Skeletal muscle atrophy induced during sepsis syndrome produced by endotoxin in the form of LPS (lipopolysaccharide), is a pathological condition characterized by the loss of strength and muscle mass, an increase in MHC (myosin heavy chain) degradation, and an increase in the expression of atrogin-1 and MuRF-1 (muscle-specific RING-finger protein 1), two ubiquitin E3 ligases belonging to the ubiquitin–proteasome system. Ang-(1–7) [Angiotensin-(1–7)], through its Mas receptor, has beneficial effects in skeletal muscle. We evaluated in vivo the role of Ang-(1–7) and Mas receptor on the muscle wasting induced by LPS injection into C57BL/10J mice. In vitro studies were performed in murine C2C12 myotubes and isolated myofibres from EDL (extensor digitorum longus) muscle. In addition, the participation of p38 MAPK (mitogen-activated protein kinase) in the Ang-(1–7) effect on the LPS-induced muscle atrophy was evaluated. Our results show that Ang-(1–7) prevents the decrease in the diameter of myofibres and myotubes, the decrease in muscle strength, the diminution in MHC levels and the induction of atrogin-1 and MuRF-1 expression, all of which are induced by LPS. These effects were reversed by using A779, a Mas antagonist. Ang-(1–7) exerts these anti-atrophic effects at least in part by inhibiting the LPS-dependent activation of p38 MAPK both in vitro and in vivo. We have demonstrated for the first time that Ang-(1–7) counteracts the skeletal muscle atrophy induced by endotoxin through a mechanism dependent on the Mas receptor that involves a decrease in p38 MAPK phosphorylation. The present study indicates that Ang-(1–7) is a novel molecule with a potential therapeutic use to improve muscle wasting during endotoxin-induced sepsis syndrome.
During sepsis syndrome induced by endotoxaemia, loss of strength and muscle mass occur, resulting in a pathological condition called skeletal muscle atrophy. The RAS (renin–angiotensin system) is composed of the classical and non-classical axis, which are important modulators of skeletal muscle function. The role of angiotensin-(1–7), the main peptide of non-classical RAS, has not been evaluated previously in endotoxaemia-induced muscle wasting.
Our results show that angiotensin-(1–7) through its Mas receptor and p38 MAPK (mitogen-activated protein kinase), prevents sepsis-induced muscle atrophy avoiding the loss of muscle function. Thus we found that angiotensin-(1–7) has a therapeutic potential in skeletal muscle atrophy related to the septic condition.
Because the rapid loss of muscle in sepsis is considered one of the causes of death in patients in intensive care units, the combined use of this peptide with anti-inflammatory drugs could improve the recovery of critical unit care patients.
Skeletal muscle atrophy is defined as the progressive loss of muscle mass and strength [1–5]. During atrophy, the major proteins of skeletal muscle that are degraded are structural proteins, such as MHC (myosin heavy chain) and actin, which are essential components in the process of muscle contraction . One of the main mechanisms involved in this catabolic process is the UPP (ubiquitin–proteasome pathway) . Activation of the UPP during muscle wasting is characterized by an increase in muscle-specific type E3 ubiquitin ligases, atrogin-1 (muscle atrophy F-box, MAFbx), and MuRF-1 (muscle-specific RING-finger protein 1). Atrogin-1 expression is controlled by the transcription factor FoxO3 (forkhead box O type 3) [7,8]. MuRF-1 expression is induced in atrophic conditions by activation of NF-κB (nuclear factor κB) [1,9]. Among the leading causes of muscle wasting is sepsis syndrome [8,10,11]. An important inducer of sepsis syndrome is the presence of a high amount of endotoxin in the bloodstream producing the pathological event known as endotoxaemia [12,13]. In this regard, the endotoxin LPS (lipopolysaccharide), a component of Gram-negative bacteria, is a major starting inducer of the endotoxin-induced sepsis syndrome. LPS, recognized by TLR4 (Toll-like receptor type 4) , is able to induce muscle atrophy by causing an increase in atrogin-1 and MuRF-1 levels [11,15–21]. Concomitantly, LPS also induces the activation of the p38 MAPK (mitogen-activated protein kinase) pathway [19,22,23]. Thus it has been reported that inhibitors of p38 MAPK block the expression of the E3 ubiquitin ligases atrogin-1 and MuRF-1  and muscle wasting induced by LPS .
