Skeletal muscle differentiation is a multistep process with two major steps. The first is the induction of muscle-specific gene expression by myogenic regulatory factors (MYFs), such as MYF5, MyoD/MYF3, MYF6, and myogenin (MYOG/MYF4). The second step is the commitment of myogenic cells to the skeletal muscle via irreversible withdrawal from the cell cycle, leading to a permanent G1 phase (McKinsey et al., 2001; Rescan, 2001); this cell cycle arrest requires the retinoblastoma tumor suppressor protein, Rb. During early differentiation, Rb inhibits DNA synthesis by binding to E2F, resulting in the repression of cyclin A/cyclin-dependent kinase (CDK)-2, cyclin D/CDK4 and 6, and cyclin E/CDK2 expression levels, which are regulated by CDK inhibitors (Skapek et al., 1996; Guo and Walsh, 1997). Tumor suppressor genes, p53 and p21, are also believed to induce cell cycle arrest (Valente et al., 2013). p53 regulates Rb differentiation without affecting its cell cycle-related functions. However, p53-defective skeletal myoblasts do not show upregulated Rb expression, resulting in reduced myoblast differentiation (Porrello et al., 2000). Therefore, skeletal muscle differentiation requires the global reprogramming of gene expression and withdrawal from the cell cycle.

There are three major dietary n-3 polyunsaturated fatty acids (PUFAs): α-linolenic acid (ALA) obtained from linseed oil, and eicosatetraenoic acid (EPA) and docosahexaenoic acid (DHA) obtained from fish oil. PUFAs play important roles in the regulation of prostaglandin synthesis, membrane properties, and activation of transcription factors (Wathes et al., 2007). PUFAs have beneficial effects for the treatment of a wide variety of maladies, including breast, prostate, and nervous system diseases (Hussain et al., 2013; Wang et al., 2015). In mammalian cells, dietary ALA can be converted into important fatty acids, EPA and DHA, via alternating steps of elongation and desaturation (Roqueta-Rivera et al., 2011). Consequently, ALA is essential for numerous processes, including general growth and development of the brain and reproductive system as well as the improvement of vision.

Exercise and ingestion of essential amino acids (EAAs), particularly leucine, stimulate muscle protein synthesis via activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway (Walker et al., 2011). While anabolic resistance to the independent effects of exercise and EAAs or proteins is prevalent with aging (Dickinson et al., 2013), the combination of these two stimuli shows high potential in combating sarcopenia (Walker et al., 2011; Dickinson et al., 2013) due to the stimulation of mTORC1 and upregulation of the translation initiation machinery expression. Indeed, we have recently demonstrated that the intake of a leucine-rich EAA mixture following a bout of high-intensity resistance exercise (RE) stimulates mTORC1 expression and prolongs myofibrillar protein synthesis for up to 24 h post-RE in old men (Dickinson et al., 2014), whereas the absence of EAA inhibits the mTORC1 response in old adults (Fry et al., 2011).

Currently, little is known about the effects of ALA and EAA deficiencies in skeletal muscle cells. The aim of the current study was to examine the effects of ALA and EAA on skeletal muscle proliferation and differentiation by assessing their cell viability, total antioxidant capacity, and apoptosis-suppressing gene expression.

Cell culture

Mouse myoblast C2C12 cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 mg/mL streptomycin in 5% carbon dioxide (CO₂) at 37℃. The growth medium was changed every two days.

C2C12 myoblast proliferation

C2C12 cells were seeded at a density of 50000 cells/well in a 6-well plate. After 24 h, the cells were cultured in DMEM without amino acids containing 20% FBS and 0.5% EAAs (GM-Control), 20% FBS and 2% EAAs (GM-EAA), or 20% FBS and ALA (5, 10, or 25 mM) (GM-ALA). After two days, the cells were collected for further analysis.

C2C12 myoblast differentiation

For myogenic differentiation, C2C12 myoblasts at 80-90% confluence was transferred to DMEM without amino acids containing 2% FBS and 0.5% EAAs (DM-Control), 2% FBS and 2% EAAs (DM-EAA), or 2% FBS and ALA (5, 10, or 25 mM) (DM-ALA). The medium was replaced with fresh medium every two days. After 6 d of differentiation induction, the cells were collected for further analysis.

