Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein–kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.

CLINICAL PERSPECTIVES

  • As the kinin B1 receptor activates signalling pathways that are also known to be involved in skeletal muscle catabolic disorders (for example, the NF-κB and PI3K/Akt pathways), we hypothesized that this receptor could contribute to the modulation of intracellular responses in atrophying skeletal muscle.

  • Our results show that kinin B1 receptor activation induced a decrease in C2C12 myotube diameter and an increase in the mRNA levels of the E3 ligases atrogin-1 and MuRF-1. Moreover, a lack of the kinin B1 receptor impaired MuRF-1 mRNA expression after induction of muscle atrophy.

  • The present study uncovers new functions for the kinin B1 receptor and demonstrates that this receptor plays a significant role in the regulation of muscle proteolysis, which may be of functional relevance to future therapies for the control of skeletal muscle mass.

INTRODUCTION

Kinin receptors subtypes B1 and B2 belong to the superfamily of GPCRs (G-protein-coupled receptors). The endogenous agonists for these receptors are peptides called kinins, generated from the kallikreins enzymes action on kininogens substrates [1]. Kinins are well known vasoactive peptides and are classically involved in blood pressure control and hydroelectrolytic balance [2,3], but they have also been described to participate in other processes, such as inflammatory responses and cell proliferation through induction of cytokine expression and transactivation of receptor tyrosine kinases, with subsequent activation of mitogenic intracellular pathways including NF-κB (nuclear factor κB), PI3K (phosphoinositide 3-kinase)/Akt and MAPKs (mitogen-activated protein kinases) [4,5]. The local modulation (cell- or tissue-dependent) of kinins and/or kinin receptors may account for their described involvement in a variety of diseases or disorders, including those of cardiovascular, renal, and central nervous systems, cancer and inflammatory processes [68]. As some of the above-mentioned signalling pathways (i.e. NF-κB and PI3K/Akt pathways) are also described to play a relevant role in skeletal muscle physiology, we hypothesized that kinin receptors could potentially contribute to fine-tuning the modulation of those intracellular pathways in normal muscle cells, and therefore with possible relevance to atrophied muscles.

Skeletal muscle represents approximately 50% of lean body mass and exerts vital functions, such as locomotion, breathing and sustentation, besides being a source of amino acids for gluconeogenesis during starvation and manifestation of some diseases [9]. Skeletal muscle mass is determined by the balance between the synthesis and degradation of proteins, which in turn is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli [10]. Such stimuli are altered in several pathologies, such as COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in situations related to immobilization and aging (sarcopenia) [1116]. As a consequence, muscle proteolysis is increased, leading to muscle atrophy, which represents a significant contribution to patient morbidity [17]. Moreover, protein synthesis is decreased in atrophic conditions, as some signalling pathways regulate both degradation and synthesis of muscular proteins [18].

In the present study, we made use of various approaches and tools, including in vitro assays with C2C12 cell myotubes, animal treatment with selective antagonists and the use of knockout mice, to address the contribution of the kinin B1 receptor in the regulation of ubiquitin E3 ligases involved in skeletal muscle atrophy, namely atrogin-1 and MuRF-1 (muscle RING-finger protein-1). We have shown that the kinin B1 receptor is involved in the expression of muscle E3 ligases through regulation of IKK [IκB (inhibitor of NF-κB) kinase]/NF-κB and PI3K/Akt pathways revealing a new role for the KKS (kallikrein–kinin system) in muscle mass control.

MATERIALS AND METHODS

Animals

All animals used in the present study were male and sexually mature, as the atrophy model used was testosterone-dependent. Balb-C mice were obtained from the Central Animal Facility of the University of São Paulo campus in Ribeirão Preto. C57BL/6 B1R−/− (kinin B1 receptor-knockout) mice as well as the WT (wild-type) control mice were provided by Professor João B. Pesquero (Department of Biophysics, Universidade Federal de Sao Paulo, Sao Paulo, Brazil). Control mice for the B1R−/− animals were from the parental lineage used to give rise to the B1R−/− mice. The animals were kept in a 12-h light/dark cycle at a temperature of approximately 25°C, with food and water ad libitum, including during the experimental period.

The use of such animals in these experiments was authorized by the Ethics Committee on Animal Experiments of the Faculty of Medicine of Ribeirão Preto (FMRP-USP-CETEA) (protocol number 046/2006).

