Skeletal muscle accounts for about 40~45% of body mass in healthy young adults, and with age, muscle tissue is gradually lost, resulting in reduced muscle size and strength. Skeletal muscle loss in aging and secondary age-related pathological conditions has been shown to exacerbate metabolic syndromes, ultimately leading to reduced quality of life (Sanchez et al., 2013). Since myoblast differentiation is essential for the formation of muscle fibers involved in the development and regeneration of skeletal muscle, it is an important factor determining the improvement of muscle mass and strength.

Myoblast differentiation is an organized process that involves the expression of muscle-specific genes and cell fusion to form multinucleated myotubes governed by muscle-specific transcription factors such as MyoD and myogenin (Horsley and Pavlath, 2004). The myosin heavy chain (MyHC), the main structural protein of myotubes, is the most abundant muscle protein, accounting for about 35% of the protein pool, and is a major muscle-specific differentiation marker (Lluís et al., 2006). MyoD and Myf5 are essential for myoblast constitution and act early in myogenesis (Rudnicki et al., 1993). Furthermore, myogenin acts an important role in controlling the fusion of myoblasts in the late stages of myogenesis (Perry and Rudnick, 2000).

As muscle mass is regulated by the balance between muscle cell replication, protein synthesis and protein degradation, enhanced muscle mass by increasing muscle differentiation and inhibiting muscle loss can prevent muscle atrophy. Typically, two muscle-specific E3 ubiquitin ligases, muscle specific RING-finger 1 (MuRF1) and atrogin-1 (MAFbx), are markedly induced in almost all types of muscle atrophy to degrade muscle proteins (Cohen et al., 2015; Bodine et al., 2001). Therefore, inhibiting MuRF1 and atrogin1/MAFbx, which are induced by various factors that cause muscle loss, is important for prevent muscle atrophy.

Ziziphus jujuba Mill. (ZJM) is known as a functional food rich in flavonoids, vitamins C, B1, and B2. Based on physiological effects, it has been shown to lower blood pressure, treat liver disease and anemia, and inhibit the growth of tumor cells (Zhang et al., 2020; Gao et al., 2013). Additionally, several studies have shown that it has strong antioxidant effects with great benefits in preventing of diseases such as cancer, cell aging, and cardiovascular diseases (Dai and Mumper, 2010). However, the biological effects of ZJM on muscle health such as myogenic differentiation and muscle atrophy remain unknown. Therefore, we investigated the role and realted mechanism of ZJM on myoblast differentiation.

Chemicals, reagents and antibodies

The methyl alcohol extract of Ziziphus jujuba Mill. (ZJM) fruit part obtained from KPEB (Korea Plant Extract Bank, Cheongju, Korea). A stock solution (5 mg/mL) was prepared in dimethyl sulfoxide (DMSO) and stored at -20℃ before use. DMSO final concentration was added to the cell culture medium to a maximum of 0.1% (v/v). TRIzol was purchased from Life Technologies (Carlsbad, CA, USA). A monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against MyHC1, MyoD, myogenin, atrogin-1 and MuRF-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-mouse immunoglobulin secondary antibody was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Enhanced Chemiluminescence Detection System was purchased from Merck Millipore (Burlington, MA, USA).

Cell culture and myogenic differentiation

C2C12 cells were maintained in 5% CO₂ at 37℃, cultured in growth medium (GM) consisting of Dulbeccós modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (GIBCO, Grand Island, New York, USA) for 3 days until they reached 90~100% confluence. Then, GM was interchanged by differentiation medium (DM) including 2% horse serum (GIBCO, Grand Island, New York, USA) to induce the myogenic differentiation of C2C12 cells. DM was replenished every 48 h, and C2C12 cells were fixed or lysed at different time points during the differentiation process.

