mas receptor in skeletal muscle
Since AKT has been demonstrated to be a key signalling pathway in skeletal muscle atrophy [24,26], we decided to evaluate whether AKT is activated in skeletal muscle upon the treatment of mice with Ang-(17). Figure 5 shows that Ang-(17)
induces AKT phosphorylation in the gastrocnemius in a manner
dependent on the Mas receptor, as demonstrated by the decrease
in this phosphorylation upon co-treatment with the antagonist
A779.
These results suggest that Ang-(17) phosphorylates AKT
in skeletal muscle through a mechanism involving the Mas
receptor.


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Figure 4 Ang-(17)/Mas axis prevents the increase in the atrogin1 and MuRF-1 expression induced by AngII in the skeletal muscle
C57BL10 male mice were systemically treated with the vehicle, AngII,
Ang-(17), AngII + Ang-(17), A779 or AngII + Ang-(17) + A779 for
1 day as described in the Materials and methods section. At the end
of the treatment, the mRNA levels of atrogin-1 (A) and MuRF-1 (B) were
determined by RT-qPCR, using GAPDH as the reference gene in the
gastrocnemius. The expression was expressed as the fold of induction relative to vehicle and the value corresponding to the mean +
S.D.
[n = 3; P < 0.05 compared with vehicle; # P < 0.05 compared with
AngII; , P < 0.05 compared with AngII + Ang-(17)].

Figure 5 Ang-(17) induces the AKT phosphorylation through the


Mas receptor in skeletal muscle
C57BL10 male mice were systemically treated with the vehicle,
Ang-(17), A779 or Ang-(17) + A779 for 7 days as described in the
Materials and methods section. At the end of the treatment, protein
extracts were obtained from the gastrocnemius. (A) The levels of the
phospho and total-AKT were detected by Western blot analysis. (B)
The quantitative analyses of the experiments shown in (A). Levels of
phospho-AKT, normalized to the total-AKT are expressed as the fold
of induction relative to vehicle and corresponding to the mean +
S.D.
[n = 3; P < 0.05 compared with vehicle; # P < 0.05 compared with
Ang-(17)].

Ang-(17), through the Mas receptor, prevents the


atrophy induced by AngII in C2 C12 myotubes

To corroborate the results obtained in vivo, we decided to


evaluate the effects of AngII on the protein levels of MHC
in the C2 C12 myotubes. Supplementary Figure S2(A) shows
that AngII decreases the levels of MHC reaching the maximum effect at 10 M, with a fall of 35 % in the levels when
compared with the controls (Supplementary Figure S2B). In
addition, Supplementary Figure S2(C) shows that this effect
of AngII was observed from 48 h after incubation, reaching
35 % of the levels shown by the control cells in 72 h (Supplementary Figure S2D). Supplementary Figure S2(E) indicates that the decrease in MHC induced by AngII is mediated
through the AT1 R, as indicated by the prevention of this effect under the incubation of myotubes with losartan, an AT1 R
blocker.
In this context, we evaluated the effects of Ang-(17) on the
decrease in MHC induced by AngII. Figure 6(A) shows that Ang(17) prevents the diminution of the MHC induced by AngII in a
dose-dependent manner, reaching its maximum effect at 10 nM
(Figure 6B). Figures 6(C) and 6(D) show that Ang-(17) exerts
its effects through the Mas receptor, as was demonstrated by the
use of the antagonist of Mas, A779, which abolished the effect
of Ang-(17).

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Together, these results indicate that Ang-(17), through its


receptor Mas, has an antagonistic effect on the atrophy induced
by AngII in vitro.