Induction of muscular atrophy in levator ani muscle by gonadectomy

As testosterone levels may vary according to age, male animals were selected so that all the experimental groups were ~75 days old and sexually mature at the end of the experiment. Each experimental group was composed of 6–8 animals. Balb-C mice, B1R−/− mice and the corresponding WT mice were anaesthetized with ketamine (65 mg/kg of body weight) and gonadectomized by a longitudinal incision in the middle region of the scrotum. The scrotum was cleaned with 70% ethanol and after incision in the tunica vaginalis of the testis and its expulsion, the vascular pedicles were tied above the head of the epididymis and sectioned. The remaining structures were reinserted into the cavity, the scrotum was sutured and the operated area was repeatedly cleaned with 70% ethanol, as described previously [19]. At 2, 7, 15 and 30 days after gonadectomy, experimental and control animals were killed by cervical dislocation and the LA (levator ani) muscles were extracted and washed in PBS containing 0.1% DEPC (diethyl pyrocarbonate). The EDL (extensor digitorum longus) muscles from both hindlimbs were also removed and used as experimental controls, as these muscles do not lose mass as quickly as LA muscle in the absence of testosterone. All muscles were weighed, frozen in liquid nitrogen and subsequently stored at −70°C. The animals were also weighed before and at the various time points after gonadectomy.

Treatment of mice with the kinin B1 receptor antagonist R-715

To evaluate the effects of kinin B1 receptor blockade in mice subjected to the atrophy of LA muscles, we used the specific antagonist R-715, which was kindly provided by Professor João Pesquero. The antagonist was administered i.p. (intraperitoneally) at 0.5 mg/kg of body weight per day. Control groups received vehicle (PBS). In these experiments, we used four experimental groups of Balb-C mice that were approximately 70 days old at the beginning of the experiment as follows: (i) gonadectomized and treated for 6 days with the antagonist, (ii) gonadectomized and treated for 6 days with PBS, (iii) non-gonadectomized animals treated for 6 days with the antagonist or (iv) non-gonadectomized animals treated for 6 days with PBS. Each experimental group was composed of 6–8 animals. The gonadectomized animals received the first dose of antagonist or PBS immediately after surgery. The animals were killed at the same age, approximately 75 days old, and received treatment with the antagonist or PBS for the same period of time.

Measurement of the diameter of C2C12 myotubes

C2C12 mouse myoblasts were grown in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS. Cells were induced to differentiate by placing them in DMEM containing 2% horse serum. Full differentiation was achieved 6 days after induction of differentiation. Differentiation was evaluated by morphological appearance of the myotubes. The images of the myotubes untreated and treated with DABK (des-Arg9-bradykinin) were analysed using ImageJ software (http://rsb.info.nih.gov/ij/). At least 20 fields in each plate were recorded and the diameters of all myotubes in each image were measured. Graphs were plotted using GraphPad Prism software.

Real-time PCR

Total RNA was isolated using TRIzol® reagent (Invitrogen) from individual skeletal muscles and C2C12 myotubes. ImProm II reverse transcriptase (Promega) was used to generate cDNAs. Samples were used for analysis of MuRF-1, atrogin-1 and kinin B1 receptor transcript expression levels by quantitative real-time PCR using the Platinum SYBR Green qPCR Super-Mix UDG system (Invitrogen). All real-time PCRs were performed in duplicate using an Applied Biosystems 7500 Real-Time PCR system at 95°C for 15 min, followed by 40 cycles of 15 s denaturation at 94°C and 1 min at 60°C for annealing and elongation. Transcripts of interest were normalized to cyclophilin B levels. The level of target transcript expression was calculated using the standard curve method [20].