Cytotoxicity assay

Cell viability was evaluated by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay. C2C12 cells (1×10³ cells) seeded in 96-well plates and cultured in DMEM supplemented with 10% FBS and 1% penicillin streptomycin, and the cells were treated with or without ZJM (0, 0.5, 1 and 5 ng/mL) for 2 days until they reached 90~100% confluence. After that, the cell viability was evaluated by treated 50 μL of XTT reagent to each one, and then incubation for 4 h. the absorbance was assessed at 450 nm using a multi-detection microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Giemsa staining

C2C12 cells were washed with PBS and fixed with 100% methanol for 20 min and dried. The Giemsa staining solutions (Sigma, St. Louis, MO, USA) were diluted with distilled water and cells were incubated with diluted staining solution for 30 min and washed with distilled water. The images of myotubes were captured using an inverted microscope (Nikon, Tokyo, Japan).

Immunofluorescence staining

C2C12 cells were washed with PBS and fixed with 3.7% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% bovine serum albumin in PBS for 3 h. Myotubes were incubated with MyHC antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4℃. After that, myotubes were stained with Alexa Fluor 488 (Invitrogen, Waltham, MA, USA) for 2 h. The nucleus were stained with DAPI. The short-axis diameters of myotubes were analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA).

Real-time quantitative RT-PCR

RNA was extracted by Trizol Reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's manual. Complementary DNA was made from 1 μg of total RNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). The Real-time quantitative RT-PCR reaction was performed with Accupower Green Star qRT-PCR master mix using an Excycler^TM 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea). The following sequences of mouse primers were used for real-time RT-PCR: GAPDH Forward: 5'-TCAAGAAGGTGGTGAAGCAG-3'; reverse: 5'-AGTGGGAGTTGCTGTTGAAGT-3'; MyHC1 forward: 5'-GCCCAGTGGAGGACAAAATA-3'; reverse: 5'-TCTACGTGCTCCTCAGCAT-3'; MyoD forward: 5'-CGCTCCAACTGCTCTGATG-3'; reverse: 5'-TAGTAGGCGGTGTCGTAGCC-3'; myogenin forward: 5'-CTACAGGCCTTGCTCAGCTC-3'; reverse: 5'-AGATTGTGGGCGTCTGTAGG-3'. The amplification parameters were as follows: initial denaturation at 95℃ for 5 min, followed by 40 cycles of 3-step PCR: denaturation at 95℃ for 1 min, annealing at 60℃ for 30 sec, and a final extension at 72℃ for 1 min. The expression levels of mRNA were calculated as fold changes by evaluating real-time quantitative RT-PCR data using the 2^-ΔΔCt method. The data were normalized to the mRNA levels of GAPDH.

Western blotting

For western blotting, cells were lysed using RIPA buffer. Then, centrifugation (13,500 rpm) at 4℃ for 15 min, the supernatant was collected, and the protein concentration was quantified. Equal amount of protein sample (20~30 μg) were boiling at 105℃ for 5 min in sample buffer. The proteins were loaded on 8~10% SDS-PAGE gels and electrophoretically transferred onto polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA) by using electrophoresis. The membranes were blocked with 5% nonfat dry milk for 1 h and incubated with primary antibodies for 4℃ overnight. Then, membranes were incubated for 1 h with secondary antibodies. The specific signals on blots were discovered using Enhanced Chemiluminescence Detection System (Merck Millipore, Burlington, MA) and were visualized under Supernova-Q1800 ChemiDoc system (ENBIO Lab, Daejeon, Korea).

Statistical Analysis

All experiments were conducted at least three times, and data are presented as the mean ± standard deviation (SD). Statistical differences were confirmed using one-way or repeated-measures ANOVA followed by Tukey's HSD test. Statistical significance was set at P < 0.05.

ZJM promotes myogenic differentiation without cytotoxicity

To determine whether the ZJM used in the experiment induces cytotoxicity during myoblast differentiation, we first tested cell viability according to the concentration of ZJM. C2C12 cells were cultured in GM with various concentrations of ZJM (0, 0.5, 1 and 5 ng/mL) for 3 days. There was no effect on cytotoxicity at any concentration of ZJM used in this study (Fig. 1A). Subsequently, the effect of ZJM on morphological changes and myotube formation was performed by Giemsa staining (Fig. 1B) and MyHC immunofluorescence staining (Fig. 1C). After 5 days of differentiation, the cells lose their myoblast morphology and the cell shape gradually changed into a new elongated and tubular shape (Fig. 1B). As shown in Fig. 1C and D, ZJM treatment resulted in a higher percentage of larger MyHC-expressing cells containing more than three nuclei compared to the non-treated control. These data suggest that ZJM enhanced the myogenic differentiation of C2C12 cells.