The Ang-(17)/Mas axis decreases the expression


of atrogin-1 and MuRF-1 induced by AngII in
myotubes in vitro
We decided to evaluate the effects of Ang-(17) on the expression of the E3 ubiquitin ligases atrogin-1 and MuRF-1 induced by
AngII in the C2 C12 myotubes. For this, the myotubes were incubated with AngII in the absence or presence of Ang-(17) for 6 h
for RNA extraction. Figures 7(A) and 7(B) show that the increases
of 4-fold and 3-fold in the expression of atrogin-1 and MuRF-1
induced by AngII, respectively, were blunted by co-incubation
with Ang-(17) in a dose-dependent manner, reaching the maximum effect at 10 nM of Ang-(17). In addition, Figures 7(C)
and 7(D) show that the diminution mediated by Ang-(17) on the
expression of atrogin-1 and MuRF-1 induced by AngII is abolished when the A779 was used, suggesting the participation of
the Mas receptor.
In this context, we evaluated the specificity of the effect mediated by Ang-(17) relative to AT1 R, AT2 R and Mas receptor.
First, we observed in Supplementary Figure S3 that AngII produces a decrease in AT1 R but increase in AT2 R levels which is
prevented by Ang-(17) in vitro and in vivo. Then, we evaluated the specificity of Ang-(17) on the induction dependent on
AngII of atrogin-1 and MuRF-1. Supplementary Figure S4 shows
that the preventive effect of Ang-(17) on atrogin-1 and MuRF-1

Ang-(17), through the receptor Mas, prevents the decrease in the myosin heavy chain induced by AngII in the
C2 C12 myotubes
C2 C12 cells were differentiated for 5 days, and then incubated as indicated. At the end of the treatment, the levels of the
myosin heavy chain (MHC) and tubulin were detected by Western blot analysis (A, C). The levels of MHC normalized to
tubulin are expressed as percentages relative to cells treated with vehicle and the values correspond to the means +
S.D.
(B, D). (A) C2 C12 myotubes were pre-incubated with several concentrations of Ang-(17) for 1 h, and then incubated with
10 M AngII for 72 h. (B) Quantitative analysis of the experiments shown in (A). (C) The myotubes were pre-incubated with
Ang-(17) (10 nM) in absence or presence of 10 M A779, and then incubated with 10 M AngII for 72 h. (D) Quantitative
analysis of the experiments shown in (C) [n = 3; P < 0.05 compared with vehicle; # P < 0.05 compared with AngII +
Ang-(17)].

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Figure 6

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Ang-(17) prevents skeletal muscle atrophy induced by AngII

Figure 7

Ang-(17)/Mas axis prevents the increase in atrogin-1 and MuRF-1 expression induced by AngII in the C2 C12
myotubes
The C2 C12 myotubes from day 5 were pre-incubated with several concentrations of Ang-(17) for 1 h, and then incubated
with 10 M AngII for 6 h (A, C). To determine the participation of Mas, myotubes were pre-incubated with Ang-(17) (10 nM)
in the absence or presence of 10 M of A779 for 1 h and then incubated with 10 M AngII for 6 h (B, D). At the end of
this treatment, the mRNA levels of the atrogin-1 (A, C) and MuRF-1 (B, D) were determined by RT-qPCR and expressed

as the fold of induction relative to cells treated with vehicle. The values correspond to the mean +
S.D. [n = 3; P < 0.05
relative to cells treated with vehicle; # P < 0.05 compared with AngII; P < 0.05 compared with AngII + Ang-(17)].


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Figure 8

Ang-(17) induces AKT phosphorylation through the Mas receptor in the C2 C12 myotubes
The C2 C12 cells were differentiated for 5 days, and then incubated as indicated. At the end of the treatment, the levels of
the phospho and total-AKT were detected by Western blot analysis. Levels of phospho-AKT, normalized to the total-AKT are
expressed as the fold of induction relative to vehicle. The value of the quantification is expressed as the fold of induction
relative to the control cells. (A) The C2 C12 myotubes at day 5 were incubated with 100 nM Ang-(17) for several times. (B)
The quantitative analysis of the experiments shown in A. (C) The myotubes were incubated with several concentrations of
Ang-(17) for 15 min. (D) The graphic bar depicts the quantitative analysis of experiments shown in (C). (E) The myotubes
were pre-incubated with 10 M A779 and then incubated with 10 nM Ang-(17) for 15 min. (F) The quantification from
experiments is show in (E) [n = 3; P < 0.05 compared with vehicle; # P < 0.05 compared with Ang-(17)]. The molecular
masses are shown in kDa.

expression was not modified by the AT2 R blocker PD123319.