An important modulator of skeletal muscle function and their pathological processes is the RAS (renin–angiotensin system) . RAS can be functionally separated into a classical and a non-classical axis. The main peptide of the classical RAS pathway is AngII (angiotensin II) which is associated with a deleterious effect in skeletal muscle [26–30]. The main peptide of the non-classical axis is Ang-(1–7) [angiotensin-(1–7)], which produces its cellular effects through the G-protein-coupled receptor Mas [31–34]. The Mas receptor is expressed in several tissues, including skeletal muscle [35,36]. Ang-(1–7) has opposite effects to those of AngII in skeletal muscle in that it reduces fibrosis  and insulin resistance . We have demonstrated that the Mas receptor is up-regulated in skeletal muscle wasting ; in addition, the Mas receptor is located in the sarcolemma of muscle fibres, indicating that atrophic muscle has the ability to respond to Ang-(1–7) . In this context, we have shown recently that Ang-(1–7), through its Mas receptor, abolishes the AngII-induced skeletal muscle atrophy in vitro and in vivo [40,41]; however, the role of Ang-(1–7) in the skeletal muscle atrophy induced by a mechanism independent of the classical RAS system has not yet been evaluated. In the present study, we evaluated the effects of Ang-(1–7) and the mechanism involved in skeletal muscle atrophy in a model of endotoxaemia-induced sepsis syndrome through the administration of LPS. Using in vivo experiments in mice injected with LPS and in vitro approaches in C2C12 myotubes and isolated skeletal muscle fibres, we determined that administration of Ang-(1–7) decreases muscle atrophy induced by LPS. We observed that Ang-(1–7) prevents the diminution of muscular strength and diameter of myofibres and myotubes. These changes occur concomitantly with the prevention of decreased MHC levels and a lower induction of atrogin-1 and MuRF-1 expression. Ang-(1–7) also inhibited LPS-induced p38 MAPK phosphorylation in vitro and in vivo. The effect on Ang-(1–7) over the increase in atrogin-1 and MuRF-1 expression induced by LPS was lost when p38 MAPK phosphorylation was maintained [by overexpression of constitutively active MKK3 (MAPK kinase kinase 3)], which corroborated the participation of p38 MAPK downstream of Ang-(1–7).
These experiments revealed the involvement of Ang-(1–7) in the maintenance of muscle mass and its potential therapeutic use against skeletal muscle atrophy during endotoxin-induced sepsis syndrome.
MATERIALS AND METHODS
Male C57BL/10J (12-weeks-old) strain mice were used. The animals were randomized and separated into experimental groups, and three independent experiments were performed. Eight experimental groups (five to seven animals/group) were designed: vehicle (water), LPS (1 mg/kg), Ang-(1–7) (100 ng/kg per min), LPS plus Ang-(1–7), A779 (100 ng/kg per min), A779 plus LPS, A779 plus Ang-(1–7), and LPS plus Ang-(1–7) plus A779. A single dose of LPS was injected i.p. (intraperitoneally) [11,19]. The treatments were performed at 18 h and 14 days depending on the parameter being evaluated. The peptides or antagonists were osmotically infused through micropumps (Alzet-Durect) as described previously . At the end of the experiment, the animals were killed under anaesthesia and the GA (gastrocnemius) and TA (tibialis anterior) muscles were dissected, removed, 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.
Cells of the mouse skeletal muscle cell line C2C12 (A.T.C.C., Manassas, VA, U.S.A.) were grown and differentiated until day 4 as described previously . The myotubes were incubated with the following treatments: LPS (500 ng/ml) from Escherichia coli 0127:B8 (Sigma), Ang-(1–7) (10 nM) (Phoenix Pharmaceuticals), A779 (10 μM) (CPC Scientific), and SB203580 (10 μM) (Tocris), for the times indicated in the Figures.
Isolation and culture of single-myofibre explants
Single myofibres were isolated from the EDL (extensor digitorum longus) muscles of C57BL/10J mice (n=5 for each group) as described by Pasut et al. . Briefly, the EDL muscle was dissected and digested in DMEM (Dulbecco's modified Eagle's medium) containing 750 units/ml collagenase type I (Worthington Biochemical) at 37°C for 1 h with gentle agitation. Single myofibres were then carefully dissociated by flushing the muscle with DMEM containing 15% (v/v) HS (horse serum). Using a dissecting microscope, single myofibres were extracted individually using fire-polished pipettes and were transferred serially into fresh DMEM/15% HS. Approximately 20–50 myofibres were transferred to 24-well plates and incubated for 24 h at 37°C in a humidified 5% CO2 incubator. The next day, the medium was aspirated and exchanged with fresh medium in which the different treatments were performed.