Cell proliferation assay

The effects of EAAs and ALA on cell proliferation were assessed using a cell counting kit (CCK)-8 assay (Cho and Kim, 2021). Briefly, the cells were dispersed into a 96-well plate at a density of 5000 cells/well. After 24 h of incubation, the cells were treated with different groups (GM-control/GM-EAA/GM-ALA). The CCK-8 solution (10 μL) was then pipetted into each well. The cells were incubated for 2 h at 37℃ in a 5% CO₂ atmosphere. The optical density at 450 nm (OD₄₅₀) was measured using a microplate reader.

Immunofluorescence analysis

Briefly, C2C12 cells were cultivated in a 96-well plate and differentiation was induced as previously reported. For immunofluorescence microscopy, the cells were fixed in 4% paraformaldehyde for 30 min and treated with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 30 min at room temperature. After blocking in 5% normal goat serum for 1 h, the cells were incubated with the anti-proliferating cell nuclear antigen (anti-PCNA), anti-PPARγ coactivator 1α (anti-PGC1α), anti-sirtuin 1 (anti-Sirt1), phosphorylated H2A.X variant histone (p-H2AX), and anti-MYOG at 4℃ for 24 h. Then, the cells were incubated with secondary antibodies (Bioss) at 37℃ for 1 h. Nuclei were stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Beyotime) for 10 min. Finally, the cells were viewed under a fluorescence microscope (Olympus, Kyoto, Japan) and three replicates of each sample were analyzed to determine the fusion index using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

TUNEL assay

The intracellular apoptosis level of cells was measured in the TUNEL assay using the In Situ Cell Death Detection Kit (11684795910; Roche, Basel, Switzerland). After washing three times with PBS, cells were fixed in 3.7% paraformaldehyde for 30 min at room temperature and subsequently permeabilized by incubation in 0.5% Triton X-100 for 30 min at room temperature. The cells were incubated with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase enzyme for 2 h and then washed three times with PBS. Cells were treated with 10 mg/mL Hoechst 33342 for 5 min, washed three times with PBS. Images were captured using a confocal microscope (LSM 710 Meta; Zeiss, Oberkochen, Germany). The apoptosis index was calculated as the percentage of TUNEL-positive nuclei.

Western blotting analysis

According to previous article (Lee et al., 2019), the cells were rinsed with PBS and lysed in a buffer containing 50 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan), and phosphatase inhibitor cocktail (Nacalai Tesque, Inc.). Equal amounts of protein (40 mg) were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). A blocking step was performed for 1 h at room temperature using 5% skimmed milk. The following primary antibodies diluted in 5% skim milk were used for immunoblotting: anti-PCNA (1:1,000 dilution), anti-LC-3 (1:1,000 dilution), anti-MYOG (1:1,000 dilution), anti-α tubulin (1:1000), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (1:1,000 dilution) antibodies. Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000 dilution, ab205718; Abcam, Cambridge, UK) was used as the secondary antibody. After incubation with the secondary antibody, the membrane was incubated with Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc.) to visualize the bands, and ImageJ software was used to quantify the band intensities.

Real-time polymerase chain reaction (PCR)

Total RNA was extracted using the TRIzol reagent, according to the manufacturer’s protocol (Park et al., 2014). The integrity of the RNA was checked via electrophoresis and the concentration was assessed using the OD absorption ratio, OD₂₆₀/OD₂₈₀. Total RNA (1 mg) was reverse-transcribed, and real-time PCR was performed to determine the related gene expression levels. Quantitative real-time PCR was performed using the SYBR Premix Ex Taq kit (TaKaRa) and CFX96 Real-Time PCR detection system (Bio-Rad, Richmond, CA, USA). The specificity of the PCR products was confirmed using a melting curve analysis. The PCR conditions were as follows: initial denaturation at 95℃ for 5 min, then 35 cycles of denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s. Relative expression levels of mRNA were calculated using the 2^-ΔΔCt method. The results were normalized to the expression of GAPDH. All primer sequences are shown in Table 1.

Table 1. Primer sequences for RT-PCR (F: forward, R: reverse).