However, the pre-incubation with AT1 R blocker losartan completely abolished the effect of AngII, and therefore any further
effect of Ang-(17).
Together, these results suggest that Ang-(17) decreases the
expression of the atrogin-1 and MuRF-1 induced by AngII, with
the participation of the Mas receptor.

Ang-(17) induces the phosphorylation of AKT


through the Mas receptor in C2 C12 myotubes
Since Ang-(17) decreases the atrophy induced by AngII in vitro,
we decided to explore if AKT signalling is involved in this process. Figure 8(A) shows that Ang-(17) induces the AKT phosphorylation in a time-dependent fashion. The quantification depicted in the Figure 8(B) shows that the effect of Ang-(17) is at
a maximum at 15 min, and then falls, reaching the basal levels at
120 min after the incubation of Ang-(17). Figures 8(C) and 8(D)
show that the optimal dose of Ang-(17) was 10 nM, in which
the maximum effect in AKT phosphorylation was reached. To

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C 2015 Biochemical Society

evaluate the participation of the Mas receptor on AKT phosphorylation, the antagonist A779 was used. Figures 8(E) and
8(F) show that A779 prevents the phosphorylation of the AKT
induced by Ang-(17) in myotubes. Supplementary Figure S5
shows that Ang-(17) induces the AKT phosphorylation specifically through Mas receptor without participation of the AngII
receptors, since AT1 R or AT2 R blockers did not changed the AKT
phosphorylation induced by Ang-(17).
Together, these results suggest that Ang-(17) induces the
AKT phosphorylation in a dose- and time-dependent manner,
through a mechanism involving the Mas receptor.

The anti-atrophic effect of Ang-(17) is dependent


on AKT signalling
To evaluate the participation of AKT in the anti-atrophic effects
shown by Ang-(17), we used the inhibitor of AKT activity,
MK-2206, and evaluated the MHC, atrogin-1 and MuRF-1 upon
incubation with AngII and Ang-(17). Figure 9(A) shows that
the incubation with MK-2206 abolished the preventive effects

The inhibition of AKT activity abolishes the prevention mediated by the Ang-(17) of the muscle atrophy induced
by AngII in the C2 C12 myotubes
(A) The C2 C12 myotubes from day 5 were pre-incubated with 1 mM MK-2206 (inhibitor of AKT) for 1 h, and further with 10 nM
Ang-(17) for 1 h, and then incubated with 10 M AngII for 72 h. At the end of the treatment, the levels of the myosin heavy
chain (MHC) were detected by Western blot analysis. The levels of the tubulin are shown as the loading control. (B) The
quantitative analysis of the experiments is shown in A. The values are expressed as the percentage of the MHC normalized

#
to tubulin, and correspond to the means +
SD [n = 3; P < 0.05 compared with vehicle; P < 0.05 compared with AngII;

P < 0.05 compared with AngII + Ang-(17)]. (C, D) The myotubes were pre-incubated with MK-2206 and Ang-(17) as
described in (A). Then, the cells were incubated with 10 M AngII for 6 h. The expressions of atrogin-1 (C) and MuRF-1
(D) were detected as described in the Materials and methods section. The value corresponds to the means +
S.D. [n = 3;

P < 0.05 compared with vehicle; # P < 0.05 compared with AngII; P < 0.05 compared with AngII+Ang-(17)].

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th

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Figure 9

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y

Ang-(17) prevents skeletal muscle atrophy induced by AngII

of Ang-(17) over the decrease in MHC induced by AngII. Figure 9(B) depicts the quantification of the experiments shown
in Figure 9(A). Next, we evaluated the effects of MK-2206 on
the expression of atrogin-1 and MuRF-1. Pre-incubation with
MK-2206 blunted the prevention dependent on Ang-(17) in the
increase in the expression of atrogin-1 (Figure 9C) and MuRF-1
(Figure 9D) induced by AngII.
These results suggest that AKT activity participates in the
anti-atrophic effects mediated by Ang-(17).

