Preventive Effects of Collagen-Derived Dipeptide Prolyl-Hydroxyproline against Dexamethasone-Induced Muscle Atrophy in Mouse C2C12 Skeletal Myotubes

Abstract

Glucocorticoids, commonly used to manage inflammatory diseases, can induce muscle atrophy by accelerating the breakdown of muscle proteins. This research delves into the influence of Prolyl-hydroxyproline (Pro-Hyp), a collagen-derived peptide, on muscle atrophy induced with dexamethasone (DEX), a synthetic glucocorticoid, in mouse C2C12 skeletal myotubes. Exposure to DEX (10 μM) for 6 days resulted in a decrease in myotube diameter, along with elevated mRNA and protein levels of two muscle-atrophy-related ubiquitin ligases, muscle atrophy F-box (MAFbx, also known as atrogin-1) and muscle ring finger 1 (MuRF-1). Remarkably, treatment with 0.1 mM of Pro-Hyp mitigated the reduction in myotube thickness caused by DEX, while promoting the phosphorylation of Akt, mammalian target of rapamycin (mTOR), and forkhead box O3a (Foxo3a). This led to the inhibition of the upregulation of the ubiquitin ligases atrogin-1 and MuRF-1. These findings indicate the potential significance of Pro-Hyp as a promising therapeutic target for countering DEX-induced muscle atrophy.

Keywords: Prolyl-hydroxyproline; collagen-derived peptide; mouse C2C12 skeletal myotubes; muscle atrophy; ubiquitin ligases.

Figure 1

Effects of Pro-Hyp on myotube diameter in DEX-stimulated C2C12 myotubes. (A) Representative images of C2C12 myotubes treated with 10 μM of DEX and Pro-Hyp (0.01 mM and 0.1 mM). Fixed cells were reacted with an anti-MHC antibody and a fluorescence-labeled secondary antibody (green). The nuclei were stained with DAPI (blue). The scale bar represents 250 μm. (B) Comparison of myotube diameters among the four treatment groups. Data are expressed as means ± SD. * p < 0.05 vs. non-treated controls, # p < 0.05 vs. DEX-treated groups. p < 0.05.

Figure 2

Effects of Pro-Hyp on muscle-atrophy-associated genes and myotube-differentiation-related genes in DEX-stimulated C2C12 myotubes. C2C12 myotubes were treated with 10 µM of DEX and Pro-Hyp (0.01 mM and 0.1 mM) for 24 h. (A) Atrogin-1, (B) MuRF-1, (C) MyoD, and (D) Myogenin mRNA levels were examined with qPCR. GAPDH was used as an internal control. Data are expressed as means ± SD. * p < 0.05 vs. non-treated controls, # p < 0.05 vs. DEX-treated groups. p < 0.05.

Figure 3

Effects of Pro-Hyp on protein levels of muscle-atrophy-associated ubiquitin ligases and ubiquitinated proteins in DEX-induced myotube atrophy. C2C12 myotubes were treated with 10 µM of DEX and Pro-Hyp (0.01 mM and 0.1 mM) for 24 h. Western blot assay examined atrogin-1, MuRF-1, and ubiquitinated protein level. β-actin was used as an internal control. (A) Representative Western blot of total forms of atrogin-1, MuRF-1, ubiquitinated proteins, and β-actin. (B) The ratio of total atrogin-1 and β-actin normalized to the control. (C) The ratio of total MuRF-1 and β-actin normalized to the control. (D) The ratio of ubiquitinated proteins and β-actin normalized to the control. Data are expressed as means ± SD. * p < 0.05 vs. non-treated controls, # p < 0.05 vs. DEX-treated groups. p < 0.05.

Figure 4

Effects of Pro-Hyp on Akt-mTOR-Foxo3a signaling in DEX-induced myotube atrophy. C2C12 myotubes were treated with 10 μM of DEX and Pro-Hyp (0.01 mM and 0.1 mM) for 24 h. Western blot assay examined Akt, phosphorylated Akt, mTOR, phosphorylated mTOR, Foxo3a, and phosphorylated Foxo3a protein levels. β-actin was used as an internal control. (A) Representative Western blot of phosphorylated and total forms of Akt, phosphorylated and total forms of Foxo3a, phosphorylated and total forms of mTOR, and β-actin. (B) The ratio of phosphorylated and total Akt normalized to the control. (C) The ratio of phosphorylated and total mTOR normalized to the control. (D) The ratio of phosphorylated and total Foxo3a normalized to the control. Data are expressed as means ± SD. * p < 0.05 vs. non-treated controls, # p < 0.05 vs. DEX-treated groups. p < 0.05.