Mesenchymal Stem Cells Promote IL-6 Secretion and Suppress the Gene Expression of Proinflammatory Cytokines in Contractile C2C12 Myotubes

Abstract

Sarcopenia is not only a major cause of disability but also a risk factor for obesity and diabetes in elderly persons. Exercise is an effective method for improving the sarcopenic condition by inducing the secretion of interleukin (IL)-6, which has the capacities to both promote muscle hypertrophy and regulate lipid metabolism and glucose homeostasis, by skeletal muscle. We previously showed that mesenchymal stem cells (MSCs) promote IL-6 secretion by lipopolysaccharide-stimulated C2C12 mouse skeletal muscle myotubes via paracrine mechanisms. Therefore, in this study, we investigated the effect of paracrine actions of MSCs on IL-6 and proinflammatory cytokine expression in contractile C2C12 myotubes by applying electrical stimulation. IL-6 secretion by C2C12 myotubes was increased by electrical stimulation, and a more significant increase in IL-6 secretion was observed in electrically stimulated C2C12 myotubes cultured in conditioned medium from MSCs. The activation of nuclear factor-κB in C2C12 myotubes was also promoted by the combination of conditioned medium from MSCs and electrical stimulation. Moreover, the increases in tumor necrosis factor-α and IL-1β mRNA expression in C2C12 myotubes induced by electrical stimulation were suppressed by culture in conditioned medium from MSCs. The present findings suggest that MSCs transplantation or injection of their extracellular vesicles improve the therapeutic effect of exercise against sarcopenia without exacerbating inflammation.

INTRODUCTION

Sarcopenia is an age-related syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength.^1,2) Sarcopenia is a recognized major cause of disability, falls, hospitalization, and social isolation, all of which lead to decreases in QOL and activities of daily living in elderly persons. In addition, sarcopenia is also a significant risk factor for metabolic disorders, such as obesity and diabetes, resulting in an increase in mortality.³⁾ Therefore, many studies have been carried out to establish a means of effectively preventing and improving the sarcopenic condition.

Exercise is globally considered to be the most effective intervention for the prevention and treatment of sarcopenia.^4,5) During exercise, the production and secretion of several myokines, such as interleukin (IL)-6, IL-15, and irisin, is induced by contracting skeletal muscle, and these myokines contribute to not only the maintenance of muscle structure and function but also the regulation of whole-body physiological function and metabolism.^6,7) Among myokines, IL-6 plays crucial roles in both skeletal muscle regeneration and lipid and glucose metabolism.^8–11) IL-6 promotes the proliferation of skeletal muscle satellite cells and their differentiation.^8,9) Moreover, IL-6 also enhances glucose metabolism,¹⁰⁾ fatty acid oxidation and lipolysis in the whole body.¹¹⁾ Therefore, the efficient induction of IL-6 secretion by skeletal muscle is important to achieve the significant therapeutic benefit of exercise in patients with sarcopenia.

Previously, our group showed that mesenchymal stem cells (MSCs), which are multipotent adult progenitor cells used for tissue engineering and regenerative therapy,^12,13) have the capacity to increase protein synthesis, ubiquitination and autophagosome formation in mouse muscle by their intramuscular injection.¹⁴⁾ We also demonstrated that MSCs are capable of promoting IL-6 secretion by C2C12 mouse skeletal muscle myotubes via paracrine mechanisms under lipopolysaccharide (LPS) stimulation.¹⁵⁾ Moreover, we confirmed that the expression of the proinflammatory cytokines, tumor necrosis factor (TNF)-α and IL-1β, in LPS-stimulated C2C12 myotubes is suppressed by the presence of MSCs. These observations suggest that MSCs have the potential to increase exercise-induced muscle growth by promoting IL-6 secretion by skeletal muscle cells without inducing inflammatory responses.