Immunocytochemistry and confocal microscopy

After treatment with 200 nM DABK for 6, 12 or 24 h, C2C12 myotubes were fixed with 2% paraformaldehyde solution in PBS for 15 min at 37°C. After washing three times with PBS, coverslips were blocked with 250 mM glycine in PBS for 15 min and 1% BSA in PBS for 45 min. Anti-MuRF-1 (Santa Cruz Biotechnology), anti-atrogin-1 (ECM Biosciences) and anti-(p50 NF-κB subunit) (Cell Signaling Technology) primary antibodies diluted 1:100 in PBS containing 1% BSA were added for 1 h at room temperature (25°C). After three washes with PBS, Alexa Fluor®594-conjugated anti-(mouse IgG) for MuRF-1 and Alexa Fluor® 488-conjugated anti-(rabbit IgG) for atrogin-1 and p50 NF-κB subunit (both secondary antibodies from Invitrogen), diluted 1:500 in PBS, were added and incubated for 1 h at room temperature in the dark. After three washes with PBS, coverslips were rinsed with distilled water, dried and mounted with ProLong® Gold antifade reagent with DAPI (Invitrogen). Images of the cells were analysed using a confocal microscope TCS SP5 (Leica Microsystems). Fluorescence images were captured using Leica Application Suite Advanced Fluorescence Software.

Protein extraction and Western blot analysis

Frozen muscles were pulverized and immediately resuspended in 0.3 ml of lysis buffer [20 mM Tris/HCl (pH 7.5), 2.5 mM CaCl2 and 0.25% Triton X-100, containing a cocktail of protease inhibitors (1 mM benzamidine, 10 mg/ml leupeptin, 10 mg/ml pepstatin, 1 mg/ml aprotinin, 1 mM PMSF, 1 mM EDTA, 100 mM sodium orthovanadate and 10 mM sodium fluoride)]. C2C12 myotubes in six-well plates were scrapped in the presence of 100 μl of the same lysis buffer. After mixing and incubating on ice for 30 min, samples were centrifuged at 20000 g for 30 min at 4°C and the supernatants were collected and stored at −20°C. Samples were quantified for total protein by the Bradford method using a commercial protein quantification kit (Bio-Rad Laboratories). A portion (70 μg) of each sample was loaded on to 12% acrylamide gels, separated by SDS/PAGE and transferred on to Hybond nitrocellulose membranes (GE Healthcare). After blocking with 3% BSA in TBST (TBS containing 1% Tween 20) for 1 h, membranes were incubated overnight at 4°C with the following primary antibodies diluted in TBST containing 3% BSA: anti-(phospho-Akt Ser473) (1:500 dilution; Cell Signaling Technology), anti-atrogin-1 (1:1000 dilution; ECM Biosciences), anti-MuRF-1 (1:1000 dilution; Santa Cruz Biotechnology), anti-(phospho-IκB) (1:500 dilution; Cell Signaling Technology), anti-β-actin (1:10000 dilution; Millipore) or anti-(cyclophilin B) (1:500 dilution; Santa Cruz Biotechnology). Membranes were washed three times with TBST and incubated for 1 h at room temperature with HRP (horseradish peroxidase)-conjugated goat anti-(rabbit IgG) (1:5000 dilution; Kirkegaard & Perry Laboratories), HRP-conjugated goat anti-(mouse IgG) (1:3000 dilution; Kirkegaard & Perry Laboratories) or HRP-conjugated donkey anti-(goat Ig) (1:10000 dilution; Santa Cruz Biotechnology) diluted in TBST. Secondary antibody binding was detected with ECL Plus (GE Healthcare) using an ImageQuant 350 detection system (GE Healthcare).

Each band was quantified densitometrically using ImageJ software. Ratios between densitometric values for the bands of interest and normalization bands were plotted using GraphPad Prism software.

Statistical analyses

Statistical analyses of data with more than two experimental groups were performed by one-way ANOVA with a Newman–Keuls post-hoc test, using the GraphPad Prism software. Values are means±S.E.M. In the case of only two experimental groups, an unpaired Student's t test was performed. *P<0.05 compared with control groups.

RESULTS

Activation of the kinin B1 receptor up-regulates atrogin-1 and MuRF-1 E3 ligases and decreases C2C12 myotube diameter

As a first step to evaluate the possible involvement of the kinin B1 receptor in the regulation of proteolysis and muscle mass, C2C12 myotubes were stimulated with the kinin B1 receptor agonist DABK (1 μM). Figure 1(A) shows that DABK evoked a significant reduction in myotube diameter at 24 and 48 h after stimulation. Subsequently, we investigated the molecular events related to atrophy in C2C12 myotubes stimulated with DABK. We analysed the expression levels of atrogin-1 and MuRF-1, two E3 ubiquitin ligases that are known to be critical in muscle protein breakdown processes [21]. Figure 1(B) shows that the expression levels of atrogin-1 and MuRF-1 mRNA are increased in C2C12 myotubes after stimulation with 200 nM DABK. MuRF-1 mRNA levels were increased after 2 h of treatment and atrogin-1 was increased after 24 h. These findings were corroborated by immunocytochemical analysis in C2C12 myotubes (Figures 1C–1E).