Measurement of myotube and isolated myofibre diameter
Myotube and myofibre cultures were photographed under a phase-contrast microscope at ×40 magnification after the indicated treatment. The minimal Feret diameters were measured in a total of 100 myotubes from ten random fields and in 20–30 myofibres from each experimental condition in a blind fashion, using computerized image analysis (ImageJ, NIH). Myotubes and myofibres were measured at three points along their length.
C2C12 cells were grown and treated on plastic coverslips. At the end of the experiments, cells were washed twice in ice-cold PBS, fixed in 4% paraformaldehyde, blocked for 1 h in 10% (v/v) goat serum in PBS, and incubated for 1 h at room temperature with a specific antibody against mouse anti-MHC. (1:100 dilution) (MF-20, Developmental Studies, Hybridoma Bank, University of Iowa, Iowa City, IA, U.S.A.). For staining, isolated myofibres were washed and fixed in a floating solution . The bound antibodies were detected by incubating the cells for 60 min with 1:100 affinity-purified Alexa Fluor® dye-conjugated goat anti-mouse antibody (Life Technologies). Nuclear staining was performed with Hoechst 33258. After rinsing, the cells were mounted with a fluorescent mounting medium (Dako Corporation) under a glass slide and viewed and photographed using the Motic BA310 epifluorescence microscope.
Fresh-frozen GA and TA muscles were sectioned, and cryosections (7 μm) were stained with H&E (haematoxylin and eosin) according to standard procedures. Fibre sizes were determined using ImageJ software on seven randomly captured images of the muscles. Fibres were manually selected, and the minimal Feret diameter of each fibre was computed using the software.
After treatment, the mice were anaesthetized and killed, the GA and TA muscles were removed, and the muscle contractile properties were measured. The maximum isometric tetanic force was determined. The muscle mass and the optimum muscle length (Lo) were used to calculate the specific net force [force normalized per total muscle fibre CSA (cross-sectional area), mN/mm2] [25,43,44].
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 and before treatments, the mice were subjected to training (once per day for 2 weeks). To perform the test, the mouse grasps (with its forepaws) the different weights and a score is assigned. The final score was calculated as the summation of the product between the link weight and the time held. The average of three measures from each mouse was normalized by the body weight.
C2C12 transient transfection was performed using Lipofectamine™ 2000 (Life Technologies) and constitutively active MKK3 (MKK3-Glu) (Addgene plasmid 14670) . Briefly, C2C12 was trypsinized, and 30000 cells/cm2 were plated. The Lipofectamine/DNA mixture was immediately added, and the cells were incubated for 24 h. The medium was then changed, and the treatments were performed.
RNA isolation, reverse transcription and quantitative real-time PCR
Total RNA was isolated from the GA and TA muscles using TRIzol® (Invitrogen). The total RNA (1 μg) was reverse-transcribed to cDNA using random hexamers and 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, AT1R (AngII type 1 receptor), Mas receptor and the housekeeping gene 18S (TaqMan Assays-on-Demand; Applied Biosystems). The mRNA expression was quantified using the comparative ΔCT method (2−ΔΔCT), with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the reference gene. The mRNA levels were expressed relative to the mean expression in the vehicle-treated mice .
The GA 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, Iowa City, IA, U.S.A.), mouse anti-atrogin-1 (1:500 dilution), rabbit anti-MuRF-1 (1:500 dilution) and mouse anti-tubulin (1:5000 dilution; Santa Cruz Biotechnology), rabbit anti-phospho-p38 MAPK (1:1000 dilution) and rabbit anti-total p38 MAPK (1:1000 dilution; Cell Signaling Technology) and anti-FLAG M2 (1:1000 dilution; Sigma). All immunoreactions were visualized by ECL (Thermo Scientific).
For statistical analysis, we used the one- or two-way ANOVA with a post-hoc Bonferroni multiple-comparison test (Prisma). A difference was considered statistically significant at P<0.05.
