Notch Inhibition via GSI Treatment Elevates Protein Synthesis in C2C12 Myotubes

Abstract

The role of Notch signaling is widely studied in skeletal muscle regeneration but little is known about its influences on muscle protein synthesis (MPS). The purpose of this study was to investigate whether Notch signaling is involved in the regulation of MPS. C2C12 cells were treated with a γ-secretase inhibitor (GSI), to determine the effect of reduced Notch signaling on MPS and anabolic signaling markers. GSI treatment increased myotube hypertrophy by increasing myonuclear accretion (nuclei/myotube: p = 0.01) and myonuclear domain (myotube area per fusing nuclei: p < 0.001) in differentiating C2C12 cells. GSI treatment also elevated myotube hypertrophy in differentiated C2C12s (area/myotube; p = 0.01). In concert, GSI treatment augmented pmTOR Ser2448 (p = 0.01) and protein synthesis (using SUnSET method) in myotubes (p < 0.001). Examining protein expression upstream of mTOR revealed reductions in PTEN (p = 0.04), with subsequent elevations in pAKT Thr308 (p < 0.001) and pAKT Ser473 (p = 0.05). These findings reveal that GSI treatment elevates myotube hypertrophy through both augmentation of fusion and MPS. This study sheds light on the potential multifaceted roles of Notch within skeletal muscle. Furthermore, we have demonstrated that Notch may modulate the PTEN/AKT/mTOR pathway.

Keywords: GSI; hypertrophy; notch; protein synthesis.

Figure 1

GSI reduces Notch signaling in differentiating C2C12 myotubes. (A) NICD/β-Actin; (B) Hes1/β-Actin expression (Integrated optical density, IOD) in 96-h myotubes treated with or without 4 µm γ-secretase inhibitor (GSI) every 12 h. Thirty minutes prior to collection all cells were treated with 1µm puromycin. For representative image: lanes 1 and 2 are Con; lanes 3 and 4 are GSI. Data were analyzed using a Student’s T-test. *** p < 0.001 vs. Control (Con). **** p < 0.0001 vs. Con (n = 3 experiments). Data are mean ± SD.

Figure 2

GSI increases myotube formation in differentiating C2C12 myotubes. (A) Indices of myotube fusion. Graph order, top left to right: Fused nuclei per field, Non-fused nuclei per field, and Total nuclei per field. Graph order, bottom left to right: Myotube number per field, Nuclei per myotube per field, Fusion index per field. (B) Indices of myotube hypertrophy. Graph order: Myotube area (µm) per field, Area (µm) per myotube per field, Myotube area (µm) per nuclei per field. (C) Representative image of 96-h myotubes co-stained with myosin heavy chain (MHC:red) and DAPI:blue. Images were taken at 20× magnification and the scale bar = 50 µm. (D) Myogenin/β-Actin; (E) MHC/β-Actin expression (Integrated optical density, IOD) in 96-h myotubes treated with or without 4 µm γ-secretase inhibitor (GSI) every 12 h. For Western blot representative images: lanes 1 and 2 are Con; lanes 3 and 4 are GSI. Thirty minutes prior to collection all cells were treated with 1µm puromycin. Data were analyzed using a Student’s T-test. * p ≤ 0.05 vs. Control (Con); ** p < 0.01 vs. Con; *** p < 0.001 vs. Con (n = 3 experiments). Data are mean ± SD.

Figure 3

GSI increases protein synthesis and mTOR signaling in C2C12 myotubes. (A) Puromycin/β-Actin; (B) Phospho (p)-mTOR Ser2448/Total mTOR; (C) p-mTOR Ser2481/Total mTOR; (D) p-4EBP1 Thr37/46/Total 4EBP1; (E) p-p70S6K Thr389/Total p70S6K; (F) p-eEF2 Thr56/Total eEF2 expression (Integrated optical density, IOD) in 96-h myotubes treated with or without 4 µm γ-secretase inhibitor (GSI) every 12 h. Thirty minutes prior to collection all cells were treated with 1 µm puromycin. For representative images: lanes 1 and 2 are Con; lanes 3 and 4 are GSI. Data were analyzed using a Student’s T-test. * p ≤ 0.05 vs. Control (Con); ** p < 0.01 vs. Con; **** p < 0.0001 vs. Con (n = 3 experiments). Data are mean ± SD.

Figure 4

GSI increases signaling upstream of mTOR in C2C12 myotubes. (A) PTEN/β-Actin; (B) p-AKT Thr308/Total AKT; (C) p-AKT Ser473/Total AKT; (D) p-TSC2 Ser939/Total TSC2; (E) p-TSC2 Thr1462/Total TSC2 expression (Integrated optical density, IOD) in 96-h myoblasts treated with or without 4 µm γ-secretase inhibitor (GSI) every 12 h. Thirty minutes prior to collection all cells were treated with 1 µm puromycin. For representative image: lanes 1 and 2 are Con; lanes 3 and 4 are GSI. Data were analyzed using a Student’s T-test. ** p < 0.01 vs. Control (Con); *** p < 0.001 vs. Con; **** p < 0.0001 (n = 3 experiments). Data are mean ± SD.