Effects of kinin B1 receptor activation on C2C12 myotubes

Figure 1
Effects of kinin B1 receptor activation on C2C12 myotubes

(A) The kinin B1 receptor agonist DABK (1 μM) induced a decrease in C2C12 myotube diameters after 24 and 48 h. (B) DABK (200 nM) induced the expression of MuRF-1 and atrogin-1 mRNAs. (C) Immunocytochemistry for atrogin-1 and MuRF-1 in C2C12 myotubes treated with DABK (200 nM) for 12 and 24 h. Scale bar, 10 μm. (D and (E) Quantification of the immunocytochemical analysis for atrogin-1 and MuRF-1. (F) DABK (200 nM) induced a decrease in IGF-1 mRNA expression levels. A.U., arbitrary units.

Figure 1
Effects of kinin B1 receptor activation on C2C12 myotubes

(A) The kinin B1 receptor agonist DABK (1 μM) induced a decrease in C2C12 myotube diameters after 24 and 48 h. (B) DABK (200 nM) induced the expression of MuRF-1 and atrogin-1 mRNAs. (C) Immunocytochemistry for atrogin-1 and MuRF-1 in C2C12 myotubes treated with DABK (200 nM) for 12 and 24 h. Scale bar, 10 μm. (D and (E) Quantification of the immunocytochemical analysis for atrogin-1 and MuRF-1. (F) DABK (200 nM) induced a decrease in IGF-1 mRNA expression levels. A.U., arbitrary units.

We also found that C2C12 myotubes stimulated with 200 nM DABK had decreased expression levels of IGF-1 (insulin growth factor-1) mRNA after 2 h, which persisted until at least 24 h after stimulation (Figure 1F). To confirm that the DABK-induced decrease in IGF-1 mRNA levels had an effect on the signalling pathways that control protein breakdown in muscles, we analysed Akt phosphorylation levels in protein extracts of C2C12 myotubes before and after stimulation with DABK. We found that phosphorylation of Akt at Ser473 was decreased by more than 50% at 2 h after DABK treatment and this persisted until at least 24 h (Figure 2A).

Activation of the kinin B1 receptor regulates PI3K/Akt and NF-κB signalling pathways

Figure 2
Activation of the kinin B1 receptor regulates PI3K/Akt and NF-κB signalling pathways

(A) Upper panel, DABK (200 nM) treatment for 2, 8 and 24 h induced a consistent decrease in Akt (Thr308) and Akt (Ser473) phosphorylation. Lower panel, quantification of the Western blots. (B) Upper panel, immunocytochemistry for the p50 subunit NF-κB. DABK (200 nM) treatment for 6 h induced the translocation of p50 to the nuclei of myotubes. Lower panel, quantification of the immunocytochemical analysis. Scale bar, 10 μm.

Figure 2
Activation of the kinin B1 receptor regulates PI3K/Akt and NF-κB signalling pathways

(A) Upper panel, DABK (200 nM) treatment for 2, 8 and 24 h induced a consistent decrease in Akt (Thr308) and Akt (Ser473) phosphorylation. Lower panel, quantification of the Western blots. (B) Upper panel, immunocytochemistry for the p50 subunit NF-κB. DABK (200 nM) treatment for 6 h induced the translocation of p50 to the nuclei of myotubes. Lower panel, quantification of the immunocytochemical analysis. Scale bar, 10 μm.

As NF-κB activation can be potentially responsible for induction of MuRF-1 expression in muscles independently of atrogin-1 induction [11], we investigated whether DABK stimulation led to activation of this transcription factor in C2C12 myotubes. Indeed, Figure 2(B) shows that the p50 NF-κB subunit was translocated to myotube nuclei 6 h after DABK stimulation.