DISCUSSION
This is the first study to demonstrate the counteracting effects of Ang-(17), through receptor Mas, on the wasting
induced by AngII in vivo and the skeletal muscle atrophy
in vitro. We observed that Ang-(17) prevents several events
induced by AngII associated with wasting, such as the decrease in the muscle strength, fibre diameter and the content
of MHC, and the increase in atrogin-1 and MuRF-1 expression. In addition, we demonstrated that Ang-(17) induced AKT
phosphorylation. Finally, our results suggest that Mas and AKT
activity participate in the anti-atrophic effects mediated by
Ang-(17).

Ang-(17) is a peptide that belongs to the non-classical axis


of RAS, which has the opposite effects of classical RAS, especially with respect to AngII. Our data suggest that in skeletal
muscle, the atrophy induced by AngII can be prevented by Ang(17) in vitro and in vivo. Thus, our data are in line with several studies that show the contrary effects of Ang-(17) and
AngII [2832]. Our results also show that Ang-(17) produces
its effects through the Mas receptor, since the use of antagonist A779 abolishes the protection dependent on Ang-(17) of
the muscle atrophy induced by AngII. Interestingly, we also
demonstrated that the effect of Ang-(17) is specifically mediated by Mas and not for the AT2 R as has been suggested by
some reports [50,51]. Comparably, several investigators have
described that Ang-(17) acts in skeletal muscle through the
Mas [35,37,38,40].
Interestingly, in the present report, we demonstrated that Ang(17) is able to induce the phosphorylation and activation of
AKT via the Mas receptor. Other investigators have also described the activation of AKT under treatment with Ang-(17)
in several tissues, including skeletal muscle [38,5254]. Whether
the effects of Ang-(17) are directly on the AKT or indirectly
through the secretion of other growth factors that activate the
AKT, such as insulin-like growth factor-I (IGF-1), must be evaluated. Interestingly, results not shown indicate that Ang-(17)
induces the expression of the IGF-1 and IGF-1 receptors in the


C The Authors Journal compilation 
C 2015 Biochemical Society

315

F. Cisternas and others

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y

forms of FoxO were used indicated that atrogin-1 and MuRF-1


were under the control of transcriptional FoxO [20,24,26]. In addition, FoxO activity is modulated by AKT since the overexpression of AKT produces the inactivation of FoxO [65]. When AKT
is activated by phosphorylation (e.g. by IGF-1), it is able to hyperphosphorylate the FoxO proteins, and thus, they are maintained in
the cytosol [64,66]. The inhibition of AKT phosphorylation (e.g.
under atrophic conditions) produces FoxO hypophosphorylation
and activation carrying the FoxO into the nucleus [65]. Thus,
treatment with Ang-(17) activates the AKT by phosphorylation
and could, in this manner, hypophosphorylate FoxO and keeps
it inactive, producing a decrease in the atrogin-1 and MuRF-1
expression.
Our study demonstrates for the first time that Ang-(17) has
the ability to prevent skeletal muscle atrophy, and it can be considered as a possible therapeutic tool to prevent this pathological
status with respect to chronic diseases or aging.

