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-(1–7) [Ang-(1–7)], through its receptor Mas, produces the opposite effects than AngII. We assessed the effects of Ang-(1–7) on the skeletal muscle atrophy induced by AngII. Our results show that Ang-(1–7), 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-(1–7) also induces AKT phosphorylation. In addition, our analysis in vitro using C2C12 myotubes shows that Ang-(1–7), 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-(1–7) induces AKT phosphorylation in myotubes; additionally, we demonstrated that the inhibition of AKT with MK-2206 decreases the anti-atrophic effects of Ang-(1–7). Thus, we demonstrate for the first time that Ang-(1–7) 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-(1–7) is novel molecule with a potential therapeutical use to improve muscle wasting associated, at least, with pathologies that present high levels of AngII.
We have studied the role of non-classical RAS in skeletal muscle wasting.
We observed that Ang-(1–7), via Mas receptor, prevents skeletal muscle atrophy, at least, via AKT phosphorylation.
Our results suggest that Ang-(1–7) could be therapeutically used as an anti-atrophic peptide in several muscle disorders that present with skeletal muscle atrophy.
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 [3–7]. 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 renin–angiotensin 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 [8–10]. 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 increase in protein breakdown, although an inhibitory component on protein synthesis has also been reported [12–16]. The enhanced protein degradation induced by AngII is dependent on the activation of the ubiquitin–proteasome 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 [17–20]. 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 . 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 [24–27].
There is an alternative axis to AngII in which the main peptide is angiotensin-(1–7) [Ang-(1–7)]. This peptide exerts a number of opposite actions to AngII, including the inhibition of cell proliferation, vasodilation and antihypertensive effects [28–32]. Ang-(1–7) produces its effects through the G-protein-coupled transmembrane receptor Mas [33,34]. Among the effects of Ang-(1–7) 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)-β signalling induced by AngII [35–41].
The purpose of the present study was to assess the effects of Ang-(1–7) on skeletal muscle atrophy induced by AngII in vitro and in vivo. Our results have demonstrated that Ang-(1–7) prevents the atrophic effects mediated by AngII in the gastrocnemius muscle and C2C12 myotubes, which are dependent on the Mas receptor. We also demonstrated that Ang-(1–7), 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-(1–7) in the myotubes.
This is the first report that demonstrates the anti-atrophic effects of Ang-(1–7) in skeletal muscle, counteracting the atrophy induced by AngII, through a mechanism dependent on the Mas receptor and AKT activity.
MATERIALS AND METHODS
The C57BL/10J (12 weeks old) strain of mice was used, and the animals were kept at room temperature with a 24 h night–day 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 4–6 animals/group. Six experimental groups were designed: those treated with the vehicle (water), AngII (1 μg/kg per min), Ang-(1–7) (100 ng/kg per min), AngII plus Ang-(1–7), A779 (100 ng/kg per min) and AngII plus Ang-(1–7) plus A779. The peptides or antagonists were osmotically infused through micropumps (Alzet-Durect) implanted under ketamine/xylazine anaesthesia into the dorsal area of the animal . 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 Andrés Bello.
The skeletal muscle cell line C2C12 (American Type Culture Collection) was grown and differentiated until day 5 as described previously . The myotubes were incubated with the following peptides or antagonists: AngII (Sigma), Ang-(1–7) (Phoenix Pharmaceuticals), A779 (10 μM; CPC Scientific), MK-2206 (10 μM; Selleckchem), losartan (5 μm; Tocris) and PD123319 (5 μm; Tocris).
RNA isolation, reverse transcription and quantitative real-time PCR
The total RNA was isolated from the diaphragm muscles using TRIzol (Invitrogen) according to the manufacturer's instructions. The total RNA (1 μg) 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 atrogin-1, MuRF-1, angiotensin type 1 receptor (AT1R), angiotensin type 2 receptor (AT2R), the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (TaqMan Assays-on-Demand; Applied Biosystems). The mRNA expression was quantified using the comparative ΔCt method (2−ΔΔCT), with GAPDH as the reference gene. The mRNA levels are expressed relative to the mean expression in the normal mice .