In this study, to reveal the effect of paracrine actions of MSCs on exercise-induced IL-6 secretion by skeletal muscle, we evaluated IL-6 secretion and proinflammatory cytokine mRNA expression in contractile C2C12 myotubes cultured in conditioned medium from MSCs. We previously developed and reported a personal computer (PC)-controllable cell and tissue system that automates electrical stimulation, sampling, dispensing, and imaging.¹⁶⁾ This system enables automatic and high-frequency processing for stimulation and monitoring as well as feedback control processing. This study used electrical stimulation and imaging subsystems to induce contraction of C2C12 myotubes.

MATERIALS AND METHODS

Cell Lines

The C2C12 mouse myoblast cell line (CRL-1772™) and C57BL/6 mouse MSCs derived from the bone marrow (MUBMX-01101) were purchased from American Type Culture Collection (Manassas, VA, U.S.A.) and Cyagen Biosciences Inc. (Santa Clara, CA, U.S.A.), respectively. C2C12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Nichirei Biosciences Inc., Tokyo, Japan), penicillin G (100 U/mL) and streptomycin (100 µg/mL) (Nacalai Tesque) at 37 °C with 5% CO₂. When C2C12 cells were induced to differentiate into myotubes, the cells were maintained in DMEM supplemented with 2% heat-inactivated horse serum (HS) (Sigma-Aldrich, St. Louis, MO, U.S.A.) for 4 d. MSCs were maintained in Mouse MSC Growth Medium (Cyagen Biosciences Inc.).

Electrical Stimulation of C2C12 Myotubes

C2C12 cells were plated in 6-well culture plates at 2 × 10⁴ cells/cm² and differentiated into myotubes. Then, the culture medium was replaced with fresh DMEM supplemented with 2% HS or conditioned medium from MSCs supplemented with 2% HS. Conditioned medium from MSCs was the supernatants of MSCs cultured in 100-mm dishes for 1 d. The IL-6 concentration in conditioned medium from MSCs was not detectable (less than 8 pg/mL) by enzyme-linked immunosorbent assay (ELISA) (Thermo Fischer Scientific, Waltham, MA, U.S.A.). For electrical stimulation of myotubes, we used the PC-controllable cell and tissue system we previously developed.¹⁴⁾ Briefly, two electrodes were set in each well, and an electric pulse (1 Hz, 2 ms, 25–45 V/20 mm as an output voltage value of a power supply) was applied to the cells for 24 h according to the previous reports.^17,18) After electrical stimulation was performed, cell images were obtained, and cell viability was measured using Cell Count Reagent SF (Nacalai Tesque). The results are expressed as the % viability.

Measurement of IL-6 Secretion, Nuclear Factor-κB (NF-κB) Phosphorylation and Proinflammatory Cytokine mRNA Expression in C2C12 Myotubes under Electrical Stimulation

Differentiated C2C12 myotubes in 6-well culture plates were electrically stimulated (1 Hz, 2 ms, 35 V/20 mm), and the culture supernatants were collected at 0, 6, 12, and 24 h after electrical stimulation. An ELISA was used for measuring the IL-6 concentration in each sample. At 24 h after electrical stimulation was applied, myotubes were collected, and a portion of each myotube sample was analyzed for measuring phosphorylated and total NF-κB p65 levels using an InstantOne™ ELISA kit (Thermo Fischer Scientific). The phosphorylated NF-κB p65 level was normalized to the total NF-κB p65 level. In addition, Sepasol^®-RNA I Super G (Nacalai Tesque) was used to extract total RNA from the rest of each myotube sample, and ReveTra Ace^® qPCR RT kit (Toyobo, Osaka, Japan) was used for reverse transcription into cDNA. Real-time PCR was carried out using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on a StepOne Real-Time PCR System (Thermo Fischer Scientific). The relative expression levels of target mRNAs were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Table 1 listed the primers for Il-6, Tnf-α, Il-1β, and Gapdh.