Ang-(1–7) inhibits the decrease in muscle force and myofibre diameter in LPS-induced skeletal muscle atrophy
We evaluated the effect of Ang-(1–7) and the Mas receptor on a model of skeletal muscle atrophy induced by LPS. Figure 1(a) shows that Ang-(1–7) prevented the decrease in maximal isometric force of the GA muscle induced by LPS; a recovery in muscle strength of 37.7% was seen compared with mice treated with LPS alone. This effect was reversed by A779, the antagonist of the Mas receptor, evident by a decrease in strength (38.4%) that was similar to mice treated with LPS (33.1%). Ang-(1–7) and A779 had no effect by themselves on muscle strength. This finding was confirmed in TA muscle (Supplementary Figure S1).
Ang-(1–7), through the Mas receptor, improves the muscle strength decrease caused by LPS
To determine whether the changes observed in isolated muscle strength can be extrapolated to living mice, we performed the forelimb strength test . Figure 1(b) shows that the reduction in muscle strength of mice treated with LPS (42.5% compared with control group) was recovered in the LPS plus Ang-(1–7) group. Finally, the effect of Ang-(1–7) on muscle strength was reversed in the presence of A779 by reaching similar values of mice treated with LPS.
To evaluate the diameter of muscle fibres, H&E staining was performed in GA muscle. Figure 2(a) shows that Ang-(1–7) strongly attenuated the decrease in muscle fibre diameter induced by LPS, and A779 blocked this effect (Figure 2b). The calculation and quantification of the Feret diameter showed that Ang-(1–7) alone did not modify the diameter (Figure 2c), whereas LPS displaced the major proportion of fibres towards 25–30 μm compared with control muscle (40–45 μm, dotted line) (Figures 2c and 2d). Figure 2(d) shows that co-treatment of LPS with Ang-(1–7) produced a significant displacement towards larger fibres, with a peak ranging between 35 and 50 μm. Figure 2(e) shows that A779 with or without Ang-(1–7) did not have any effect on fibre muscle diameter and Figure 2(f) shows that A779 inhibited the displacement of the curve observed for Ang-(1–7) in mice treated with LPS, keeping the diameter peak between 25 and 30 μm. These results were also corroborated in the TA muscle as shown in Supplementary Figure S2.
Ang-(1–7), through the Mas receptor, increases muscle fibre diameter in LPS-induced skeletal muscle atrophy
Together, these results suggest that Ang-(1–7) through the Mas receptor decreased the functional and anatomical parameters of the skeletal muscle atrophy induced by LPS in vivo.
Ang-(1–7) inhibits the decrease in MHC by abolishing the expression of ubiquitin E3 ligases in LPS-induced skeletal muscle atrophy
MHC is one of the major proteins whose levels decrease in skeletal muscle atrophy through a mechanism that involves the UPP. Figures 3(a) and 3(b) show that the diminution of 53.3% in MHC levels induced by LPS in GA muscles was reversed by treatment with Ang-(1–7) and LPS. This effect of Ang-(1–7) was reversed by the antagonism of Mas with A779, reaching a response similar to LPS treatment (48.6% of MHC compared with control).
Ang-(1–7) prevents a decrease in the MHC levels and the increase in atrogin-1 and MuRF-1 induced by LPS in skeletal muscle, via the Mas receptor
To assess the participation of the UPP, we evaluated the protein levels of atrogin-1 and MuRF-1, two key enzymes involved in the process of skeletal muscle atrophy. Figures 3(c) and 3(d) show that LPS increased both atrogin-1 and MuRF-1, 1.97- and 2.52-fold respectively, compared with the vehicle group, whereas the administration of Ang-(1–7) resulted in a decrease of 25.8% for atrogin-1 and 70.3% for MuRF-1 compared with mice treated with LPS. The effects of Ang-(1–7) on the decreased levels of atrogin-1 and MuRF-1 induced by LPS were inhibited in the presence of A779.
The effects mediated by Ang-(1–7) and Mas also were observed for atrogin-1 and MuRF-1 gene expression in both GA and TA (Supplementary Figures S3a and S3b)
These results indicate that Ang-(1–7) through its Mas receptor inhibited the decrease in MHC, atrogin-1 and MuRF-1 induced by LPS in vivo through a mechanism dependent on Mas.
We have shown previously that, in LPS-induced muscle wasting, there is an increase in Mas receptor gene expression , so we evaluated the effect of Ang-(1–7) on the expression of Mas receptor and AT1R in LPS-induced muscle atrophy. As we have shown, LPS induced the up-regulation of Mas receptor in vitro (Supplementary Figure S4a) and in vivo (Supplementary Figure S4b), but Ang-(1–7) did not have any effect on the expression of Mas receptor. By the other hand, LPS induced the down-regulation of AT1R in vitro (Figure S4c) and in vivo (Figure S4d), and Ang-(1–7) did not modify the gene expression of AT1R. These results indicate that Ang-(1–7) did not modify the expression of Mas receptor and AT1R induced by LPS in vitro and in vivo.
