IGF-1 Signaling Regulates Mitochondrial Remodeling during Myogenic Differentiation

Abstract

Skeletal muscle is essential for locomotion, metabolism, and protein homeostasis in the body. Mitochondria have been considered as a key target to regulate metabolic switch during myo-genesis. The insulin-like growth factor 1 (IGF-1) signaling through the AKT/mammalian target of rapamycin (mTOR) pathway has a well-documented role in promoting muscle growth and regeneration, but whether it is involved in mitochondrial behavior and function remains un-examined. In this study, we investigated the effect of IGF-1 signaling on mitochondrial remodeling during myogenic differentiation. The results demonstrated that IGF-1 signaling stimulated mitochondrial biogenesis by increasing mitochondrial DNA copy number and expression of genes such as Cox7a1, Tfb1m, and Ppargc1a. Moreover, the level of mitophagy in differentiating myoblasts elevated significantly with IGF-1 treatment, which contributed to mitochondrial turnover. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) were identified as two key mediators of IGF-1-induced mitochondrial biogenesis and mitophagy, respectively. In addition, IGF-1 supplementation could alleviate impaired myoblast differentiation caused by mitophagy deficiency, as evidenced by increased fusion index and myosin heavy chain expression. These findings provide new insights into the role of IGF-1 signaling and suggest that IGF-1 signaling can serve as a target for the research and development of drugs and nutrients that support muscle growth and regeneration.

Keywords: IGF-1; energy metabolism; mitochondrial biogenesis; mitochondrial remodeling; mitophagy; muscle regeneration; myogenic differentiation.

Figure 1

IGF-1 signaling regulates myoblast differentiation. C2C12 myoblasts or satellite cells were cultured in the DM supplemented with 0.1% DMSO, 10 ng/mL IGF-1, or 200 nM BMS for 5 days. (A) Representative immunoblots of IGF-1R, pIGF-1R, mTOR, pmTOR, AKT, pAKT, or GAPDH in C2C12 cells treated with 0.1% DMSO (NC), 10 ng/mL IGF-1, or 200 nM BMS. (B) Representative images of myotube formation and quantitative analysis of fusion index in C2C12 cells. Cells were stained with DAPI (blue) and myosin heavy chain (MHC, green) to visualize nuclei and myosin, respectively. Scale bar = 100 μm. (C) Representative immunoblots and quantitative analysis of MyoG and α-tubulin in C2C12 myotubes. (D) Representative images of myotube formation and quantitative analysis of fusion index in satellite cells. (E) Representative immunoblots and quantitative analysis of MyoG and α-tubulin in satellite cells. Data represent means ± SD. * p < 0.05 and ** p < 0.01. Abbreviations: IGF-1, Insulin-like growth factor 1; BMS, BMS754807; NC, vehicle control; mTOR, mammalian target of rapamycin; AKT, protein kinase B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MyoG, myogenin.

Figure 2

Inhibition of IGF-1 signaling leads to mitochondrial dysfunction and damage. (A) Representative flow charts and quantitative analysis of mitochondrial ROS levels in DMSO, IGF-1, and BMS-C2C12 that were stained with MitoSoX cells during differentiation. (B) Quantitative analysis of apoptotic (Annexin V+) cells during differentiation. (C) Quantitative analysis of the JC-1 red: green fluorescence ratio with FACS during differentiation. (D) Electron micrographs of differentiating C2C12 cells. Scale bar = 1 μm. Data represent means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. Abbreviations: ROS, reactive oxygen species; JC-1, 5,5′,6,6′-tetrachloro-1,1′-3,3′-tetraethyl-benzimidazolylcarbocyanine iodide.