Table 1. Primer Sequences for Real-Time RT-PCR

Gene	Primer sequence (5′-3′)
Il-6	Forward:	5′-CCG GAG AGG AGA CTT CAC AG-3′
	Reverse:	5′-CAG AAT TGC CAT TGC ACA AC-3′
Tnf-α	Forward:	5′-CCC CAA AGG GAT GAG AAG TT-3′
	Reverse:	5′-CAC TTG GTG GTT TGC TAC GA-3′
Il-1β	Forward:	5′-GGA TGA GGA CAT GAG CAC CT-3′
	Reverse:	5′-AGC TCA TAT GGG TCC GAC AG-3′
Gapdh	Forward:	5′-ACA GCA GTG TGC AGT TGA TGA-3′
	Reverse:	5′-GGC AGT CAT GTC CGG TGA TG-3′

Statistical Analysis

Results are presented as the mean ± standard deviation (S.D.) of three experiments. ANOVA was used to test the statistical significance of differences among groups. Multiple comparisons between all groups were performed with the Tukey–Kramer test.

RESULTS AND DISCUSSION

Effect of Conditioned Medium from MSCs on the Secretion of IL-6 and Cell Viability of Contractile C2C12 Myotubes

Previous reports utilized contractile C2C12 myotubes with electrical stimulation application as an in vitro exercise model.^19,20) They also demonstrated that contraction of C2C12 myotubes induced by electrical stimulation efficiently induces IL-6 production. We developed a cell and tissue system described previously.¹⁶⁾ This study utilized the PC-controllable electrical stimulation of cultured cells and automatic capture of dynamic images functions from among the potential functions of this system (Fig. 1). We observed that C2C12 myotubes contracted in response to electrical stimulation in this system (data not shown). Therefore, we used this system to evaluate IL-6 secretion by contractile myotubes cultured in conditioned medium from MSCs.

Fig. 1. Photographs of the PC-Controllable Cell and Tissue System in an Incubator

The PC-controllable cell and tissue system (A) was composed of a microscope, camera, and automatic stage in an incubator (B) and a PC and function generator for signal processing outside the incubator. Four 6-well culture plates could be placed side-by-side on the stage, and electrical stimulation was applied to each well in the left column (C).

IL-6 secretion by C2C12 myotubes was significantly enhanced by culturing C2C12 myotubes in conditioned medium from MSCs (Fig. 2A). However, IL-6 secretion was not changed by electrical stimulation at 25 V whether C2C12 myotubes were cultured in control medium or conditioned medium from MSCs. When electrical stimulation was applied to C2C12 myotubes cultured in control medium at 35 V for 24 h, IL-6 secretion was increased by approximately 2.0-fold, although there was no change in IL-6 secretion until 12 h after the electrical stimulation (Fig. 2C). This result is in accordance with a previous report that demonstrated that significant induction of IL-6 production by C2C12 myotubes requires electrical stimulation for more than 20 h.¹⁷⁾ As shown in Fig. 2C, we further observed that electrical stimulation at 35 V for 24 h induced approximately 3.8-fold increase in the secretion of IL-6 by C2C12 myotubes cultured in conditioned medium from MSCs. These findings indicate that MSCs have the potential to synergistically promote contraction-induced IL-6 secretion by skeletal muscle via paracrine mechanisms. It remains unclear which paracrine factors from MSCs are involved in this phenomenon. However, hepatocyte growth factor (HGF) is secreted from MSCs,^{21, 22)} and Perdomo et al. demonstrated that HGF significantly enhances glucose uptake and decreases fatty acidoxidation in L6 rat myotubes.²³⁾ Moreover, other group reported that HGF regulates proliferation and differentiation of human skeletal muscle myoblasts and C2C12 cells in a dose-dependent manner.²⁴⁾ In addition, insulin-like growth factor-1 (IGF-1) is also secreted from MSCs,²²⁾ and IGF-1 has been reported to increase the proliferation of L6 myoblasts and diameter of C2C12 myotubes.^25,26) Thus, these growth factors have the capacity to exert direct effects on skeletal muscle cells, and therefore might be contribute to MSCs-induced synergistic enhancement of IL-6 secretion from contractile skeletal muscle myotubes.