In vivo functional impairment of the kinin B1 receptor by pharmacological blockade or genetic ablation reveals its role in muscle atrophy by modulating MuRF-1-related pathways

Next, we investigated the possible role of the kinin B1 receptor in an in vivo model of muscle atrophy not associated with invasive neuromuscular lesions, such as denervation, or systemic metabolic disturbance, as caused by cachexia or sepsis. We used the testosterone-dependent fast-twitch LA muscle model. LA muscle from rats and mice has a high expression of androgen receptors compared with other skeletal muscles, for example EDL [22,23]. Therefore gonadectomy induces a rapid and massive loss of LA muscle mass, which is associated with the increased expression of atrogin-1 and MuRF-1 [24,25]. At 7 days after gonadectomy, LA mass was reduced by ~30% (Figure 3A). Relative EDL muscle mass was also analysed as a control, and, as expected was shown not to be altered (Figure 3A). Interestingly, we found that the expression level of kinin B1 receptor mRNA was increased in the LA muscles at 7 days after gonadectomy (Figure 3D), which was paralleled by the increase in atrogin-1 and MuRF-1 mRNA expression (Figures 3B and 3C). We then treated gonadectomized mice with R-715, a peptidic kinin B1 receptor antagonist [26]. Blockade of the kinin B1 receptor reduced the atrogin-1 and MuRF-1 protein levels in LA muscles from gonadectomized mice (Figures 4A and 4B). We also analysed the phosphorylation levels of Akt and IκB in LA muscles from R-715-treated or control animals. Interestingly, Figure 4(C) shows that Akt phosphorylation was not regulated in this model (at least within the time period analysed) and that the kinin B1 receptor blockade had no effect on this target either. On the other hand, analysis of IκB phosphorylation levels showed that it increased in animals at 7 days after gonadectomy and that treatment with R-715 impaired its phosphorylation (Figure 4D), suggesting that the kinin B1 receptor is able to regulate NF-κB in LA muscles. Despite the effect of R-715 on the expression of atrogin-1 and MuRF-1 in LA muscles at 7 days after gonadectomy, we did not detect any effect of this compound on relative LA muscle mass, as muscles from the control and treated groups lost muscle mass to the same extent after 7 days of treatment (Figure 4E).

Kinin B1 receptor mRNA expression is increased similarly to muscle-specific E3 ligases in an LA muscle atrophy model

Figure 3
Kinin B1 receptor mRNA expression is increased similarly to muscle-specific E3 ligases in an LA muscle atrophy model

(A) After 7 days of gonadectomy, relative LA muscle mass was significantly decreased, whereas relative EDL muscle mass was not altered. (B–D) After gonadectomy, atrogin-1, MuRF-1 and kinin B1 receptor mRNA levels were increased in LA muscles.

Figure 3
Kinin B1 receptor mRNA expression is increased similarly to muscle-specific E3 ligases in an LA muscle atrophy model

(A) After 7 days of gonadectomy, relative LA muscle mass was significantly decreased, whereas relative EDL muscle mass was not altered. (B–D) After gonadectomy, atrogin-1, MuRF-1 and kinin B1 receptor mRNA levels were increased in LA muscles.

Treatment of mice with the kinin B1 receptor antagonist R-715 impairs atrogin-1 and MuRF-1 up-regulation and IκB phosphorylation, but has no effect on Akt activation and relative mass in LA muscles

Figure 4
Treatment of mice with the kinin B1 receptor antagonist R-715 impairs atrogin-1 and MuRF-1 up-regulation and IκB phosphorylation, but has no effect on Akt activation and relative mass in LA muscles

After treatment of gonadectomized Balb-C mice with R-715, up-regulation of atrogin-1 (Atg-1) (A) and MuRF-1 (B) protein was impaired. Akt (Ser473) phosphorylation levels (C) were unaffected, but IκB phosphorylation (D) was significantly reduced. (E) R-715 had no effect on LA relative mass of gonadectomized mice. NC, non-gonadectomized; C7d, 7 days after gonadectomy; Cyp B, cyclophilin B.