Figure 3

IGF-1 signaling regulates mitochondrial biogenesis during myogenic differentiation. (A) Mitochondrial DNA (mtDNA) content in C2C12 myotubes after 3 days of differentiation. (B–E) mRNA expression of Cox7a1 (B), Tfb1m (C), Tfam (D), and Ppargc1a (D) genes in C2C12 cells treated with DMSO (NC), 10 ng/mL IGF-1, or 200 nM BMS after 3 days of differentiation. (F) The Mito Tracker Green (MTG) staining of C2C12 cells after 3 days of differentiation. (G) The mitochondrial mass evaluation by MTG staining and quantified by FACS. (H) Representative immunoblots and quantitative analysis of PGC-1α and GAPDH in C2C12 cells after 3 days of differentiation. (I) Immunofluorescent staining of MHC (green) and DAPI (blue) and quantitative analysis of the fusion index for siNeg and siPGC1α C2C12 cells with or without IGF-1 treatment after 5 days of differentiation. Scale bar = 100 μm. Data represent means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

IGF-1 signaling regulates mitophagy during myogenic differentiation. (A) Representative immunoblots and quantitative analysis of SQSTM1/p62 (p62), ATG5, and GAPDH in differentiating cells. (B) Representative immunoblots and quantitative analysis of LC3B and GAPDH in differentiating cells with or without chloroquine (CQ) treatment. In the experiments for monitoring the autophagic flux, cells were treated with 50 μM CQ for 4 h as CQ treatment leaded to LC3B-II aggregation. The autophagic flux was analyzed through the ratio of LC3B-II expression in CQ-treated and untreated cells. (C) Representative fluorescent images and quantitative analysis of colocalization (yellow) in ad-GFP-LC3 (green) transfected cells and stained with anti-TOMM20 (red) at day 1 of differentiation. Scale bar = 10 μm. (D) mRNA expression of mitophagy receptors in differentiating C2C12 myoblasts with or without treatment of IGF-1. (E) Representative immunoblots and quantitative analysis of BNIP3 and GAPDH in C2C12 cells after 3 days of differentiation. Data represent means ± SD. * p < 0.05 and ** p < 0.01.

Figure 4

IGF-1 signaling regulates mitophagy during myogenic differentiation. (A) Representative immunoblots and quantitative analysis of SQSTM1/p62 (p62), ATG5, and GAPDH in differentiating cells. (B) Representative immunoblots and quantitative analysis of LC3B and GAPDH in differentiating cells with or without chloroquine (CQ) treatment. In the experiments for monitoring the autophagic flux, cells were treated with 50 μM CQ for 4 h as CQ treatment leaded to LC3B-II aggregation. The autophagic flux was analyzed through the ratio of LC3B-II expression in CQ-treated and untreated cells. (C) Representative fluorescent images and quantitative analysis of colocalization (yellow) in ad-GFP-LC3 (green) transfected cells and stained with anti-TOMM20 (red) at day 1 of differentiation. Scale bar = 10 μm. (D) mRNA expression of mitophagy receptors in differentiating C2C12 myoblasts with or without treatment of IGF-1. (E) Representative immunoblots and quantitative analysis of BNIP3 and GAPDH in C2C12 cells after 3 days of differentiation. Data represent means ± SD. * p < 0.05 and ** p < 0.01.

Figure 5

IGF-1 induces PGC-1α and BNIP3 accumulation dose-dependently. (A–D) Representative immunoblots and quantitative analysis of PGC-1α (B), BNIP3 (C), MHC (D), and α-tubulin in C2C12 cells after 5 days of differentiation. (E) Immunofluorescent staining of MHC (green) and DAPI (blue) of the fusion index in C2C12 cells treated with 0~50 ng/mL of IGF-1 after 5 days of differentiation. (F) Quantitative analysis of the fusion index. Scale bar = 100 μm. Data represent means ± SD. * p < 0.05 and ** p < 0.01.

Figure 6

IGF-1 increases the differentiation potential in mitophagy- and autophagy-deficient myoblasts. (A–C) Representative immunoblots and quantitative analysis of BNIP3, MyoG, and α-tubulin in C2C12 cells after 5 days of differentiation. (D) Immunofluorescent staining of MHC (green) and DAPI (blue) and quantitative analysis of the fusion index for siNeg and siBNIP3 C2C12 cells with or without IGF-1 treatment after 5 days of differentiation. (E) Representative immunoblots analysis of MHC, p62, BNIP3, and GAPDH in C2C12 cells after 5 days of differentiation in the presence of 3-MA and/or IGF-1. (F) Immunofluorescent staining of MHC (green) and DAPI (blue) of differentiating C2C12 cells with treatment of 3-MA and/or IGF-1. (G) Quantitative analysis of the fusion index. Scale bar = 100 μm. Data represent means ± SD. * p < 0.05 and ** p < 0.01.