Effects of Rosemary Extract on C2C12 Myoblast Differentiation and 5-Aminoimidazole-4-carboxamide Ribonucleoside (AICAR)-Induced Muscle Cell Atrophy

Abstract

Sarcopenia is an aging-related disease that involves the gradual loss of muscle mass and function. However, no suitable therapeutic drugs are currently available. Accordingly, the purpose of the present study was to evaluate the effectiveness of rosemary extract (RE) in inducing myotube differentiation and inhibiting 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced muscle atrophy in mouse C2C12 cells. Morphological changes associated with the onset of RE-induced differentiation were evaluated by measuring myotube diameter, and the expression of proteins related to muscle differentiation and atrophy was measured using western blot analysis. Treatment with RE increased myotube thickness and the expression of the myogenic differentiation 1 (MyoD) and myogenin proteins. The effect of RE treatment on 5′-adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), MyoD, myogenin, muscle atrophy factors forkhead box O3a (FoxO3a), MAFbx/atrogin-1, and muscle RING finger-1 (MuRF-1) protein expression in AICAR-induced muscle-atrophied C2C12 cells was evaluated using western blot analysis. Treatment with RE reduced FoxO3a, MAFbx/atrogin-1, and MuRF-1 expression and significantly increased MyoD and myogenin expression. These findings suggest that RE has the potential to be used as an active ingredient in sarcopenia treatments.

1. Introduction

Sarcopenia is a chronic condition that develops during the natural process of aging. However, although its clinical significance has been discussed for a long time, it has recently been officially classified as a disease. In 2016, the USA included sarcopenia in the ICD-10-CM, which is a global classification of diseases published by the World Health Organization, and assigned the disease a specific cod (M62.84) [1]. Sarcopenia was also included in the eighth revision of the Korean Classification of Diseases (KCD) and was assigned an ICD-10-CM code (M62.5) [2]. Sarcopenia is defined as persistent muscle atrophy characterized by a slow loss of skeletal muscle mass and function, physical disability, poor quality of life, and death [3]. Sarcopenia is generally diagnosed by measuring muscle mass, strength, and function. Regular exercise is the best treatment for muscle atrophy [4]. As no drugs have been approved to date, new therapeutic agents must be developed to prevent sarcopenia.

Rosemary (Rosmarinus officinalis L., Lamiaceae), which is native to the Mediterranean region, has many culinary and medicinal uses. To date, there are more than 20 varieties of rosemary, and the herb has been reported to exert a variety of physiological effects, including the improvement of memory [5], antioxidant status [6], inflammation [7], osteoporosis [8], as well as promoting the differentiation of mesenchymal stem cells into osteoblasts and adipocytes [9] and hypertrophy in human skeletal muscle cells [10]. Rosemary contains a variety of phytochemicals, including rosmarinic acid, carnosol, carnosic acid, oleanolic acid, and ursolic acid (UA), which itself has been reported to exhibit physiological activities, including cytotoxicity to tumor cells [11], anticancer effects [12], and hypouricemic [13]. Many research results have been reported on the effects of various phytochemicals in rosemary on skeletal muscle cells. Rosmarinic acid increases glucose uptake in skeletal muscle cells and activates 5′-adenosine monophosphate-activated protein kinase (AMPK) [14]. Carnosol attenuated C2C12 myotube atrophy induced by tumor-derived exosomal miR-183-5p via inhibition of Smad3 pathway activation and maintenance of mitochondrial respiration [15]. Carnosic acid weakens insulin resistance induced by free fatty acids in muscle cells and adipocytes [16]. Ursolic acid ameliorates indoxyl sulfate-induced disorder of mitochondrial biogenesis in C2C12 cells [17]. Together, UA and leucine have been reported to potentiate the differentiation of C2C12 murine myoblasts through the mTOR signaling pathway [18]. Indeed, an ethanol extract of loquat (Eriobotrya japonica), which contained UA, was reported to prevent dexamethasone-induced muscle atrophy by inhibiting the muscle degradation pathway in Sprague Dawley rats [19], and loquat leaf extract has been reported to enhance both myogenic differentiation and muscle function in aged Sprague Dawley rats [20] and healthy human adults [21]. Therefore, it is necessary to study the inhibitory effects of ethanolic rosemary extracts on sarcopenia.