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 anti-phospho-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).
Fibre diameter determination and quantification
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 μm) 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 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 Feret's diameter of each fibre was quantified by the ImageJ software .
Skeletal muscle histology
Freshly frozen gastrocnemius muscles were sectioned and cryosections (7 μm) were placed on glass slides. Haematoxylin and eosin staining was performed according to standard procedures.
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 frequency–force 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].
At the end of the treatment, the mice were subjected to a measurement of muscle strength by a weightlifting test as described previously . 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 link's weight and the timed hold. The average of three measures by each mouse was normalized by the body weight.
The statistical analysis was evaluated using the one-way or two-way ANOVA with a post-hoc Bonferroni multiple-comparison test (Sigma Stat). A difference was considered statistically significant at a P value < 0.05.
Ang-(1–7) prevents the decrease in the muscle strength and fibre diameter induced by AngII in skeletal muscle
We evaluated the effects of Ang-(1–7) and the Mas receptor on the AngII-induced atrophy in skeletal muscle. For this, the mice were infused with AngII, Ang-(1–7), 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-(1–7), but maintained when Ang-(1–7) and A779 were co-administrated 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 μm, 11% compared with 5%; 20–30 μm, 55% compared with 21%). Concomitantly, AngII produces a decrease in the proportion of bigger fibres than control (30–40 μm, 32% compared with 61%; >40 μm, 2% compared with 13%). Figure 1(B, panel b) shows that the administration of Ang-(1–7) does not change the diameter relative to control. Interestingly, Figure 1(B, panel c) shows that the co-administration of AngII plus Ang-(1–7) prevents the displacement of the fibre size induced by AngII reaching a diameter similar to Ang-(1–7) alone (<20 μm, 4%; 20–30 μm, 28%; 30–40 μm, 57%; and >40 μm, 11%). Figure 1(B panel c) shows that the co-administration of A779 together AngII plus Ang-(1–7) abolishes the effects of Ang-(1–7), 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-(1–7) on the fibre size.
The decrease in the fibre diameter induced by AngII is prevented by Ang-(1–7) via the receptor Mas in skeletal muscle
Next, we evaluated whether the effect of Ang-(1–7) 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-(1–7), 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-(1–7) 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-(1–7), abolished the prevention mediated by Ang-(1–7), 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 than the control mice, which was prevented in the mice infused with AngII plus Ang-(1–7) (Figure 2C). The same Figure shows that the administration of A779 inhibits the preventive effects of Ang-(1–7).
Angiotensin-(1–7), through the Mas receptor, prevents the decrease in the muscle strength induced by AngII in skeletal muscle
Together, these results strongly suggest that Ang-(1–7) improves the diameter of the fibres and the muscle strength of mice treated with AngII through a mechanism that involves Mas receptor.
Systemic administration of Ang-(1–7) 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-(1–7) and A779 on the myosin heavy chain (MHC) levels in the gastrocnemius muscle upon the administration of AngII. Figure 3(A) shows that Ang-(1–7) prevents the decrease in the levels of MHC induced by AngII. This effect was abolished by the co-treatment with A779, suggesting the participation of Mas receptor. The quantification of these experiments is shown in Figure 3(B).
Ang-(1–7) prevents the decrease in the myosin heavy chain induced by AngII in skeletal muscle
To evaluate the effects of Ang-(1–7) 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-(1–7). The same Figures show that this effect mediated by Ang-(1–7) is decreased by treatment with A779, suggesting the participation of Mas receptor.
Ang-(1–7)/Mas axis prevents the increase in the atrogin-1 and MuRF-1 expression induced by AngII in the skeletal muscle
These results suggest that Ang-(1–7), 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-(1–7) 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-(1–7). Figure 5 shows that Ang-(1–7) 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.
