Scratch wound closure of myoblasts and myotubes is reduced by inflammatory mediators

Abstract

Complex interactions exist between muscle repair processes and acute inflammatory responses that are initiated by exercise‐induced muscle damage. The purpose of this study was to examine whether inflammatory mediators secreted by activated macrophages affect the migration of myogenic cells to the injury site. Migration was measured using a scratch wound closure assay in C₂C₁₂‐derived myogenic cells incubated in activated macrophage‐conditioned medium. Both myoblast and myotube migrations were significantly reduced in activated macrophage‐conditioned medium compared with control medium. Furthermore, we demonstrated that the inhibitory effect on myoblast and myotube migrations was mediated, at least in part, by the two major cytokines secreted by activated macrophages, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6. These findings suggest that the migration rate of myogenic cells may be reduced by inflammatory mediators. It may provide useful insights for future researches on the role of macrophages in the process of muscle repair and regeneration.

Introduction

Strenuous eccentric or unaccustomed exercise with prolonged duration frequently results in exercise‐induced muscle damage, and subsequently initiates skeletal muscle repair and regeneration 1, 2. The process of skeletal muscle repair includes a degeneration/inflammation phase and a regeneration phase 3, 4. Muscle damage results in an acute inflammatory response that is characterised by rapid infiltration of neutrophils and macrophages, elevated secretion of inflammatory cytokines and increased production of oxygen radicals 5, 6, 7, 8. During the degeneration/inflammation phase, quiescent myogenic precursors, referred to as satellite cells, are activated and migrate to the site of injury where they proliferate 9, 10. At the regeneration phase, myoblasts are induced to undergo terminal differentiation and eventually fuse to form myotubes 10, 11. In addition, the switch from proinflammatory (M1) to anti‐inflammatory (M2) macrophages is important for support and regulation of muscle regeneration 4, 12.

Various studies indicate that complex interactions exist between muscle and the immune system during the early degeneration/inflammation phase 4, 13. For example, Chazaud et al. demonstrated that activated satellite cells release factors that attract monocytes/macrophages to the injured site 14. However, in a macrophage–satellite cell coculture experiment, Merly et al. found that macrophages stimulate proliferation and inhibit differentiation of satellite cells 15. Recent studies further demonstrate that during the acute inflammatory response macrophage‐secreted cytokines, such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐6, may play important roles in these complex immune–muscle interactions 4, 15, 16. TNF‐α and IL‐6 transcripts have been reported to peak at 24 hours and 3 days after muscle injury, respectively 4. The increased secretion of TNF‐α and IL‐6 has been shown to be involved in myogenic proliferation and differentiation 17, 18, 19, 20. Moreover, recent studies suggest that IL‐6 plays a similar role as TNF‐α during the early stages of muscle regeneration 4. TNF‐α exerts multiple effects on myogenic cells. For example, it stimulates myogenic proliferation 21, inhibits myogenic differentiation 22, 23 and modulates the myogenic protein expression 24. However, the effects of TNF‐α on myogenic cells are selectively manifested according to the TNF‐α concentration as well as the differentiation status of myogenic cells. For example, Alvarez et al. demonstrated that treating C₂C₁₂‐derived myotubes with low TNF‐α concentration resulted in decreased protein level, whereas a high TNF‐α concentration led to an increase in protein concentration 25. Langen et al. reported that TNF‐α suppressed the expression of muscle‐specific proteins in undifferentiated myoblasts 24. However, this suppressive effect was not observed in differentiated myotubes.

Previous studies have investigated the roles of macrophages and their secreted cytokines on the myoblast migration. However, these experiments refer to situations where satellite cells are distant from the injury site. For instance, TNF‐α and other cytokines released by macrophages act as chemoattractants that enhance myoblast migration across gelatin‐coated polycarbonate filter or endothelium monolayer 26, 27. Because myoblasts and myotubes exist in muscle tissue, both cell types should be affected when muscle is injured. However, no previous studies have reported the effects of muscle injury on myotubes. Thus, this study aimed to examine the effects of activated macrophages induced by muscle injury on myoblasts and myotubes.

Materials and methods

Cell culture

Myoblast cell line C₂C₁₂ that was derived from mouse muscle satellite cells and murine macrophage cell line RAW 264·7 were obtained from Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan). Both cell lines were maintained in growth medium consisting of Dulbecco's Modified Eagle's medium (DMEM; HyClone Laboratories, Logan, UT) supplemented with 10% fetal calf serum (HyClone Laboratories), 1% glutamine and 1% penicillin (HyClone Laboratories) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. To induce differentiation, C₂C₁₂ cells were incubated in growth medium supplemented with 2% horse serum for at least 3 days as described 28. Myotubes were identified by their distinct morphology and multiple nuclei. The fusion index (FI% = number of nuclei within myotubes/total number of nuclei × 100) was 97% under this culture condition.

Preparation of macrophage‐conditioned media

Conditioned media were prepared from activated or non‐activated macrophages. Briefly, RAW 264·7 cells at a concentration of 1 × 10⁶ cells/ml were replenished with medium in the presence or absence of 2 µg/ml lipopolysaccharide (LPS; Sigma‐Aldrich, St. Louis, MO) and 10 U/ml of interferon (IFN)‐γ (Sigma‐Aldrich), and cultured at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The medium supplemented with LPS and IFN‐γ was incubated for 24 hours at 37°C in the absence of cells and was used as a control. After 24 hours, supernatants were harvested, centrifuged and filtered to remove cell debris. Activated or control macrophage‐conditioned medium was mixed with fresh growth medium at a ratio of 1:5 for the following scratch would closure assay.