skeletal muscle and myotubes. Thus, we can explain that Ang-(1


7) could have a biphasic effect on AKT phosphorylation, since
we observed an early induction of AKT phosphorylation in vitro,
but a phosphorylation at 7 days in the gastrocnemius, when
Ang-(17) was administrated in vivo. Further studies must be
done to explore this issue.
Interestingly, Ang-(17) not only induces AKT phosphorylation, but also AKT activity, participating in the mechanism
through Ang-(17) exerting its anti-atrophic effects. Thus, our
results suggest that the inhibition of AKT through the inhibitor
MK-2206 abolishes the effects of Ang-(17). In skeletal muscle,
three AKT isoforms have been demonstrated: AKT-1 is involved
in embryonic development, post-natal survival and growth; AKT2 is essential in the glucose homoeostasis and regulation of insulin; and AKT-3 plays a main role in central nervous system
development [55]. Further studies to analyse which AKT isoform(s) is (are) activated in the effects of Ang-(17) on skeletal
muscle atrophy will be performed.
In skeletal muscle, we observed a restoration of the strength
towards a normal value, after treatment with Ang-(17). This observation is in agreement with recently published results, where
the muscle strength in dystrophic mice is increased, albeit the
genetic defects are present [35]. A possible cause is that Ang(17) can influence satellite cell function, improving its proliferation and/or differentiation ability. It has been described
that the alteration on the biology of satellite cells has a high
impact on the skeletal muscle atrophy associated with cancer
cachexia [56]. Along this line, it has also been described that
AngII modulates the activity of satellite cells, producing an inhibition of skeletal muscle regeneration by the suppression of
the proliferation in satellite cells, which could be a key factor
in the mechanism leading to skeletal muscle atrophy under
chronic disease [57]. Thus, the mechanism involved in the restoration of the strength is not clear, and further studies will be done
to address this point.
Our data clearly indicate that Ang-(17) prevents the decrease
in the MHC induced by AngII. In the literature, it has been suggested that there is a decrease in the sarcomeric proteins, such as
MHC and myosin light chain, in several forms of atrophy [1,58
60]. Thus, Ang-(17) could protect muscles from alterations in
the sarcomere structure, which has a direct influence on muscle
strength.
The results obtained in the present study also demonstrated the
prevention of Ang-(17) on the increase in atrogin-1 and MuRF1 induced by AngII. This observation is in agreement with the
down-regulation of the sarcomeric myosin protein levels which
are a main target of MuRF-1; although, it has also been suggested
that it is a target of atrogin-1, carrying it to proteasomal degradation [61]. Another target of atrogin-1 is the eukaryotic translation factor eIF3-f [62,63]. Thus, the atrogin-1-dependent eIF3-f
degradation decreases the protein synthesis, and the treatments
with Ang-(17) could prevent this pathway, favouring its antiatrophic effect. One possible mechanism involved in the decrease
in atrogin-1 and MuRF-1 expression by Ang-(17) is the regulation of the transcriptional activity of forkhead box O (FoxO).
This transcriptional factor induces the expression of atrogin-1 and
MuRF-1 [64]. Evidence from studies where constitutively active

CLINICAL PERSPECTIVES

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C 2015 Biochemical Society

r
r

We have studied the role of non-classical RAS in skeletal


muscle wasting.
We observed that Ang-(17), via Mas receptor, prevents
skeletal muscle atrophy, at least, via AKT phosphorylation.
Our results suggest that Ang-(17) could be therapeutically
used as an anti-atrophic peptide in several muscle disorders
that present with skeletal muscle atrophy.

AUTHOR CONTRIBUTION

Franco Cisternas, Mara Gabriela Morales, Johanna Abrigo, Yaneisi


Vazquez and Carla Meneses were responsible for carrying out
the experiments, and analysing and interpreting the data. Enrique
Brandan gave support for the force measurements in isolated
muscle. Claudio Cabello-Verrugio, Felipe Simon and Mara Gabriela Morales were involved in drafting the manuscript for publication. Claudio Cabello-Verrugio was responsible for conceiving all of
the experiments and was involved in analysing the data, preparing
them for publication and drafting the manuscript.

ACKNOWLEDGEMENT

We thank Darling Vera for the technical assistance.

FUNDING

The present study was supported by the Association-Francaise


Contre Les Myopathies [grant number AFM 16670 (to C.C.V.)], the
Fondo Nacional de Desarrollo Cientfico y Technologico (FONDECYT)
[grant numbers 1120380 (to C.C.V.), 3130593 (to M.G.M.),
1121078 (to F.S.), 1110426 (to E.B.)], the Millennium Institute
on Immunology and Immunotherapy [grant number P09-016-F (to
F.S.)], the CARE [grant number PFB12/2007 (to E.B.)], Fundacion
Chilena para Biologa Celular [grant number MF-100 (to E.B.)],

Ang-(17) prevents skeletal muscle atrophy induced by AngII

and Proyecto Interno UNAB [grant number UNAB-DI-281-13/R (to


C.C.V.)].

17
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