Fig. 2. IL-6 Secretion Level and Cell Viability of Electrically Stimulated C2C12 Myotubes Cultured in Conditioned Medium from MSCs

The IL-6 concentration in supernatants of C2C12 myotubes cultured in control medium or conditioned medium from MSCs with or without electrical stimulation (ES) at 25 V (A), 35 V (C), and 45 V (E). The cell viability of MSCs was measured after ES at 25 V (B), 35 V (D), and 45 V (F). Each value represents the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01; compared with the corresponding group without ES. ^† p < 0.05, ^†† p < 0.01; compared with the corresponding group with control medium.

Increase in IL-6 secretion by C2C12 myotubes was also observed by electrical stimulation at 45 V for 24 h (Fig. 2E). However, the degree of increase in IL-6 secretion by C2C12 myotubes induced by electrical stimulation was lower at 45 V than at 35 V. This could be due to the damage to C2C12 myotubes caused by the electrical stimulation. The cell viability of C2C12 myotubes was not changed with electrical stimulation at 25 V or 35 V (Figs. 2B, D), whereas a reduction in cell viability was observed with electrical stimulation at 45 V (Fig. 2F). In addition, images of C2C12 myotubes showed that electrical stimulation at 45 V caused significant damage to the myotubes (Supplementary Fig. S2). Therefore, subsequent experiments were carried out with electrical stimulation at 35 V.

Participation of NF-κB Activation in the Promotion of IL-6 Secretion by Contractile C2C12 Myotubes by Conditioned Medium from MSCs

To reveal the mechanisms underlying the synergistic increase in IL-6 secretion by C2C12 myotubes induced by the combination of the paracrine actions of MSCs and electrical stimulation, we next investigated the NF-κB activation in electrically stimulated C2C12 myotubes cultured in conditioned medium from MCSs. It has been reported that the exercise-induced increase in the production of IL-6 in skeletal muscle is mediated by activation of NF-κB.^27,28) In addition, we previously demonstrated that NF-κB is involved in the MSCs-induced enhancement of IL-6 secretion by C2C12 myotubes via paracrine mechanisms.¹⁵⁾ As shown in Fig. 3, the relative phosphorylated NF-κB p65 level in C2C12 myotubes was increased approximately 1.6-fold by culturing C2C12 myotubes in conditioned medium from MSCs. We observed a similar increase in the relative level of phosphorylated NF-κB p65 by applying electrical stimulation to C2C12 myotubes cultured in control medium. Moreover, phosphorylated NF-κB p65 level in C2C12 myotubes cultured in conditioned medium from MSCs was increased approximately 3.2-fold by electrical stimulation. This increased level of phosphorylated NF-κB p65 in C2C12 myotubes induced by the combination of conditioned medium from MSCs and electrical stimulation corresponds to the increased IL-6 secretion by C2C12 myotubes (Fig. 2C), suggesting that the synergistic induction of IL-6 secretion by C2C12 myotubes is mainly regulated by NF-κB.

Fig. 3. Relative Expression Levels of Phosphorylated NF-κB p65 in Electrically Stimulated C2C12 Myotubes Cultured in Conditioned Medium from MSCs

Electrical stimulation (ES) at 35 V was applied to C2C12 myotubes cultured in control medium or conditioned medium from MSCs for 24 h, and the phosphorylated NF-κB p65 levels were measured and normalized to the total NF-κB p65 levels. Each value represents the mean ± S.D. (n = 3). ** p < 0.01; compared with the corresponding group without ES. ^† p < 0.05, ^†† p < 0.01; compared with the corresponding group with control medium.