Fig. 1. ZJM promotes myogenic differentiation without cytotoxicity.
(A) C2C12 cells were cultured for 3 days at the indicated concentration of ZJM. Cell viability was analyzed by XTT assay. (B) Giemsa staining was performed on the 5 day of C2C12 cells differentiation to measure changes in myotube morphology, such as myotube length and diameter. (C) Immunofluorescence staining of MyHC (green color), and the nuclei marker, DAPI (blue color), showed myotube formation in the C2C12 cells. (D) Relative myotube diameters were measured. **P < 0.01 vs. DMSO control.

ZJM upregulates myogenic differentiation markers in C2C12 muscle cells

Since it was confirmed that myotube formation was morphologically stimulated by ZJM, we tested the effects of ZJM on the expression of myogenic differentiation markers. The effects of various concentrations ZJM (0, 0.5, 1 and 5 ng /mL) on muscle differentiation markers, including MyHC1, MyoD and myogenin were evaluated by real-time quantitative RT-PCR (Fig. 2A) and western blotting (Fig. 2B). Real-time quantitative RT-PCR analysis indicated that ZJM treatment significantly increased the expression of MyHC1, MyoD and myogenin in a concentration-dependent manner (Fig. 2A). Consistent with these results, western blot and quantitative analysis confirmed that ZJM clearly induced the expression of MyHC1, MyoD and myogenin (Fig. 2B and C). Next, we identified the time at which ZJM had the greatest effect the expression of myogenic markers. As shown in Figs. 3, the mRNA (Fig. 3A) and protein levels (Fig. 3B and C) of MyHC1, MyoD and myogenin peaked at day 7 compared to control. Consequently, the ZJM considerably enhance in the expressions of MyHC1, MyoD and myogenin on day 7 of differentiation.

Fig. 2. ZJM upregulates myogenic differentiation markers in C2C12 muscle cells.
C2C12 myoblasts, cultured with ZJM (0.5, 1 and 5 ng/mL) for 7 days. (A) mRNA expression levels of MyHC1, MyoD and myogenin were analyzed by real-time quantitative RT-PCR. (B) The cell lysates were analyzed by western blotting with antibodies. β-actin was used as internal control. (C) Quantification of relative ratio of band intensity was performed using Image J software. Data represent means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. DMSO control.

Fig. 3. ZJM stimulates C2C12 muscle cells on day 7 of myogenic differentiation.
C2C12 myoblasts were cultured in a DM with ZJM (5 ng/mL) for indicated different time points. (A) mRNA expression levels of MyHC1, MyoD and myogenin were examined by real-time quantitative RT-PCR. (B) The cell lysates were examined by Western blotting with antibodies. β-actin was used as internal control. (C) Quantification of relative ratio of band intensity was performed using Image J software. Data represent means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. DMSO control at 0 min; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 as compared with DMSO control at the indicated time.

ZJM down-regulates muscle protein degradation in C2C12 muscle cells

To determine whether ZJM treatment inhibited critical marker associated with muscle protein degradation, we examined the protein levels of atrogin-1 and MuRF-1 as two muscle-specific E3 ubiquitin ligases. The effects of various concentrations of ZJM (0, 0.5, 1 and 5 ng/mL) on muscle atrophy markers were assessed by western blotting (Fig. 4A and B). These data indicated that ZJM appreciably decrease the expression of atrogin-1 and MuRF-1 at a concentration of 5 ng/mL, which was protected concentration for muscle atrophy. Next, to determine when ZJM affects protein degradation, we cultured in the presence of ZJM at 5 ng/mL for 3, 5 and 7 days after the start of C2C12 myoblasts differentiation. As shown in Figs. 5A and B, cells treated with ZJM showed significantly inhibition on atrogin-1 and MuRF-1 expression compared to control (Fig. 5A and B). These results suggest that 5 ng/mL of ZJM substantially reduces the expressions of atrogin-1 and MuRF-1 on day 7 of dif-ferentiation.

