Angiotensin (1-7) Decreases Myostatin-Induced NF-κB Signaling and Skeletal Muscle Atrophy

Abstract

Myostatin is a myokine that regulates muscle function and mass, producing muscle atrophy. Myostatin induces the degradation of myofibrillar proteins, such as myosin heavy chain or troponin. The main pathway that mediates protein degradation during muscle atrophy is the ubiquitin proteasome system, by increasing the expression of atrogin-1 and MuRF-1. In addition, myostatin activates the NF-κB signaling pathway. Renin-angiotensin system (RAS) also regulates muscle mass. Angiotensin (1-7) (Ang-(1-7)) has anti-atrophic properties in skeletal muscle. In this paper, we evaluated the effect of Ang-(1-7) on muscle atrophy and signaling induced by myostatin. The results show that Ang-(1-7) prevented the decrease of the myotube diameter and myofibrillar protein levels induced by myostatin. Ang-(1-7) also abolished the increase of myostatin-induced reactive oxygen species production, atrogin-1, MuRF-1, and TNF-α gene expressions and NF-κB signaling activation. Ang-(1-7) inhibited the activity mediated by myostatin through Mas receptor, as is demonstrated by the loss of all Ang-(1-7)-induced effects when the Mas receptor antagonist A779 was used. Our results show that the effects of Ang-(1-7) on the myostatin-dependent muscle atrophy and signaling are blocked by MK-2206, an inhibitor of Akt/PKB. Together, these data indicate that Ang-(1-7) inhibited muscle atrophy and signaling induced by myostatin through a mechanism dependent on Mas receptor and Akt/PKB.

Keywords: Akt/PKB; Angiotensin-(1-7); NF-κB signaling; RAS; muscle atrophy.

Figure 1

Angiotensin (1-7) (Ang-(1-7)) prevented the myostatin-induced decrease of the myotube diameter. (A) C₂C₁₂ myoblasts differentiated for five days (myotubes) were pre-incubated in the absence or presence of Ang-(1-7) (10 nM) for 60 min, and then incubated with myostatin (Mstn, 1 µg/mL) for 72 h. Myosin heavy chain (MHC; green) was detected through indirect immunofluorescence (IFI). Hoechst was used to stain the nuclei (blue). The bar scale represents 100 μm. (B) The graphics show the distribution of the myotube diameters. The values are expressed as a percentage of the total myotubes and correspond to the mean ± standard deviation (SD) from three independent experiments (n = 3; * p < 0.05 vs. control).

Figure 2

Ang-(1-7) decreased the myostatin-dependent decline in the myofibrillar protein levels in the myotubes. The C₂C₁₂ myotubes were incubated with myostatin (Mstn, 1 µg/mL) for 72 h in the presence or absence of Ang-(1-7) (10 nM; pre-incubated for 1 h). (A) The MHC, troponin, and β-actin protein levels were determined by Western blot analysis. The molecular weights are shown in kDa. Densitometric analysis for the MHC (B) and troponin (C) protein levels with values normalized to β-actin and expressed as a fold of change relative to the control (myotubes without treatment). All of the values correspond to the mean ± SD from three independent experiments (n = 3; * p < 0.05 vs. control without treatment).

Figure 3

Ang-(1-7) avoided the myostatin-dependent induction of atrogin-1 and MuRF-1 in the myotubes. The C₂C₁₂ cells differentiated for five days were pretreated or not with Ang-(1-7) (10 nM) for 1 h, and further with myostatin (Mstn, 1 µg/mL) for 12 or 24 h. The mRNA levels of atrogin-1 (A) and MuRF-1 (B) were determined by RT-qPCR. (C) The protein levels of atrogin-1 and MuRF-1 were detected by Western Blot analysis. β-actin was used as the loading control. The molecular weights are shown in kDa. Quantitative analysis of atrogin-1 (D) and MuRF-1 (E) levels with values normalized to β-actin. All of the values correspond to the mean ± SD from three independent experiments (n = 3; * p < 0.05 vs. control without treatment).