Ang-(1–7) inhibits the decrease in skeletal muscle atrophy induced by LPS
To assess the effect of Ang-(1–7) on LPS-induced atrophy on the muscle cell, we used the models of C2C12 myotubes and muscle myofibres isolated from the EDL muscle. Figures 4(a) and 4(b) show that the atrophic effect mediated by LPS (32% diameter decrease) were inhibited by co-incubation with Ang-(1–7). This effect mediated by Ang-(1–7) was reversed by the use of A779. Similar results were observed in EDL-isolated myofibres (Figures 4c and 4d), where treatment with Ang-(1–7) attenuated the decrease in the diameter of the myofibres induced by LPS and A779 inhibited the Ang-(1–7) effect.
Ang-(1–7), through the Mas receptor, increases fibre diameter in LPS-induced skeletal muscle atrophy
Next, we evaluated the mechanism involved in the inhibition of decrease in myotube diameter in vivo. Figures 5(a) and 5(b) show that Ang-(1–7) inhibited 78% of the LPS-induced decrease in MHC levels observed in C2C12 myotubes, reaching similar levels to those observed in the control cells. This effect was dependent on the Mas receptor since the use of A779 in conjunction with LPS and Ang-(1–7) showed a fall by 81.2% similar to the LPS group. To evaluate the participation of the UPP, we determined the protein levels of atrogin-1 and MuRF-1. Figures 5(c) and 5(d) show that co-incubation of myotubes with LPS and Ang-(1–7) caused a decline in the levels of atrogin-1 and MuRF-1 (68% and 57% respectively compared with LPS-treated cells). This effect was completely inhibited by the use of A779. The changes observed at the protein level were related to the changes observed at the mRNA level (Supplementary Figures S3c and S3d) where Ang-(1–7), through its Mas receptor, decreased the expression of atrogin-1 and MuRF-1 in LPS-treated myotubes.
The Ang-(1–7)/Mas axis prevents the decrease in MHC levels and the increase in the ubiquitin E3 ligases atrogin-1 and MuRF-1 induced by LPS
These results indicate that Ang-(1–7), through its Mas receptor, inhibited the decrease in MHC protein levels through a mechanism that involved inhibition of the UPP in LPS-induced muscle atrophy in vitro.
Ang-(1–7) prevents p38 MAPK phosphorylation in LPS-induced skeletal muscle atrophy
in vivo and in vitro
The increase in E3 ligases by LPS administration is dependent on the p38 MAPK pathway [19,22,23]. Therefore we tested the participation of p38 MAPK in the anti-atrophic effects shown by Ang-(1–7) in vivo and in vitro. We observed that use of SB203580, a p38 MAPK inhibitor, abolished the decrease in myotube diameter (Supplementary Figures S5a and S5b) and MHC levels (Supplementary Figure S5c) induced by LPS in vitro. Figures 6(a) and 6(b) show that Ang-(1–7) prevented an LPS-induced increase in p38 MAPK phosphorylation in GA muscles. A779 blocked the effect of Ang-(1–7) by reversing the LPS-induced increase in p38 MAPK phosphorylation. The in vivo results were substantiated further in C2C12 myotubes. Figures 6(c) and 6(d) show that the phosphorylation of p38 MAPK induced by LPS was inhibited by Ang-(1–7) co-treatment, and this effect was depended on the Mas receptor because this inhibition was lost when the A779 antagonist was used.
Ang-(1–7), through the Mas receptor, inhibits the p38 MAPK phosphorylation induced by LPS
in vivo and in vitro
To demonstrate further the involvement of the p38 MAPK pathway in the anti-atrophic effect of Ang-(1–7) on LPS-induced skeletal muscle atrophy, we overexpressed a constitutively active form of MKK3 (MKK3-Glu), a p38 MAPK activator . Figure 7(a) shows that transfection with MKK3-Glu induced the p38 MAPK phosphorylation in C2C12 cells without LPS. Ang-(1–7) did not have any effect upon p38 MAPK phosphorylation induced by MKK3-Glu. Finally, we evaluated the effect of Ang-(1–7) in cells with constitutively active p38 MAPK activation on the expression of atrogin-1 and MuRF-1. Figure 7(b) shows that cells transfected with MKK3-Glu had increased basal levels of atrogin-1 similar to control cells treated with LPS, and Ang-(1–7) did not prevent the induction in the expression of atrogin-1, indicating that Ang-(1–7) did not modify the activity of the p38 MAPK downstream kinase activator MKK3. Although there are conflicting reports in relation to the regulation of MuRF-1 by p38 MAPK [19,47], we found that constitutive activation of p38 MAPK induced MuRF-1 gene expression. In addition, treatment with Ang-(1–7) alone or in conjunction with LPS did not modify MuRF-1 gene expression in cells transfected with MKK3-Glu. These results suggest that the effect of Ang-(1–7) on p38 MAPK could be the modulation of activators upstream of MKK3.