Gene	Forward primer	Reverse primer	TM/℃
CAT	5’-GCAGATACCTGTGAACTGTC-3’	5’-GTAGAATGTCCGCACCTGAG-3’	56
SOD	5’-AAAGCGGTGTGCGTGCTGAA-3’	5’-CAGGTCTCCAACATGCCTCT-3’	60
BAX	5’-TGAAGACAGGGGCCTTTTTG-3’	5’-AATTCGCCGGAGACACTCG-3'	58
BCL2	5’-ATGCCTTTGTGGAACTATATGGC-3’	5’-GGTATGCACCCAGAGTGATGC-3'	58
MYF5	5’-ATGCCATCCGCTACATTGAGAG-3’	5’-CCGTCAGAGCAGTTGGAGGT-3'	58
MRF4	5’-CCTCAGCCTCCAGCAGTCTT-3’	5’-TACTTCTCCACCACCTCCTCCA-3'	58
p53	5’-GAACCGCCGACCTATCCTTA-3’	5’-GGCAGGCACAAACACGAAC-3'	58
Beclin-1	5’-ATGGAGGGGTCTAAGGCGTC-3’	5’-TCCTCTCCTGAGTTAGCCTCT-3	58
PCNA	5’-GAT GCC GTC GGG TGA ATT TG-3’	5’-CCA TTG CCA AGC TCT CCA CT-3'	58
MYOG	5’-AGC CAC ACT GAG GGA GAA G-3’	5’-GTT GAG GGA GCT GAG CAA G-3'	58
Ki-67	5’-GAG CTA ACT TGC GCT GAC T-3’	5’-TGT CAC ATT CAA TAC TCC TTC CA-3'	56
GAPDH	5’-TGA AGG TCG GAG TGA ACG GAT TT-3’	5’-CCA TTT GAT GTT GGC GGG AT-3'	58

Statistical analyses

All values are expressed as the mean ± standard error of the mean. Differences were tested using analysis of variance. Results were compared using the student’s paired t-test. Statistical significance was set at p < 0.05. Significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

Proliferation of C2C12 myoblasts is affected by ALA and EAAs

First, safe concentrations of ALA and EAAs were determined as they may exert cytotoxic effects on C2C12 cells. Therefore, C2C12 cells were incubated with either synthetic ALA or EAAs at concentrations ranging from 5-25 mM of ALA and 0.5-2% EAAs (Fig. 1A). CCK-8 assay revealed that treatment with 10 mM of ALA increased the C2C12 cell viability compared to the controls (Fig. 1B and 1C). However, ALA concentrations greater than 50 mM were toxic (data not shown). EAA treatment showed a similar trend and 2% of EAA caused a significant increase in C2C12 cell viability (Fig. 1B and 1C). Based on these data, concentrations of 10 mM of ALA and 2% EAAs were used in further experiments.

Figure 1.Effects of α-linolenic acid (ALA) and essential amino acids (EAAs) on C2C12 cell proliferation. The control growth medium (GM) contained 0.5% EAA. (A) Schematic diagram of the proliferation experiment. (B) Images of morphological changes were obtained using phase contrast photomicrography after 48 h of treatment. Scale bars represent 100 μm. (C) Cell viability of C2C12 cells was measured using the cell counting kit (CCK)-8 assay. Cells were treated with ALA (0, 5, 10, and 25 μM) or EAA (1 and 2%) for 48 h. (D, E) Expression levels of proliferating cell nuclear antigen (PCNA) were determined using western blotting and immunofluorescence assays. Scale bars represent 20 μm. (F) mRNA expression levels of PCNA and Ki-67 were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). *p < 0.05, **p < 0.01, ***p < 0.005 vs. the control group.

ALA and EAAs affect PCNA and Ki-67 proliferative marker

The effects of ALA and EAA on the proliferation of C2C12 cells were examined by determining PCNA expression. Western blotting and immunofluorescence assay results showed that 10 mM of ALA and 1-2% EAAs caused ~2-fold increase in PCNA expression levels compared to the controls (Fig. 1D and 1E). Moreover, real-time qPCR results showed that 10 mM of ALA significantly increased PCNA and Ki-67 expression levels (***p < 0.001 and *p < 0.05, respectively) compared to the controls (Fig. 1F). Similarly, treatment of C2C12 cells with 2% EAAs resulted in a significant in the mRNA expression levels of PCNA and Ki-67 (**p < 0.01 and *p < 0.05, respectively) compared to the controls (Fig. 1F).

ALA and EAAs regulate the antioxidant systems in C2C12 cells

Following the treatment of C2C12 cells with ALA and EAA for 48 h, we determined the expression levels of PGC1α and Sirt1 using immunofluorescence assay. As shown in Fig. 2A and 2B, PGC1α expression levels were significantly increased after treatment with 5-25 mM of ALA and 1-2% EAA. However, there was no difference in the expression levels of Sirt1 compared to the controls (Fig. 2C).