Figure 4

Ang-(1-7) reduced the reactive oxygen species (ROS) production induced by myostatin in the myotubes. (A) C₂C₁₂ myotubes were incubated with myostatin (Mstn, 1 µg/mL) for 24 h in the presence or absence of Ang-(1-7) (10 nM; pre-incubated for 1 h). The ROS levels were then determined by the detection of fluorescence using a CM-DCF-DA probe. The nuclei were labelled with Hoechst. The scale bar represents 100 µm. (B) Quantification of the DCF-positive myotubes. The values are expressed as a fold of induction relative to the myotubes without treatment, and correspond to the mean ± SD from three independent experiments (n = 3; * p < 0.05 vs. control without treatment).

Figure 5

Ang-(1-7) reduced the myostatin-induced NF-κB signaling in myotubes. (A) C₂C₁₂ myotubes were pre-incubated with or without Ang-(1-7) (10 nM) and after 1 h with myostatin (Mstn, 1 µg/mL) for 24 h. Protein levels of IκB and β-actin were detected by Western blot analysis. Molecular weights are shown in kDa. (B) Quantitative analysis for IκB protein levels with values normalized to β-actin. (C) Luciferase activity of pNF-κB-luc. (D) The mRNA levels of TNF-α were determined by RT-qPCR. The values are expressed as a fold of induction relative to the control (myotubes without treatment), and correspond to the mean ± SD from three independent experiments (n = 3; * p < 0.05 vs. control without treatment).

Figure 6

Reduction of myostatin dependent on muscle atrophy and signaling by Ang-(1-7) is mediated through Mas receptor in myotubes. C₂C₁₂ myotubes, pretreated with or without Ang-(1-7) (10 nM), A779 (10 µM) (pre-incubated for 30 min), or both, were incubated with myostatin (Mstn, 1 µg/mL) for 12 or 24 h. The parameters measured were: (A) distribution of the myotube diameters; mRNA levels of (B) atrogin-1, (C) MuRF-1, and (E) TNF-α; (D) Luciferase activity of pNF-κB-luc; and (F) ROS levels. The values are expressed as a fold of induction relative to the myotubes without treatment and correspond to the mean ± SD from three independent experiments (n = 3; * p < 0.05 vs. control without treatment).

Figure 7

Prevention of myostatin-dependent muscle atrophy and signaling by Ang-(1-7) is mediated by the Akt/PKB activity in the myotubes. The C₂C₁₂ myotubes were incubated with myostatin (Mstn, 1 µg/mL) for 12 or 24 h in the presence or absence of Ang-(1-7) (10 nM), MK2206 (10 µM; pre-incubated for 30 min), or both. The parameters measured were as follows: (A) distribution of the myotube diameters; mRNA levels of (B) atrogin-1, (C) MuRF-1 and (E) TNF-α; (D) Luciferase activity of pNF-κB-luc; and (F) ROS levels. The values are expressed as a fold of induction relative to the myotubes without treatment, and correspond to the mean ± SD from three independent experiments (n = 3; * p < 0.05 vs. control without treatment).

Figure 7

Prevention of myostatin-dependent muscle atrophy and signaling by Ang-(1-7) is mediated by the Akt/PKB activity in the myotubes. The C₂C₁₂ myotubes were incubated with myostatin (Mstn, 1 µg/mL) for 12 or 24 h in the presence or absence of Ang-(1-7) (10 nM), MK2206 (10 µM; pre-incubated for 30 min), or both. The parameters measured were as follows: (A) distribution of the myotube diameters; mRNA levels of (B) atrogin-1, (C) MuRF-1 and (E) TNF-α; (D) Luciferase activity of pNF-κB-luc; and (F) ROS levels. The values are expressed as a fold of induction relative to the myotubes without treatment, and correspond to the mean ± SD from three independent experiments (n = 3; * p < 0.05 vs. control without treatment).