Ang-(1–7) induces the AKT phosphorylation through the Mas receptor in skeletal muscle
These results suggest that Ang-(1–7) phosphorylates AKT in skeletal muscle through a mechanism involving the Mas receptor.
Ang-(1–7), through the Mas receptor, prevents the atrophy induced by AngII in C2C12 myotubes
To corroborate the results obtained in vivo, we decided to evaluate the effects of AngII on the protein levels of MHC in the C2C12 myotubes. Supplementary Figure S2(A) shows that AngII decreases the levels of MHC reaching the maximum effect at 10 μM, 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 AT1R, as indicated by the prevention of this effect under the incubation of myotubes with losartan, an AT1R blocker.
In this context, we evaluated the effects of Ang-(1–7) on the decrease in MHC induced by AngII. Figure 6(A) shows that Ang-(1–7) 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-(1–7) 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-(1–7).
Ang-(1–7), through the receptor Mas, prevents the decrease in the myosin heavy chain induced by AngII in the C2C12 myotubes
Together, these results indicate that Ang-(1–7), through its receptor Mas, has an antagonistic effect on the atrophy induced by AngII in vitro.
The Ang-(1–7)/Mas axis decreases the expression of atrogin-1 and MuRF-1 induced by AngII in myotubes
We decided to evaluate the effects of Ang-(1–7) on the expression of the E3 ubiquitin ligases atrogin-1 and MuRF-1 induced by AngII in the C2C12 myotubes. For this, the myotubes were incubated with AngII in the absence or presence of Ang-(1–7) 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-(1–7) in a dose-dependent manner, reaching the maximum effect at 10 nM of Ang-(1–7). In addition, Figures 7(C) and 7(D) show that the diminution mediated by Ang-(1–7) 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.
Ang-(1–7)/Mas axis prevents the increase in atrogin-1 and MuRF-1 expression induced by AngII in the C2C12 myotubes
In this context, we evaluated the specificity of the effect mediated by Ang-(1–7) relative to AT1R, AT2R and Mas receptor. First, we observed in Supplementary Figure S3 that AngII produces a decrease in AT1R but increase in AT2R levels which is prevented by Ang-(1–7) in vitro and in vivo. Then, we evaluated the specificity of Ang-(1–7) on the induction dependent on AngII of atrogin-1 and MuRF-1. Supplementary Figure S4 shows that the preventive effect of Ang-(1–7) on atrogin-1 and MuRF-1 expression was not modified by the AT2R blocker PD123319. However, the pre-incubation with AT1R blocker losartan completely abolished the effect of AngII, and therefore any further effect of Ang-(1–7).
Together, these results suggest that Ang-(1–7) decreases the expression of the atrogin-1 and MuRF-1 induced by AngII, with the participation of the Mas receptor.
Ang-(1–7) induces the phosphorylation of AKT through the Mas receptor in C2C12 myotubes
Since Ang-(1–7) 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-(1–7) induces the AKT phosphorylation in a time-dependent fashion. The quantification depicted in the Figure 8(B) shows that the effect of Ang-(1–7) is at a maximum at 15 min, and then falls, reaching the basal levels at 120 min after the incubation of Ang-(1–7). Figures 8(C) and 8(D) show that the optimal dose of Ang-(1–7) was 10 nM, in which the maximum effect in AKT phosphorylation was reached. To 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-(1–7) in myotubes. Supplementary Figure S5 shows that Ang-(1–7) induces the AKT phosphorylation specifically through Mas receptor without participation of the AngII receptors, since AT1R or AT2R blockers did not changed the AKT phosphorylation induced by Ang-(1–7).
Ang-(1–7) induces AKT phosphorylation through the Mas receptor in the C2C12 myotubes
Together, these results suggest that Ang-(1–7) induces the AKT phosphorylation in a dose- and time-dependent manner, through a mechanism involving the Mas receptor.