Skeletal muscle differentiation is a strictly regulated process involving the specification of mesodermal precursors in myoblasts and subsequent differentiation and fusion into multinucleated myotubes. During the process of muscle differentiation, myogenic regulatory factors (MRFs), including the basic helix-loop-helix (bHLH) transcription factors MyoD, Myf5, myogenin, and MRF4, play important roles in muscle formation [21]. In particular, MyoD is essential to the formation of skeletal muscle and plays unique roles in the development of epaxial and hypaxial muscle [22,23,24].

AMPK is an energy-sensing and signaling protein that, when activated, increases ATP production by stimulating glucose uptake and fatty acid oxidation and simultaneously inhibits ATP-consuming processes such as protein synthesis. In the skeletal muscles of both rodents and humans, AMPK is strongly activated in skeletal muscles by repeated muscle contraction [25] and exercise [26,27,28], and in many tissues, including skeletal muscle, AMPK is activated by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) [28,29,30]. The 26S proteasome degrades proteins that are tagged for destruction through the covalent attachment of ubiquitin chains, which is catalyzed by three enzymes (E1, E2, and E3). The E3 enzymes include multiple ubiquitin ligases, each of which is specific for the degradation of particular proteins. In skeletal muscle, the E3 enzymes MAFbx/atrogin-1 and muscle RING finger-1 (MuRF-1) play important roles in the proteasomal protein breakdown that occurs during muscle cell atrophy. The expression of the atrophy-related genes MAFbx/atrogin-1 and MuRF-1 is regulated by forkhead box (FoxO) transcription factors, especially FoxO3a, which is activated by AMPK and promotes skeletal muscle cell atrophy [31].

In order to investigate the possibility of using RE as a functional material by suppressing aging-related muscle cell atrophy, the present study evaluated the effects of RE on myoblast differentiation and AICAR-induced muscle cell atrophy in C2C12 myoblasts. As far as I know, this is the first report on the induction of differentiation of C2C12 myoblasts using RE.

2. Materials and Methods

2.1. Chemicals and Reagents

RE was provided by KINDS BIOTIX Inc. (Hanam, Republic of Korea). Whole rosemary leaves were authenticated by Min Hye Yang (Ph.D. in Pharmacognosy, Department of Pharmacy, College of Pharmacy, Pusan National University, Busan, Republic of Korea). A voucher specimen (PNU-0039) was deposited at the College of Pharmacy, Pusan National University. RE was extracted with 20% ethanol and contained UA (25.31% w/w) (Supplementary Table S1). The RE stock solution (100 mg/mL) was prepared using dimethyl sulfoxide (DMSO; Generay Biotech, Shanghai, China), stored at −20 °C, and then diluted in cell culture medium with a final DMSO concentration of 0.1% (v/v) so as not to inhibit cell growth. 3-(4,5-Dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromide (MTT) was obtained from Invitrogen (Waltham, MA, USA), and AICAR was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies against MyoD (SC-398608), myogenin (SC-52903), FoxO3a (SC-48348), MuRF-1 (SC-377460), ACC (SC-137104), AMPK (SC-74461), and MAFbx/atrogin-1 (SC-166806) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and antibodies against p-FoxO3a (9465s), p-ACC (3661s), and p-AMPK (2535s) were purchased from Cell Signaling Technology (Danvers, MA, USA). β-Actin (BS6007M) was purchased from Bioworld Technology, Inc. (Nanjing, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Welgene Inc. (Daegu, Republic of Korea), and horse serum (HS) was obtained from Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS) and penicillin–streptomycin were purchased from HyClone (Logan, UT, USA).

2.2. Cell Culture and Induction of Differentiation

Murine C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in growth medium (DMEN with 10% FBS and 1% penicillin-streptomycin; 37 °C, 95% relative humidity, 5% CO₂) and subcultured at 80–90% confluence. To induce the differentiation of myoblasts into myotubes, C2C12 cells were seeded in six-well plates (1.0 × 10⁵ cells/well) that contained DMEM with 10% FBS and grown to 70–80% confluence (37 °C, 24 h), after which the medium was replaced with differentiation medium (DMEM with 2% HS), which was subsequently replaced every 2 d. After 4 d in differentiation medium, 90% of the cells had fused into myotubes, and cells cultured in differentiation medium for 4–6 d were used for further experiments.