The p38 MAPK pathway is involved in the anti-atrophic effect of Ang-(1–7) in LPS-induced skeletal muscle atrophy
In the present study, we showed for the first time that the activation of the non-classical RAS through Ang-(1–7) and the Mas receptor decreased skeletal muscle atrophy induced by LPS in vitro and in vivo. We observed that the Ang-(1–7)/Mas axis prevented key events induced by LPS in skeletal muscle atrophy, including the decrease in muscle strength, fibre diameter and the levels of MHC, as well as the up-regulation of atrogin-1 and MuRF-1 expression. In addition, we demonstrated that the anti-atrophic effect of Ang-(1–7) involved the inhibition of p38 MAPK phosphorylation.
Few studies have demonstrated that non-classical RAS axis is active in skeletal muscle [26,37,48,49]. Our study showed that Ang-(1–7) recovers muscle strength that was reduced by LPS. To date, the only studies that demonstrated an effect of Ang-(1–7) on skeletal muscle function were performed in the muscular dystrophy mice models, which showed that the Ang-(1–7)/Mas axis improves muscle fibrosis and muscle strength [37,49]. Although our results indicated that Ang-(1–7) increases muscle strength, the mechanism by which this peptide affects the process of muscle contraction is unknown. In skeletal muscle atrophy, specific fibre types are affected that involve slow type I or fast type II muscle fibres that alter the metabolism and generation of strength . In the model of endotoxin-induced sepsis syndrome caused by LPS injection, a fast-to-slow fibre type shift with preferential atrophy of fast glycolytic fibres is observed . A possible mechanism of Ang-(1–7) that leads to an increase in muscle strength is the modification of the type of muscle fibres. Thus future experiments must be performed to evaluate the effect of Ang-(1–7) on the type of fibres in skeletal muscle atrophy and possible pathways involved in this process.
In the present study we used male mice to evaluate the effect of Ang-(1–7) on LPS-induced muscle wasting; however, there is evidence that indicates sex-dependent differences in: (i) muscle composition and its functionality [51,52]; (ii) prognosis in sepsis [53,54]; and (iii) the expression and activation of the Ang-(1–7)/Mas receptor axis in cardiovascular pathologies [55–58]. For this reason, further studies could be performed in female mice to elucidate the different sex-related response to Ang-(1–7) on muscle wasting induced by LPS.
Several studies have shown that LPS induces skeletal muscle atrophy through UPP activation by increasing the expression of ubiquitin ligase atrogin-1 and MuRF-1, starting 6 h after LPS treatment [11,19,59], which is in agreement with the results of the present study. It is important to note that the early increase in UPP is consistent with a late decrease in MHC in response to LPS as has been reported [19,41]. We determined that Ang-(1–7) prevents the induction of atrogin-1 and MuRF-1 expression. Therefore the effect of Ang-(1–7) on the decrease in UPP can explain its preventive activity in the decrease in sarcomeric protein levels, such as MHC.
It has been reported that regulation of atrogin-1 and MuRF-1 gene expression depends on the activation of different signalling pathways in muscle wasting, among them p38 MAPK [22,23]. In our model of endotoxin-induced muscle wasting, it was demonstrated previously that LPS induces the phosphorylation of p38 MAPK, which is necessary and sufficient for the up-regulation of atrogin-1, resulting in myotube atrophy . Although the induction of MuRF-1 expression mediated by LPS in vitro and in vivo is mainly due to the NF-κB signalling pathway [9,23], other reports indicate that the expression of MuRF-1 is also induced through a p38 MAPK-dependent mechanism . These antecedents show that the inhibition of p38 MAPK phosphorylation by treatments with Ang-(1–7) may be an important therapeutic target against muscle wasting.