The anti-atrophic effect of Ang-(1–7) is dependent on AKT signalling
To evaluate the participation of AKT in the anti-atrophic effects shown by Ang-(1–7), we used the inhibitor of AKT activity, MK-2206, and evaluated the MHC, atrogin-1 and MuRF-1 upon incubation with AngII and Ang-(1–7). Figure 9(A) shows that the incubation with MK-2206 abolished the preventive effects of Ang-(1–7) 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-(1–7) in the increase in the expression of atrogin-1 (Figure 9C) and MuRF-1 (Figure 9D) induced by AngII.
The inhibition of AKT activity abolishes the prevention mediated by the Ang-(1–7) of the muscle atrophy induced by AngII in the C2C12 myotubes
These results suggest that AKT activity participates in the anti-atrophic effects mediated by Ang-(1–7).
This is the first study to demonstrate the counteracting effects of Ang-(1–7), through receptor Mas, on the wasting induced by AngII in vivo and the skeletal muscle atrophy in vitro. We observed that Ang-(1–7) 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-(1–7) induced AKT phosphorylation. Finally, our results suggest that Mas and AKT activity participate in the anti-atrophic effects mediated by Ang-(1–7).
Ang-(1–7) 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-(1–7) in vitro and in vivo. Thus, our data are in line with several studies that show the contrary effects of Ang-(1–7) and AngII [28–32]. Our results also show that Ang-(1–7) produces its effects through the Mas receptor, since the use of antagonist A779 abolishes the protection dependent on Ang-(1–7) of the muscle atrophy induced by AngII. Interestingly, we also demonstrated that the effect of Ang-(1–7) is specifically mediated by Mas and not for the AT2R as has been suggested by some reports [50,51]. Comparably, several investigators have described that Ang-(1–7) acts in skeletal muscle through the Mas [35,37,38,40].
Interestingly, in the present report, we demonstrated that Ang-(1–7) 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-(1–7) in several tissues, including skeletal muscle [38,52–54]. Whether the effects of Ang-(1–7) 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-(1–7) induces the expression of the IGF-1 and IGF-1 receptors in the 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-(1–7) was administrated in vivo. Further studies must be done to explore this issue.
Interestingly, Ang-(1–7) not only induces AKT phosphorylation, but also AKT activity, participating in the mechanism through Ang-(1–7) exerting its anti-atrophic effects. Thus, our results suggest that the inhibition of AKT through the inhibitor MK-2206 abolishes the effects of Ang-(1–7). In skeletal muscle, three AKT isoforms have been demonstrated: AKT-1 is involved in embryonic development, post-natal survival and growth; AKT-2 is essential in the glucose homoeostasis and regulation of insulin; and AKT-3 plays a main role in central nervous system development . Further studies to analyse which AKT isoform(s) is (are) activated in the effects of Ang-(1–7) 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-(1–7). This observation is in agreement with recently published results, where the muscle strength in dystrophic mice is increased, albeit the genetic defects are present . A possible cause is that Ang-(1–7) 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 . 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 . 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-(1–7) 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-(1–7) 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-(1–7) on the increase in atrogin-1 and MuRF-1 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 . 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-(1–7) could prevent this pathway, favouring its anti-atrophic effect. One possible mechanism involved in the decrease in atrogin-1 and MuRF-1 expression by Ang-(1–7) is the regulation of the transcriptional activity of forkhead box O (FoxO). This transcriptional factor induces the expression of atrogin-1 and MuRF-1 . Evidence from studies where constitutively active 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 . 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 . Thus, treatment with Ang-(1–7) 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-(1–7) 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.
angiotensin type 1 receptor
angiotensin type 2 receptor
forkhead box O
insulin-like growth factor-I
myosin heavy chain
wheat germ agglutinin
Franco Cisternas, María 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 María 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.
We thank Darling Vera for the technical assistance.
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 Científico y Technológico (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.)], Fundación Chilena para Biología Celular [grant number MF-100 (to E.B.)], and Proyecto Interno UNAB [grant number UNAB-DI-281-13/R (to C.C.V.)].
These authors contributed equally to the present work