Figure 4
Treatment of mice with the kinin B1 receptor antagonist R-715 impairs atrogin-1 and MuRF-1 up-regulation and IκB phosphorylation, but has no effect on Akt activation and relative mass in LA muscles

After treatment of gonadectomized Balb-C mice with R-715, up-regulation of atrogin-1 (Atg-1) (A) and MuRF-1 (B) protein was impaired. Akt (Ser473) phosphorylation levels (C) were unaffected, but IκB phosphorylation (D) was significantly reduced. (E) R-715 had no effect on LA relative mass of gonadectomized mice. NC, non-gonadectomized; C7d, 7 days after gonadectomy; Cyp B, cyclophilin B.

To investigate the in vivo role of the kinin B1 receptor in muscle proteolysis, we used B1R−/− mice [8] that were subjected to the LA muscle atrophy model protocol. Strikingly, the results showed that LA muscles from gonadectomized B1R−/− mice did not have an increased expression of MuRF-1 mRNA (Figures 5A and 5B). Additionally, atrogin-1 mRNA was up-regulated 4-fold compared with LA muscle from WT mice with the same genetic background (Figures 5C and 5D). As further evidence for the role of the kinin B1 receptor in muscle protein catabolism, it is important to note that, despite the observed loss of LA muscle mass after gonadectomy in both WT and B1R−/− animals, the relative muscle mass in the B1R−/− mice was actually significantly higher than that observed in the WT group (Figure 5E).

Induction of LA muscle atrophy in B1R−/− mice impairs MuRF-1 up-regulation, but has no effect on protection of muscle mass

Figure 5
Induction of LA muscle atrophy in B1R−/− mice impairs MuRF-1 up-regulation, but has no effect on protection of muscle mass

MuRF-1 mRNA expression was increased in LA muscles from WT mice (A), but not in B1R−/− mice (B). Atrogin-1 mRNA expression was increased in LA muscles from WT mice (C) and was augmented even more in B1R−/− mice (D). (E) Comparison of relative LA muscle mass from WT and B1R−/− mice.

Figure 5
Induction of LA muscle atrophy in B1R−/− mice impairs MuRF-1 up-regulation, but has no effect on protection of muscle mass

MuRF-1 mRNA expression was increased in LA muscles from WT mice (A), but not in B1R−/− mice (B). Atrogin-1 mRNA expression was increased in LA muscles from WT mice (C) and was augmented even more in B1R−/− mice (D). (E) Comparison of relative LA muscle mass from WT and B1R−/− mice.

DISCUSSION

As the signalling pathways triggered by the kinin B1 receptor (e.g. NF-κB and PI3K/Akt) have also been described to be implicated in skeletal muscle catabolic disorders, we hypothesized that this receptor could play a possible role in muscular atrophy. The results reported in the present paper show that activation of the kinin B1 receptor might indeed play an important role in muscle mass regulation.

The first part of our study shows that stimulation of C2C12 myotubes with the kinin B1 receptor agonist DABK leads to (i) a decrease in myotube diameter (see Figure 1A), (ii) an increase in atrogin-1 and MuRF-1 mRNA and protein levels (see Figure 1B–E), (iii) a decrease in IGF-1 mRNA (see Figure 1F) and Akt phosphorylation levels (see Figure 2A), and (iv) translocation of NF-κB to myotube nuclei (see Figure 2B). When muscle tissue loses mass during atrophy, a decrease in cross-sectional area is observed in the muscle fibres, which is the macroscopic and prominent evidence of muscle atrophy. In C2C12 myotubes, it is possible to evaluate this parameter by measuring the diameter of the myotubes. Several molecules that induce muscle atrophy in vivo, for example dexamethasone, lead to a decrease in the diameter of C2C12 myotubes. Atrogin-1 and MuRF-1 are E3 ubiquitin-ligases specifically expressed in muscle tissue and are directly involved in the substantial protein degradation observed during muscle atrophy by activation of the UPS (ubiquitin–proteasome system) [21,27]. The increase in the expression level of these molecules in muscle is an early marker of muscle atrophy in different models [28]. As DABK induced the expression of atrogin-1 and MuRF-1 mRNAs in C2C12 myotubes, our findings suggest a role for the kinin B1 receptor in muscle atrophy by regulating signalling pathways such as the PI3K/Akt and IKK/NF-κB pathways.