2.3. Cell Viability Assay

Cell viability was evaluated by the MTT assay to measure the metabolic activity of mitochondria in living cells. In the MTT assay, yellow tetrazolium MTT is reduced to purple MTT formazan by mitochondrial enzymes. C2C12 myoblasts were seeded into six-well plates (1.0 × 10⁵ cells/well). After 24 h, the cells were incubated (37 °C) in medium that contained different concentrations of RE (0–200 μg/mL) or AICAR (0–2 mM). After another 24 h, MTT (0.5 mg/mL) was added to the culture medium in the dark. Absorbance at 540 nm was measured using a microplate reader (Molecular Devices, San Jose, CA, USA), and relative cell viability was calculated as follows: (treated group/control group) × 100%.

2.4. Measurement of Myotube Diameter

Myotube cultures were photographed using a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany; 200× magnification) for 4–6 d following the induction of differentiation. Four fields were chosen randomly, and the diameters of 150 myotubes were measured using ImageJ (version 4.16; National Institutes of Health, Bethesda, MD, USA). The mean diameter of each myotube was calculated from three short axis measurements taken along each myotube’s length.

2.5. Western Blot Analysis

Both RE- and AICAR-treated cells were harvested after treatment using a cell scraper, incubated in lysis buffer [50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 40 mM β-glycerol phosphate, 120 mM NaCl, 25 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 mM microcystin-LR, and 1 mM benzamidine] for 30 min at 4 °C, and centrifuged at 13,000× g for 30 min. The protein contents of the resulting supernatants were quantified using protein assay reagents (Bio-Rad, Hercules, CA, USA). Equal amounts of quantified proteins were denatured by boiling at 100 °C for 5 min in sample buffer (Bio-Rad). Proteins in the supernatants were separated via 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked using 5% non-fat dry milk in Tris-buffered saline with Tween-20 (1 h, room temperature, with shaking). The blocked membranes were washed three times with TBS (10 min each), incubated overnight (4 °C) in TBS-T containing desired primary antibodies, and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). After washing, protein bands on the membranes were visualized using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Chicago, IL, USA).

2.6. Statistical Analysis

All results are presented as the mean ± standard deviation (SD) values of at least three independent experiments. The significance of differences between treatment groups (p < 0.05) was evaluated by one-way analysis of variance (ANOVA), followed by Turkey’s test, using GraphPad Prism (version 5.0; GraphPad Software).

3. Results

3.1. Effects of RE on Cytotoxicity in C2C12 Myoblasts

To evaluate the cytotoxicity of RE to murine C2C12 myoblasts, cell viability was examined using the MTT assay. Treatment with RE did not affect cell viability up to a concentration of 40 μg/mL (Figure 1). However, treatment with concentrations greater than 40 μg/mL reduced cell viability. Therefore, further experiments were conducted using a RE concentration of 0–40 μg/mL.

3.2. Effects of RE on C2C12 Myotube Differentiation

To evaluate the effect of RE on myotube differentiation, C2C12 myoblasts cultured in growth medium were subsequently cultured in differentiation-inducing medium in the presence or absence of RE, and the diameters of C2C12 myotubes under different treatments were measured after 4 d. Treatment with RE increased C2C12 myotube diameter in a concentration-dependent manner (Figure 2a), and myotube length was increased by promoting myotube differentiation capacity. In addition, myotube diameter and length exhibited a positive relationship. However, under treatment with 40 μg/mL RE, the C2C12 myoblasts failed to differentiate, and the cobblestone shape of the original C2C12 myoblasts was maintained. The positive effect of RE on myotube diameter was concentration-dependent up to 20 μg/mL RE (Figure 2b).

Next, the effects of RE treatment on the expression of MRF-related proteins were evaluated using western blot analysis. Treatment with RE had a positive and concentration-dependent effect on the expression of the differentiation markers MyoD and myogenin up to 20 μg/mL RE (Figure 2c). Furthermore, neither MyoD nor myogenin were expressed at substantial levels in cells that failed to differentiate (Figure 2c). Based on these observations, the experiment confirmed that RE induces the differentiation of C2C12 myoblasts into myotubes.