There is abundant evidence that Ang-(1–7) inhibits p38 MAPK phosphorylation by decreasing the deleterious effects of AngII in renal vasculature  and spinal nociceptive transmission , vascular smooth muscle cell proliferation  and vascular remodelling . We have shown recently that Ang-(1–7), through the Mas receptor, can counteract the signalling induced by AngII in skeletal muscle fibrosis by decreasing p38 MAPK phosphorylation . Despite these effects on p38 MAPK activity, the mechanism through which Ang-(1–7) inhibits p38 MAPK phosphorylation is unknown. A possibility is that this mechanism could involve the induction of phosphatases to dephosphorylate p38 MAPK. In this regard, Ang-(1–7) has been reported to increase DUSP1 (dual-specificity phosphatase 1) levels in cardiac fibroblasts . In addition, Ang-(1–7) increased the expression of SHP1 (Src homology 2 domain-containing protein tyrosine phosphatase 1) in renal cells, preventing p38 MAPK phosphorylation . Thus further studies must be performed to evaluate the participation of these or other phosphatases in the effect of Ang-(1–7) on p38 MAPK inhibition during muscle wasting.
Several studies have shown that an increase in ROS (reactive oxygen species) production is involved in skeletal muscle atrophy [66–70]. Intraperitoneal injection of LPS in mice resulted in a higher level of ROS in skeletal muscle and a greater expression of atrogin-1 and MuRF-1 . Interestingly, it has been reported that the induction of atrogin-1 and MuRF-1 by oxidative stress is dependent on p38 MAPK signalling . We have recently demonstrated that Ang-(1–7) decreases ROS production and p38 MAPK activation induced by AngII . In addition, ROS increase p38 MAPK phosphorylation through the activation of upstream kinases MKK3/6 . Our results indicate that Ang-(1–7) could be acting upstream of these kinases because the activation constitutive of MKK3 inhibits the anti-atrophic effect of Ang-(1–7), at least in the induction of atrogin-1 and MuRF-1 gene expression. Thus the decrease in the levels of ROS and the subsequent inhibition of p38 MAPK phosphorylation could be one of the mechanisms involved in the reduction of LPS-mediated atrophy. Future experiments must focus on evaluating the effect of Ang-(1–7) on oxidative stress in muscle wasting induced by LPS and evaluating the possible antioxidant role of this peptide in this model.
LPS has been reported to induce the expression of TGF-β1 (transforming growth factor β1) in endothelial cells [74–76]. Furthermore, TGF-β1 is capable of inducing skeletal muscle atrophy  through UPP activation and the up-regulation of atrogin-1 and MuRF-1 . Ang-(1–7), through the Mas receptor, decreases the expression and activity of TGF-β1 through the inhibition of ROS production . Ang-(1–7) also decreases the TGF-β1 signalling pathway in dystrophic skeletal muscle, which leads to an improvement of muscle physiology. Although the role of TGF-β1 in the induction of atrophy has not been extensively studied, these antecedents would indicate that the Ang-(1–7)/Mas axis could affect the TGF-β signalling pathway involved in skeletal muscle atrophy.
LPS increases the levels of AngII which acts through AT1R . Thus the effect of Ang-(1–7) on LPS-induced muscle wasting may be due to the fact that Ang-(1–7) antagonizes the direct effect of AngII on skeletal muscle, as we have demonstrated recently [40,41]. Our data show that, in mice treated with LPS, AT1R expression is down-regulated in skeletal muscle, probably producing a loss of sensitivity to the increased AngII levels, as has been reported previously .