Although IGF-1 is an endocrine peptide, it can also be produced by muscle tissues in a paracrine manner [29]. It is well known that IGF-1 is involved in activation of the PI3K/Akt pathway, which in turn leads to the phosphorylation of FoxO (forkhead box O) factors in the cytoplasm, preventing their entrance into the nucleus to activate the expression of atrogin-1 and MuRF-1 mRNAs [30,31]. Indeed, our results show that DABK induced a decrease in IGF-1 mRNA levels (see Figure 1F) and Akt phosphorylation (see Figure 2A) in C2C12 myotubes and therefore provide evidence for an important role of the kinin B1 receptor in regulating muscle mass, since the PI3K/Akt/mTOR (mammalian target of rapamycin) pathway is the main signalling pathway involved in the regulation of protein synthesis and degradation in muscle.

On the other hand, it has been shown that kinin B1 receptor expression is regulated by NF-κB [32] and that activation of this receptor also leads to phosphorylation of NF-κB [33], therefore mediating inflammatory responses in different tissues. We have shown that DABK activates the NF-κB pathway in C2C12 myotubes (see Figure 2B), suggesting an additional mechanism by which the kinin B1 receptor may control MuRF-1 expression.

Taken together, our present findings show that, at the cellular level, activation of the kinin B1 receptor regulates protein metabolism, causing a significant decrease in C2C12 myotube diameter. This effect was due to an integrated regulation of the PI3K/Akt and NF-κB pathways, which ultimately leads to the induction of the expression of the ubiquitin ligases atrogin-1 and MuRF-1.

To validate these findings at the physiological level, we used an in vivo model of muscle atrophy, namely the testosterone-dependent fast-twitch LA muscle model. The LA muscle of adult rodents is a suitable model to evaluate the regulation of muscle proteolysis because it displays a rapid and progressive atrophy in response to androgen deprivation, with minor systemic or inflammatory effects associated with fasting, cachexia or surgical muscle denervation. We have shown, as expected, that loss of LA muscle mass after gonadectomy was paralleled by increases in atrogin-1 and MuRF-1 mRNA levels, but strikingly also by an increase in kinin B1 receptor mRNA levels (see Figure 3B). Such an increase in kinin B1 receptor levels suggests the participation of this receptor in the control of protein content during atrophy of LA muscles. Accordingly, we have also shown that antagonist blockade of the kinin B1 receptor impaired the increase in atrogin-1 and MuRF-1 protein levels in LA muscles from gonadectomized mice (see Figure 4A), corroborating the in vitro data obtained with C2C12 myotubes (see Figures 1B and 1C). Interestingly, treatment with the kinin B1 receptor antagonist had no effect on Akt phosphorylation levels, but impaired the phosphorylation of IκB (see Figure 4B). These results suggest that, in vivo, the NF-κB pathway is the main pathway involved in muscle protein metabolism regulated by kinin B1 receptor activation. Nevertheless, further studies using other muscle atrophy models are necessary to strengthen the impact of these important findings.

Despite the fact that the kinin B1 receptor antagonist was effective in reducing atrogin-1 and MuRF-1 content in LA muscles of gonadectomized mice, it had no effect on muscle mass loss (see Figure 4C), confirming that other compensatory factors and pathways may also be involved. Therefore the combination of kinin B1 receptor blockade with other agents/drugs also needs to be evaluated in future studies.

Our results obtained with B1R−/− mice show that these animals do not have an up-regulation of MuRF-1 mRNA levels in LA muscles after gonadectomy (see Figure 5A), whereas atrogin-1 mRNA levels were increased 4-fold compared with the WT group (see Figure 5B). It is noteworthy that LA muscles from B1R−/− mice have a higher relative mass compared with the WT group at all of the time periods analysed, including before gonadectomy (see Figure 5A). These data suggest that, in the complete absence of the kinin B1 receptor during the ontogeny of B1R−/− mice, the E3 ligases involved in muscle atrophy are differentially modulated, reinforcing the fact that MuRF-1 may play a prevailing role downstream of kinin B1 receptor activation. In this case, a probable compensatory effect was achieved via atrogin-1 overexpression. Moreover, it is also possible that such compensatory modulation of ubiquitin ligases and muscle mass involve other proteolytic pathways, such as the calcium-dependent and lysosomal proteolysis pathways. Finally, the observation that relative LA muscle mass from B1R−/− mice was higher than in WT mice suggests a role for the kinin B1 receptor in the regulation of muscle mass not only during the atrophy process, but also in physiological conditions.

