Expression of the Mas receptor is upregulated in skeletal muscle wasting

Abstract

Skeletal muscle atrophy during sepsis, immobilization, and chronic diseases is characterized by an increase in expression and activity of the muscle-specific ubiquitin 3 ligases atrogin-1 and MuRF-1. The classical renin–angiotensin system (RAS), by high level of circulating angiotensin II (AngII) is directly involved in skeletal muscle wasting associated with cardiac and renal failure. Ang (1–7), a peptide belonging to the non-classical RAS system, produces effects that are opposite to AngII. The actions of Ang (1–7) are mediated by its binding and signalling through the Mas receptor. Our purpose is to assess the effects of atrophic stimuli AngII, lipopolysaccharide (LPS), and immobilization on the expression of the Mas receptor in skeletal muscle. For that we used gastrocnemius and tibialis anterior muscles of C57BL10 mice treated with AngII, LPS or subjected to unilateral hindlimb immobilization by casting. In addition, we used C2C12 myotubes incubated with AngII or LPS. We evaluated Mas expression by quantitative real-time PCR, Western blot immunohistochemical analysis. Skeletal muscle atrophy was corroborated by the expression of atrogin-1 and MuRF-1 and the fibre diameter. Our results show that Mas receptor expression was increased by AngII or LPS in vitro and in vivo, and upregulated by immobilization. The increase of the Mas expression was concomitantly with the upregulation of atrogin-1 and MuRF-1 and the reduction of the fibre diameter. These results from studies in vitro and in vivo demonstrate for the first time that the Mas receptor is increased under atrophic stimulus and suggest that the non-classical RAS system could have an important role in muscle wasting.

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Acknowledgments

This study was supported by research grants from Association-Francaise Contre Les Myopathies AFM 16670 (CCV); FONDECYT 1120380 (CCV), 3130593 (MGM), 1121078 (FS); and the Millennium Institute on Immunology and Immunotherapy, P09-016-F (FS); UNAB-DI-281-13/R (CCV).

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Correspondence to Claudio Cabello-Verrugio.

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María Gabriela Morales and Johanna Abrigo have contributed equally to this work.

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Fig. S1: Upregulation of the Mas receptor induced by AngII in C

2 C 12 myotubes is dependent on the AT-1 receptor. C2C12 myotubes from day 5 were pre-incubated with losartan (10 μM) or PD123319 (10 μM) for 1 h prior to the incubation with AngII (500 nM) for 3 h. At the end of this treatment, the mRNA levels of Mas were determined by RT-qPCR using β-actin as the reference gene. The expression was expressed as the fold of induction, normalized to the levels in the control cells. Values correspond to the mean ± SD of three independent experiments (*, P < 0.05 relative to the control cells, #, P < 0.05 relative to AngII-vehicle) (TIFF 219 kb)

Fig. S2: The Mas receptor is increased in the tibialis anterior of AngII-induced skeletal muscle atrophy.

Skeletal muscle atrophy was induced in C57BL/10J mice by osmotic infusion of AngII (1 μg/kg/min). (a) Mas receptor was detected by indirect immunohistochemical analysis in cryosections of tibialis anterior muscles from control mice and mice treated with AngII for 14 days. Nuclei were labelled with haematoxylin. The bar corresponds to 50 μm. The images are representative of three independent experiments, using three mice for each experimental condition. (b) Histological analysis of the tibialis anterior of control mice and mice treated with AngII for 14 days. Muscle cross sections were stained with haematoxylin and eosin to visualize muscle architecture. The bar corresponds to 50 μm. The images are representative of three independent experiments, using three mice for each experimental condition. (c) Quantitative analysis of the fibre diameter from three independent experiments using three mice for each experimental condition. The values are expressed as the percentage of the total fibres quantified (*, P < 0.05 relative to the control) (TIFF 8578 kb)

Fig. S3: Upregulation of Mas receptor induced by LPS in C

2 C 12 myotubes is dependent on the TLR-4 receptor. C2C12 myotubes from day 5 were pre-incubated with CLI-095 (5 μM) for 1 h prior to the incubation with LPS (500 ng/ml) for 3 h. At the end of this treatment, mRNA levels of Mas were determined by RT-qPCR using β-actin as the reference gene. The expression was expressed as the fold of induction, normalized to the levels in the control cells. Values correspond to the mean ± SD of three independent experiments (*, P < 0.05 relative to the control cells, #, P < 0.05 relative to LPS-vehicle) (TIFF 208 kb)

Fig. S4: The Mas receptor is upregulated in the tibialis anterior of LPS-induced skeletal muscle atrophy.

Skeletal muscle atrophy was induced in C57BL/10J mice by intraperitoneal injection of LPS (1 mg/kg). (a) The Mas receptor was detected by indirect immunohistochemical analysis in cryosections of the tibialis anterior of muscles from control and LPS-treated mice for 14 days. Nuclei were labelled with haematoxylin. The bar corresponds to 50 μm. The images are representative of three independent experiments, using three mice for each experimental condition. (b) Histological analysis of the tibialis anterior of control mice and mice treated with LPS for 14 days. Muscle cross sections were stained with haematoxylin and eosin to visualize muscle architecture. The bar corresponds to 50 μm. The images are representative of three independent experiments, using three mice for each experimental condition. (c) Quantitative analysis of the fibre diameter from three independent experiments using three mice for each experimental condition. The values are expressed as the percentage of the total fibres quantified (*, P < 0.05 relative to the control) (TIFF 8007 kb)

Fig. S5: The Mas receptor is augmented in the tibialis anterior of disuse-induced skeletal muscle atrophy.

Skeletal muscle atrophy was induced in C57BL/10J mice by unilateral immobilizated hindlimb by casting. (a) The Mas receptor was detected by indirect immunohistochemical analysis in cryosections of tibialis anterior muscles from non-immobilized hindlimbs and hindlimbs immobilized for 14 days. Nuclei were labelled with haematoxylin. The bar corresponds to 50 μm. The images are representative of three independent experiments, using three mice for each experimental condition. (b) Histological analysis of tibialis anterior non-immobilized hindlimbs and hindlimbs immobilized for 14 days. Muscle cross sections were stained with haematoxylin and eosin to visualize muscle architecture. The bar corresponds to 50 μm. The images are representative of three independent experiments, using three mice for each experimental condition. (c) Quantitative analysis of the fibre diameter from three independent experiments using three mice for each experimental condition. The values are expressed as the percentage of the total fibres quantified (*, P < 0.05 relative to the non-immobilizated muscles) (TIFF 8431 kb)

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Morales, M.G., Abrigo, J., Meneses, C. et al. Expression of the Mas receptor is upregulated in skeletal muscle wasting. Histochem Cell Biol 143, 131–141 (2015). https://doi.org/10.1007/s00418-014-1275-1

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Keywords

  • Mas receptor
  • Muscle wasting
  • E3 ligases
  • Renin–angiotensin system