There is evidence that shows and compares the beneficial effect of classical RAS axis blockade using losartan, with the administration of Ang-(1–7) [81,82]. Thus, to anticipate the effect of losartan and compare it with the anti-atrophic action of Ang-(1–7) in the muscle wasting induced by LPS, the following antecedents should be considered. (i) There is a complex interaction between AT1R and Mas receptors that leads to the final Ang-(1–7) effect in the mouse heart. When AT1Rs are blocked, Ang-(1–7) produces Mas receptor-mediated effects which are not produced by Ang-(1–7) itself . Thus the possible mechanisms involved in this interaction could include a functional antagonism between these receptors . However, our data show that LPS strongly decreases the expression of AT1R and produces an increase in the Mas levels. Thus this differential regulation of AT1R and Mas receptor could increase the beneficial effect of Ang-(1–7) on LPS-induced muscle wasting and could be different from the effects produced by AT1R blockers. (ii) Losartan acts classically by blocking the AT1R; however, in our model, we show that LPS strongly decreases the expression of AT1R. Thus it could be possible to discard this mechanism of action in the case that the effect of losartan can be evaluated in LPS-induced muscle wasting. (iii) Losartan could exert some of its effect through AT1R-independent mechanisms. In this regard, it has been previously reported that losartan inhibits LPS-induced pro-inflammatory gene expression in macrophages by activating the PPARγ (peroxisome–proliferator-activated receptor γ) pathway rather than by the competitive inhibition of AT1R binding to AngII . (iv) Losartan and Ang-(1–7) improve muscle strength in animal models of Duchenne muscular dystrophy through the inhibition of the TGF-β1 signalling pathway [37,86]. Considering that TGF-β induces muscle atrophy, and that it has been recently described that LPS can induce the expression of TGF-β, this could be a common mechanism for the possible anti-atrophic effect of losartan in LPS-induced muscle wasting. (v) Losartan has been described to increase the Ang-(1–7) levels in some pathologies [81,87]. Thus a possible mechanism of action could be indirectly mediated by Ang-(1–7). Although in the present study, we did not evaluate the effect of losartan, and considering all of the possibilities of losartan's action, future experiments will be needed to compare the effect of losartan and Ang-(1–7) in the muscle wasting induced by LPS.
Recently, it has been demonstrated that Ang-(1–7) acts as an anti-inflammatory molecule in several tissues [88–90]. Ang-(1–7), through its Mas receptor, inhibits the mRNA expression of the pro-inflammatory cytokines IL-6 (interleukin 6) and TNFα (tumour necrosis factor α) increased by LPS in mouse peritoneal macrophages  and in vivo . These results suggest that Ang-(1–7) is able to reduce inflammation caused by LPS; however, further experiments are needed to determine the direct effect of the Ang-(1–7)/Mas axis on the expression of inflammatory molecules on skeletal muscle.
Our results using the A779 antagonist suggest the participation of Mas in the anti-atrophic effect of Ang-(1–7), which we have reported previously [40,41]. Interestingly, we did not observe any impairment of any muscle parameter (such as strength or diameter of fibres) or significant changes in survival rate (results not shown) of mice treated with LPS and A779 compared with LPS treatment or vehicle, contrasting with previous reports that show an impairment of basal conditions with A779 [37,93]. Our results could be explained as a consequence of LPS treatment increasing Mas receptor levels in skeletal muscle  which could have a more protective effect. Despite these findings, it would be interesting to use Mas receptor-knockout mice in future experiments to address more properly its role in LPS-induced muscle wasting.
In summary, the present study shows for the first time the anti-atrophic effect of Ang-(1–7) in LPS-induced muscle atrophy and the participation, at least, of the p38 MAPK signalling pathway.
María Gabriela Morales was responsible for carrying out the experiments, and analysing and interpreting the data. Hugo Olguín assisted with isolated muscle myofibres. Gabriella Di Capua performed the analysis of AT1R and Mas receptors, Enrique Brandan assisted with force measurements in isolated muscle. Felipe Simon assisted with LPS treatment in mice. Claudio Cabello-Verrugio and María Gabriela Morales were involved in drafting the paper for publication. Claudio Cabello-Verrugio was responsible for conceiving all the experiments and was involved in analysing the data, preparing it for publication, and drafting the paper.
We thank Darling Vera for technical assistance.
This study was supported by research grants from Association-Française Contre Les Myopathies (AFM) [grant number 16670 (to C.C.-V.)]; National Fund for Science & Technology Development (FONDECYT) [grant numbers 1120380 (to C.C.-V.), 3130593 (to M.G.M.), 1121078 (to F.S.), 1110426 (to E.B.), 1130631 (to H.O.)]; Millennium Institute on Immunology and Immunotherapy [grant number P09-016-F (to F.S.)]; Center for Aging and Regeneration (CARE) [grant number PFB12/2007 (to E.B.)], Fundación Chilena para Biología Celular [grant number MF-100]; UNAB-DI [grant number 281-13/R (to C.C.-V.)].
AngII type 1 receptor
Dulbecco’s modified Eagle’s medium
extensor digitorum longus
haematoxylin and eosin
mitogen-activated protein kinase
myosin heavy chain
MAPK kinase kinase
muscle-specific RING-finger protein 1
nuclear factor κB
reactive oxygen species
transforming growth factor β