Fig. 4. ZJM down-regulates muscle degradation markers in C2C12 muscle cells.
C2C12 muscle cells were cultured in DM with 0.5, 1 and 5 ng/mL ZJM for 7 days. (A) The cell harvest were reviewed by western blotting with antibodies. β-actin was used as internal control. (B) Quantification of relative ratio of band intensity was performed using Image J software. Data represent means ± SD (n = 3). ^*P < 0.05, ^***P < 0.001 vs. DMSO control.

Fig. 5. ZJM reduces muscle degradation in C2C12 muscle cells on day 7 of myogenic differentiation.
C2C12 myoblasts were cultured in a DM with ZJM (5 ng/mL) for indicated different time points. (A) The cell dissolved matter were tested by western blotting with antibodies. β-actin was used as internal control. (B) Quantification of relative ratio of band intensity was performed using Image J software. Data represent means ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control at 0 min; ^#P < 0.05, ^##P < 0.01 as compared with DMSO control at the indicated time.

The fundamental findings of the present study is that (A) ZJM morphologically promotes myoblast differentiation without cytotoxicity and strengthens MyHC-positive multinucleated myotubes. These results are due to (B) ZJM induces myogenic differentiation markers and (C) suppresses muscle protein degradation makers.

Muscle differentiation is multistep processes proceeding via proliferation of myoblasts, cell cycle, the formation of multinucleated myotubes by myoblast fusion (Horsley and Pavlath, 2004; Krauss, 2010). In particular, MyHC detected myogenic differentiation, as demonstrated by the increased fusion index and multinucleated by immunofluorescence microscopy. Results of the current study indicated that ZJM enhances myotube length, fusion index and number of myotubes without cytotoxicity (Fig. 1). Therefore, based on this results, we observed that ZJM could increase the expression of MyHC, MyoD, and myogenin (Fig. 2 and 3), as we expected ZJM to increase myogenic specific genes. Myoblast differentiation is controlled by the myogenic regulatory factors (MRF), such as MyoD, myogenin and Myf5, which regulate the expression of several muscle-specific genes, such as MyHC, and a cyclin-dependent kinase inhibitor (Parker et al., 2003; Tapscott, 2005; Hawke and Garry, 2001). In particular, MyoD and myogenin promotes multinucleation by myoblast fusion and strengthening of mature skeletal muscle fibers (Bentzinger et al., 2012). Also, MyHC is responsible for the intrinsic rate of muscle contraction and regulates force production by controlling the expression of structural muscle-specific genes. Taken together, the present study data indicate that ZJM stimulates myogenic differentiation by increasing the expression of myogenic differentiation-related specific factors, such as MyHC, MyoD and myogenin.

In muscle atrophy caused by rapid loss of muscle mass and muscle weakness, the balance between protein synthesis and degradation pathways acts an important role, and in particular, the severity is controlled by the increased degree of protein breakdown (Jagoe and Goldberg, 2001). These protein degradation processe shares a common mechanism by induction of the atrogin-1 and MuRF1 (Lecker et al., 1999; Lecker et al., 2004). Atrogin-1 and MuRF-1 are two muscle-specific E3 ubiquitin ligases that promote muscle atrophy and induce increased protein degradation via the ubiquitin-proteasome system (Cohen et al., 2015; Bodine et al., 2001; Lagirand-Cantaloube et al., 2009). These protein degradation-related genes serve as early marker of skeletal muscle atrophy, and therefore considered master regulators. In this study, it was confirmed that ZJM can significantly reducing the gene expression of atrogin-1 and MuRF-1 on day 7 at a concentration of 5 ng/mL (Fig. 4 and 5).

In conclusion, our finding indicate that ZJM treatment promotes skeletal muscle myoblast differentiation, particularly affecting myotube morphology and progression of the differentiation process. ZJM might be a potential functional candidate to protect skeletal muscle from muscle weakness and atrophy associated with chronic diseases by stimulating myogenic differentiation and inhibiting protein degradation.

This study was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07049679) (NRF-2022R1C1C2010740).

The authors declare that they have no conflict of interest.