Figure 2.Effects of ALA and EAAs on antioxidative factors. (A) Micrographs show the PPARγ coactivator (PGC)-1α (green) and Hoechst 33258 nuclear fluorochrome (blue). (B) Bar graph shows mean fluorescence intensity (MFI) analysis (MFI ± standard error of the mean (SEM) from n = 250-300; **** p < 0.001 vs. control). (C) Micrographs show sirtuin 1 (SIRT1) (green) and Hoechst 33258 nuclear fluorochrome (blue). (D) mRNA expression levels of catalase (CAT) and superoxide dismutase (SOD) were determined using RT-qPCR. Scale bars represent 20 μm. *p < 0.05, ***p < 0.005 vs. the control group.

SOD-2 is an effective regulator of cellular reactive oxygen species, and PGC-1α is an important regulator of SOD-2 (Qu et al., 2009; Leick et al., 2010). Thus, we explored the effects of PGC-1α on the expression levels of SOD-2 and other key components of antioxidant systems, such as CAT, in C2C12 cells. As shown in Fig. 2D, in the cells treated with 10 mM of ALA or 2% EAA, the levels of SOD-2 and CAT were significantly increased when compared to the controls. These results suggest that ALA and EAAs may regulate the antioxidant systems in C2C12 cells.

ALA and EAAs prevent DNA damage in C2C12 cells

To investigate the effects of ALA and EAAs on DNA damage, we performed immunofluorescence staining to detect DNA damage that can be marked by the presence of p-H2AX. As shown in Fig. 3, the percentage of cells with p-H2AX was significantly reduced in 10 mM of ALA and 1-2% EAA supplement group compared with the control (0.5% EAA). These results indicate that low EAA-induced genotoxic stress can be prevented by ALA and EAA supplementation.

Figure 3.Effects of ALA and EAAs on the DNA damage of C2C12 cells during proliferation. (A) Immunostaining images of the DNA damage marker, phosphorylated H2A.X variant histone (p-H2AX) (green), and the nuclear marker, Hoechst 33258 (blue), in C2C12 cells. All experiments were performed thrice and the representative result is shown. Scale bars represent 20 μm. (B) Quantification of colocalization of p-H2AX with Hoechst 33258 from random fields. Bar graph represents the mean ± SEM values from three independent experiments.

ALA and EAAs reduce apoptosis and autophagy in C2C12 cells

After 48 h of treatment, the cells were subjected to the terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) assay to determine apoptosis. Results showed that the number of positive TUNEL cells was significantly reduced after treatment with 10 mM of ALA and 1-2% EAA compared to the controls (Fig. 4A and 4B). Moreover, real-time PCR results showed that 10 mM of ALA significantly decreased Bax and p53 expression levels (*p < 0.05 and **p < 0.01, respectively), while significantly increasing Bcl-2 expression levels (*p < 0.05) compared to the controls (Fig. 4C). Similarly, treatment of C2C12 cells with 2% of EAA resulted in a significant decrease in the mRNA expression levels of Bax and p53 (**p < 0.01 and ***p < 0.005, respectively) and a significant increase in Bcl-2 expression levels (*p < 0.05) compared to the controls (Fig. 4C).

Figure 4.Effects of ALA and EAAs on the apoptosis and autophagy of C2C12 cells. (A, B) Terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) assay and apoptosis index after treatment for 48 h. Scale bars represent 20 μm. (C) mRNA expression levels of B-cell lymphoma (Bcl)-2, Bcl-2-associated X (Bax), and p53 were determined using RT-qPCR. (D) mRNA expression levels of beclin-1 were determined using RT-qPCR. (E) Representative bands of light chain 3 (LC-3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained via western blotting.

In addition, autophagy factors were determined using real-time PCR and western blotting. As shown in Fig. 4D, treatment with 10 mM of ALA and 2% EAA significantly increased beclin-1 expression levels, while decreasing LC-3 expression levels (*p < 0.05) compared to the controls. These data suggest that ALA and EAAs inhibit apoptosis and autophagy in C2C12 cells during proliferation.

ALA and EAAs improve the differentiation capacities of C2C12 cells

Cells were cultured in GM for two days after seeding, followed by supplementation with different concentrations of ALA or EAAs for up to 6 d to evaluate the differentiation C2C12 cells (Fig. 5A). The results showed that 10 mM of ALA caused increased differentiation of C2C12 cells, compared to the controls, as revealed by their cell lengths and cell widths (**p < 0.01 and ***p < 0.005, respectively) (Fig. 5B to 5D). EAA treatment showed a similar trend and 2% of EAA caused a significant increase in the differentiation of C2C12 cells (***p < 0.05 and ***p < 0.005, respectively) (Fig. 5B to 5D). Based on these data, concentrations of 10 mM of ALA and 2% EAA were used in further experiments.