IL-6 and Proinflammatory Cytokines mRNA Expression in Contractile C2C12 Myotubes in Conditioned Medium from MSCs

We also investigated the effect of paracrine actions of MSCs on the IL-6 and other proinflammatory cytokines mRNA expression in electrically stimulated C2C12 myotubes. The expression level of IL-6 mRNA in C2C12 myotubes was significantly increased by both culturing in conditioned medium from MSCs and applying electrical stimulation (Fig. 4A). Moreover, the highest increase in the expression of IL-6 mRNA was induced by the combination of conditioned medium from MSCs and electrical stimulation. This result is in accordance with the data on IL-6 protein secretion (Fig. 2C) and phosphorylated NF-κB p65 expression (Fig. 3). Moreover, we also observed that electrical stimulation significantly increased the expression levels of TNF-α and IL-1β mRNA in C2C12 myotubes cultured in control medium, whereas the increases in these proinflammatory cytokines mRNA expression induced by electrical stimulation were suppressed when C2C12 myotubes were cultured in conditioned medium from MSCs (Figs. 4B, C). These observations are in agreement with our previous report demonstrating that the elevated expression of TNF-α and IL-1β mRNA in C2C12 myotubes induced by LPS stimulation are greatly decreased by coculture with MSCs.¹⁵⁾ Keller et al. demonstrated that Ca²⁺ stimulation increased the expression of IL-6 mRNA and decreased TNF-α mRNA expression in human skeletal muscle cells.²⁹⁾ They noted that this inhibition of TNF-α expression in skeletal muscle could be due to the anti-inflammatory effects of IL-6, which was greatly increased in Ca²⁺-stimulated skeletal muscle. In addition, it has been reported that IL-6 suppressed TNF-α and IL-1β production by LPS-stimulated peripheral blood mononuclear cells.³⁰⁾ These observations suggest that the increase in IL-6 secretion by contractile C2C12 myotubes induced by the paracrine actions of MSCs contributes to the inhibition of the expression of proinflammatory cytokines.

Fig. 4. Expression Levels of Il-6 (A), Tnf-α (B), and Il-1β (C) mRNA in Electrically Stimulated C2C12 Myotubes Cultured in Conditioned Medium from MSCs

Electrical stimulation (ES) at 35 V was applied to C2C12 myotubes cultured in control medium or conditioned medium from MSCs for 24 h, and the mRNA expression levels were measured and normalized to that of Gapdh. Each value represents the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01; compared with the corresponding group without ES. ^† p < 0.05, ^†† p < 0.01; compared with the corresponding group with control medium.

In conclusion, we demonstrate that paracrine actions of MSCs promote the secretion of IL-6 by contractile C2C12 myotubes. Moreover, this response is mediated by NF-κB. We also show that MSCs suppress electrical stimulation-induced proinflammatory cytokine expression in C2C12 myotubes. It is known that MSCs exist as fibro-adipogenic progenitors (FAPs) in the interstitial space of skeletal muscle.^31–33) FAPs have been reported to respond to mechanical load and stimulate the proliferation of skeletal muscle satellite cells in a paracrine fashion.³⁴⁾ In addition, it has been demonstrated that overload induces the local production of IL-6 in skeletal muscle myofibers, and myofiber-produced IL-6 promotes satellite cell proliferation.^8,35) Taking these findings and our present observations into consideration, it is likely that FAPs promote exercise-induced IL-6 secretion by skeletal muscle via paracrine mechanisms, resulting in satellite cell proliferation during muscle growth. Moreover, we assume that MSCs transplantation or injection of their extracellular vesicles have the capacity to improve the therapeutic effect of exercise against sarcopenia by enhancing exercise-induced IL-6 secretion by skeletal muscle without exacerbating inflammatory responses.

Acknowledgments

This study was supported in part by the Ritsumeikan Global Innovation Research Organization (R-GIRO) project at Ritsumeikan University.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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