3.3. Effects of AICAR on C2C12 Myotube Atrophy

To evaluate the hypertrophic effects of RE on muscle cell atrophy, a muscle cell atrophy model was established using AICAR (an AMPK activator) to induce muscle cell atrophy. Prior to the experiment, the cytotoxicity of AICAR on C2C12 myoblasts and myotubes was determined using cell counting (Figure 3a) and the MTT assay (Figure 3b). Treatment with AICAR negatively affected both cell number and viability in a concentration-dependent manner (Figure 3a,b). Based on this, the maximum concentration of AICAR was set at 1 mM for the next experiment.

Next, the morphology and diameter of C2C12 myotubes were measured to evaluate the effect of muscle cell atrophy. Non-differentiated C2C12 myoblasts exhibited a cobblestone-shaped morphology, whereas cells that had undergone differentiation were observed as elongated myotubes (Figure 3c). Treatment with AICAR reduced myotube diameter in a concentration-dependent manner (Figure 3d). Therefore, AICAR treatment was used to establish a muscle cell atrophy model to investigate the effects of RE.

3.4. Effect of RE on AICAR-Induced C2C12 Myotube Atrophy

Before evaluating whether RE promoted hypertrophy in AICAR-induced C2C12 myotube atrophy, the cytotoxicity of simultaneous RE and AICAR treatment was evaluated using maximum RE and AICAR concentrations (40 μg/mL and 1 mM, respectively). After no serious cytotoxicity was observed, RE concentrations of up to 40 μg/mL were used (Figure 4). C2C12 myoblasts were cultured in differentiation medium for 6 d, and the resulting myotubes were treated with 1 mM AICAR for 24 h to induce muscle cell atrophy. Then, various concentrations of RE were added, and the inhibition of muscle cell atrophy and promotion of hypertrophy were evaluated after 24 h.

The C2C12 myoblasts clearly differentiated into myotubes when cultured in differentiation medium, with or without 40 μg/mL RE (Figure 5a), and clearly exhibited muscle cell atrophy when treated with 1 mM AICAR. However, treatment with RE reduced AICAR-induced muscle cell atrophy in a concentration-dependent manner and resulted in muscle cell hypertrophy (Figure 5a). Treatment with AICAR alone reduced myotube diameter by ~60% when compared with vehicle-treated control myotubes (Figure 5b). In addition, myotube diameter increased with increasing RE concentration, reaching 70–80% the diameter of vehicle-treated control myotubes at 40 μg/mL RE (Figure 5b).

The relationship between these morphological changes and the expression of muscle differentiation factors was investigated using western blot analysis. Expression levels of phosphorylated AMPK and phosphorylated ACC, which are related to muscle differentiation, increased during AICAR treatment but decreased in response to treatment with RE (Figure 5c). In addition, expression levels of muscle degradation-related transcription factors (p-FoxO3a, MAFbx/atrogin-1, and MuRF1) were increased by AICAR treatment but reduced by RE treatment (Figure 5d). Moreover, when myotubes, in which muscle cell atrophy occurred, were treated, expression levels of the muscle differentiation factors MyoD and myogenin gradually increased (Figure 5d).

4. Discussion

Skeletal muscles are involved in movement, respiration, and the maintenance of homeostasis in the body. Thus, the loss of skeletal muscle and function can be fatal. However, muscle atrophy and sarcopenia cannot be cured using current technology, and regular exercise is the only effective strategy for maintaining muscle mass and suppressing sarcopenia. Therefore, continuous prevention and more effective pharmaceutical agents are needed. In addition, it would be more useful to consume active ingredients that prevent sarcopenia daily. The RE used in the present study induced myotube differentiation, inhibited AICAR-induced muscle cell atrophy, and further promoted hypertrophy, thereby exhibiting its potential for use as a functional substance for preventing sarcopenia.