In conclusion, the results of the present study show that the kinin B1 receptor significantly contributes to the modulation of downstream signalling responses in skeletal muscle, leading to the fine-tune regulation of the UPS, and ultimately influencing the proteolysis rate balance (Figure 6). We believe that our findings open new perspectives for the investigation of skeletal muscle protein metabolism and approaches to control its balance, which is of immediate clinical relevance.

Schematic representation of the proposed role of kinin B1 receptor in a skeletal muscle cell

Figure 6
Schematic representation of the proposed role of kinin B1 receptor in a skeletal muscle cell

In the steady state, PI3K is activated by growth factors and generates PIPs (phosphatidylinositol phosphates), recruiting and activating PDK1 (phosphoinositide-dependent kinase-1), which in turn recruits Akt to this complex at the membrane. At this stage, Akt becomes phosphorylated at Ser473 by mTORC2 (mTOR complex 2) and at Thr308 by PDK1. Activated Akt phosphorylates FoxO transcription factors and prevents them from entering into the nucleus to induce atrogin-1 and MuRF-1 transcription. In addition, in physiological conditions, IκB is found constitutively unphosphorylated and interacting with RelA/p50 (NF-κB), which impairs its entry into the nucleus to induce MuRF-1 transcription. Our data suggest that activation of the kinin B1 receptor inhibits PI3K activation, consequently impairing Akt activation and phosphorylation of FoxO. In addition, kinin B1 receptor activates IKK, which phosphorylates IκB, releasing it from NF-κB, inducing its ubiquitination and then directing it to degradation by the proteasome.

Figure 6
Schematic representation of the proposed role of kinin B1 receptor in a skeletal muscle cell

In the steady state, PI3K is activated by growth factors and generates PIPs (phosphatidylinositol phosphates), recruiting and activating PDK1 (phosphoinositide-dependent kinase-1), which in turn recruits Akt to this complex at the membrane. At this stage, Akt becomes phosphorylated at Ser473 by mTORC2 (mTOR complex 2) and at Thr308 by PDK1. Activated Akt phosphorylates FoxO transcription factors and prevents them from entering into the nucleus to induce atrogin-1 and MuRF-1 transcription. In addition, in physiological conditions, IκB is found constitutively unphosphorylated and interacting with RelA/p50 (NF-κB), which impairs its entry into the nucleus to induce MuRF-1 transcription. Our data suggest that activation of the kinin B1 receptor inhibits PI3K activation, consequently impairing Akt activation and phosphorylation of FoxO. In addition, kinin B1 receptor activates IKK, which phosphorylates IκB, releasing it from NF-κB, inducing its ubiquitination and then directing it to degradation by the proteasome.

Abbreviations

     
  • B1R−/−

    kinin B1 receptor knockout

  •  
  • DABK

    des-Arg9-bradykinin

  •  
  • DMEM

    Dulbecco’s modified Eagle’s medium

  •  
  • EDL

    extensor digitorum longus

  •  
  • FoxO

    forkhead box O

  •  
  • HRP

    horseradish peroxidase

  •  
  • IGF-1

    insulin growth factor-1

  •  
  • IκB

    inhibitor of NF-κB

  •  
  • IKK

    IκB kinase

  •  
  • KKS

    kallikrein–kinin system

  •  
  • LA

    levator ani

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • MuRF-1

    muscle RING-finger protein-1

  •  
  • NF-κB

    nuclear factor κB

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • TBST

    TBS containing 1% Tween 20

  •  
  • UPS

    ubiquitin–proteasome system

  •  
  • WT

    wild-type

AUTHOR CONTRIBUTION

Lucas Parreiras-e-Silva, Rosana Reis, Geisa Santos and Marcelo Pires-Oliveira performed the experiments and interpreted the results; João Pesquero provided reagents and contributed to data interpretation; Marcelo Gomes contributed to data interpretation and discussion; Rosely Godinho contributed to experimental design, data interpretation and discussion; and Claudio Costa-Neto and Lucas Parreiras-e-Silva conceived the study, interpreted the results and wrote the paper.

FUNDING

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). L.T.P.-e-S. and R.I.R. were recipients of Ph.D. fellowships from FAPESP; C.M.C.-N., R.O.G., M.D.G. and J.B.P. hold Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) research fellowships.

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