Figure 5.Effects of ALA and EAAs on the differentiation of C2C12 cells. (A) Schematic diagram of the differentiation experiment. (B) Images of morphological changes were obtained using phase contrast photomicrography after 6 d of treatment. Scale bars represent 100 μm. (C, D) Cell lengths and widths of C2C12 cells.

ALA and EAAs regulate the differentiation marker in C2C12 cells

The effects of ALA and EAA supplementation on the differentiation marker, MYOG, and muscle generation markers, MYF4 and MYF5, were determined using real-time PCR (Fig. 6A). The results showed a significant increase in MYOG, MYF4 and MYF5 expression levels in 10 mM of ALA or 2% EAA supplemented groups compared with those in the control (0.5% EAA). In addition, the protein levels of MYOG were determined using western blotting and immunofluorescence assay, which showed that ALA and EAAs caused a significant increase in MYOG levels compared to the levels observed in the controls (Fig. 6B and 6C).

Figure 6.Regulation of ALA and EAAs on the differentiation markers. (A) mRNA expression levels of myogenin (MYOG/MYF4) and MYF5 were determined using RT-qPCR. (B, C) Expression levels of PCNA were determined using western blotting and immunofluorescence assays. Scale bars represent 20 μm.

While there are reports of ALA inhibiting ER+MCF-7 cell growth in a dose dependent manner (Roy et al., 2017), the present study focused on determining the safe concentration of ALA to improve C2C12 cell proliferation under low supplementation of EAA (0.5%) in vitro. Our results showed that ALA decreased the numbers of TUNEL-positive and DNA-damaged cells induced by EAA deficiency. Additionally, C2C12 cells under EAA-deficient conditions showed an increase in the levels of the DNA damage response marker, p-H2AX, which was rescued by ALA and EAA supplementation during proliferation (Fig. 3). Our data also suggest that EAA re-supplementation reversed the inhibition of proliferation and differentiation caused by EAA deficiency in C2C12 cells.

In the present study, ALA and EAA supplementation caused an increase in the expression levels of PGC1α, which prevented cell death under EAA-deficient conditions in C2C12 cells. Following the knockdown of PGC1α, the ratio of anti-apoptotic protein (Bcl-2) to pro-apoptotic protein (Bax) in SKOV3/DDP cells is significantly reduced (Shen et al., 2018). Several investigators (Daval et al., 2004) have proposed that insult-induced changes in Bax and Bcl-2 genes lead to an imbalance between the activators and suppressants of cell death. The optimal balance of these molecules in individual neurons acts as a deciding factor in determining the cell fate (Xia et al., 1995). Our data showed that ALA induced modulatory effects on apoptosis-associated proteins (Bax and Bcl-2) under EAA-deficient conditions by upregulating Bcl-2 expression and downregulating Bax expression, suggesting the positive influence of ALA on C2C12 cell proliferation.

The observations of the present study reveal the potential roles of ALA in the upregulation of PGC1α expression levels and BCl-2/Bax ratio as well as the cell quality status under EAA-deficient conditions, thus maintaining the proliferation and differentiation of C2C12 cells (Fig. 7). ALA-induced restoration of the optimal Bax: Bcl-2 ratio (1:2) may be the key factor in promoting cell survival, which strongly supports the views of previous investigators (Xia et al., 1995) pertaining to the importance of maintaining an optimal balance of Bcl-2 family proteins in ensuring cell survival.

Figure 7.Scheme of the apoptosis pathway modulated by ALA and EAAs in C2C12 cells. Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma-2; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha.

In conclusion, our findings indicate that EAA deficiency inhibits the proliferation and differentiation of C2C12 cells, which can be restored by supplementation with a moderate dose of ALA. Moreover, our results demonstrate the importance of ALA and EAAs in skeletal muscle cell development.

None.

X.S.C., D.Z. designed the experiment. D.Z., X.H.L. conducted the experiments, analyzed the results, and wrote the article. S.H.L. and G.H. helped with the analyses of the results and figures. X.S.C. revised the manuscript.

This result was supported by “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021RIS-001).

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No potential conflict of interest relevant to this article was reported.