Skeletal muscles are involved in the regeneration of muscle fibers that contain mechanical, chemical, or degenerative lesions through activation, differentiation, and proliferation [32,33]. The differentiation of myoblasts into myotubes is essential for the formation of muscle fibers involved in the development and regeneration of skeletal muscles [34]. These monocyte myoblasts proliferate, differentiate, and then fuse with existing muscle fibers to form multinucleated myotubes and myofibers. Similar proliferation and differentiation of muscle cells occur during development and postnatal birth [35]. Therefore, the promotion of myoblast proliferation and differentiation and the induction of myotube hypertrophy should benefit muscle regeneration and the maintenance of muscle mass.

Recent studies have reported that muscle atrophy occurs as a result of increased proteolysis, which is, in turn, caused by activation of the ubiquitin-proteasome pathway [36,37,38]. Indeed, the muscle-specific ubiquitin ligases MAFbx/atrogin-1 and MuRF-1 are expressed early in the muscle atrophy process and are directly involved in muscle protein degradation [37,38]. In addition, when muscle atrophy is induced, muscle-specific transcription factors that are involved in myogenic differentiation (e.g., MyoD and myogenin) are downregulated in response to muscle-specific gene expression [39,40]. In addition, it has been reported that carnosol, a rosemary leaf extract, can also induce skeletal muscle hypertrophy by inhibiting the ubiquitin-proteasome system-dependent protein degradation pathway via suppression of E3 ubiquitin ligase MuRF-1 [10].

The injection of AICAR into mice increased FoxO1 and FoxO3 expression, and the AICAR-induced upregulation of FoxO1 was not affected by the suppression of AMPKα2 [41]. The treatment of C2C12 myotubes with AICAR has been reported to induce both protein breakdown and the upregulation of FOXO, MAFbx/atrogin-1, MuRF-1, and two other FoxO target genes, namely microtubule-associated protein 1A/1B-light chain 3 (LC3) and Bnip3 [42]. Meanwhile, AMPK phosphorylates FoxO3a at a site known to activate the transcription factors, thereby inducing generalized protein degradation; however, this may not necessarily affect the localization of FoxO3a to the nucleus. Treatment with AICAR also increases the binding of FoxO3 to MuRF-1 and atrogin-1 promoters [43].

The regulation of FoxO3a activity is possible by very complex mechanisms such as phosphorylation, acetylation, and methylation [44]. In particular, phosphorylation modification regulates FoxO3a activity via a cytoplasmic-nuclear shuttle mechanism. FOXO3a is targeted for phosphorylation by numerous protein kinases, and phosphorylation leads to two opposing results. The first is a signaling pathway that causes a negative effect on FoxO3a activity. Phosphorylation through AKT/GSK/ERK/IKK after stimulation of insulin and growth factors increases the cytoplasmic retention of FoxO3a and further induces the degradation of FoxO3a. The second is a signaling system that causes a positive effect on FoxO3a activity. In the presence of oxidant/nutrient stress, phosphorylation occurs through JNK/MST1/AMPK, promotes nuclear translocation, and increases the activity of FoxO3a. According to our experiments, the phosphorylation of FoxO3a was increased when AICAR, an AMPK activator, was treated on C2C12 cells (Figure 5d). Moreover, when RE was treated to the cells, the phosphorylation was reduced. Therefore, the down-regulation of E3 ligase (MuRF1 and MAFbx) activity was caused by inhibition of AMPK activity and inhibition of FoxO3a nuclear translocation, which were not investigated in our experiment (Figure 6).

In the present study, AICAR-induced muscle cell atrophy was confirmed through the activation of AMPK and FoxO3a, and the atrophy-inhibiting effects of RE treatment were confirmed by the observation of increased muscle hypertrophy. To study more specific mechanisms, additional studies related to the interaction between the AMPK pathway and its down-stream transcription factors are needed. In addition, further investigation of other myogenic factors (e.g., MyoD and myogenin) is also warranted.

5. Conclusions

In the present study, RE treatment was observed to promote muscle differentiation by increasing the expression of the myoblast differentiation factors MyoD and myogenin and to inhibit muscle cell atrophy by downregulating the transcription factors FoxO3, MAFbx/atrogin-1, and MuRF1 (Figure 6). Additional animal studies are needed to elucidate the specific mechanisms underlying the action of RE and the efficacy of RE in suppressing sarcopenia.