Scratch wound closure assay

In vitro muscle wounding was performed by mechanical disruption of myoblasts as previously described 29. Confluent C₂C₁₂ cells in 10 cm culture plates were wounded manually by scraping the monolayer using a sterile pipette tip. After washing, cells were replenished with activated or control macrophage‐conditioned medium or fresh growth medium in the presence of 2, 1, 0·5 and 0 pg/ml of TNF‐α or IL‐6, individually. C₂C₁₂ myoblasts were then incubated at 37°C for 0, 4, 8 or 12 hours, and C₂C₁₂‐derived myotubes were incubated at 37°C for 0, 12, 24 and 30 hours. The distance of the gap was measured at various time points after scraping under a high power field (400× magnification), and images were captured using an inverted microscope (Zeiss Axiovert, NY). The extent of wound closure was calculated as follows: (Distance of original wound gap − Distance of remaining wound gap)/Distance of original wound gap × 100%.

Determination of the TNF‐α and IL‐6 concentrations

Concentrations of TNF‐α and IL‐6 of the activated macrophage RAW 264·7 cells‐conditioned medium as described above were measured by a sandwich enzyme‐linked immunosorbent assay (ELISA). A total of 100 µl of capture antibody for TNF‐α (KMC3011; Invitrogen, Camarillo, CA) or IL‐6 (KMC0061; Invitrogen) that had been mixed with coating buffer was added to 96‐well plates and incubated at 4°C overnight. Plates were washed once with washing buffer [0·05% Tween‐20 in 1× phosphate buffered saline (PBS)]. A total of 200 µl of assay buffer was added into the wells and incubated at 4°C for 1 hour. After washing, 100 µl of standard solution of TNF‐α or IL‐6 or supernatant collected from activated macrophage‐conditioned medium was added and incubated at 4°C for 2 hours. After washing twice, 100 µl of detection buffer containing secondary antibody was added and incubated at 4°C for 30 minutes. Wells were then washed and 100 µl of substrate solution was added and incubated for 5 minutes. The optical density at 450 nm was measured using the microplate reader (BIO‐RAD, model 3550, VA). The detection limits for TNF‐α and IL‐6 were 15·6 and 7·8 pg/ml, respectively. All samples were assayed from three separate studies.

Statistical analysis

All data are reported as mean ± SD. Statistical difference between different medium groups was determined by a two‐way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Statistical difference between different concentrations of cytokines (IL‐6 or TNF‐α) was determined by repeated measures ANOVA followed by the Bonferroni post hoc test. Statistical difference between myoblasts and myotubes at 12 hours was determined by Student's t‐test. Statistical significance was set at P < 0·05.

Results

Reduced migration in myoblasts and myotubes treated with activated macrophage‐conditioned medium

In an attempt to determine whether myoblast and/or myotube migration is affected by activated macrophages at the injury site, C₂C₁₂ myoblasts and C₂C₁₂‐derived myotubes after scraping were cultured in growth medium, control or activated macrophage (RAW 264·7 cell)‐conditioned medium many times (Figures 1A and 2A). Compared with the percentage of migrated cells incubated in growth medium, myoblast migration was significantly reduced when cultured in activated macrophage‐conditioned medium at 8 and 12 hours after scraping (Figure 1B). Also, myotube migration was significantly reduced when cells were cultured in activated macrophage‐conditioned medium at 24 and 30 hours after scraping (Figure 2B). Because activated macrophage condition medium might contain the stimulator, cells after scraping were incubated in growth medium containing LPS plus IFN‐γ to study clearly the effect of them on cell migration. The result showed that LPS plus IFN‐γ did not significantly affect myoblast and myotube migrations (Figures 1B and 2B). Myoblasts migrated and covered about 90% of the wounded area at 12 hours after scraping, whereas myotubes only filled about 30% of the wounded area at the same time (Figures 1 and 2). This finding indicates that myoblasts migrated at a significantly faster rate than myotubes (P < 0·001).

Figure 1.

The wound closure of C₂C₁₂ myoblasts was reduced by activated macrophages. After scraping, C₂C₁₂ myoblasts were cultured in growth medium, control macrophage‐conditioned medium, activated macrophage‐conditioned medium, or growth medium plus LPS (2 µg/ml) and IFN‐γ (10 U/ml). (A) Vertical lines indicate the cell margin. Horizontal lines indicate the distance between the edges of the injured monolayer. (B) The extent of wound closure was measured and calculated as (Distance of original wound gap − Distance of remaining wound gap)/Distance of original wound gap × 100%. C₂C₁₂ cells were cultured in normal growth medium (open bar), with the presence of control macrophage‐conditioned medium (hatched bar), activated macrophage‐conditioned medium (dotted bar), or LPS and IFN‐γ (solid bar). Values are mean ± SD of three independent experiments. Scale bar, 100 µm. Significant difference between the values obtained from control cells (incubated in growth medium) and those incubated in other media was set at *P < 0·05; **P < 0·01.

Figure 2.

The wound closure of C₂C₁₂‐derived myotubes was reduced by activated macrophages. After scraping, C₂C₁₂‐derived myotubes were cultured in growth medium, control macrophage‐conditioned medium, activated macrophage‐conditioned medium, or growth medium plus LPS (2 µg/ml) and IFN‐γ (10 U/ml). (A) Vertical lines indicate the cell margin. Horizontal lines indicate the distance between the edges of the injured monolayer. (B) The extent of wound closure was measured and calculated as (Distance of original wound gap − Distance of remaining wound gap)/Distance of original wound gap × 100%. C₂C₁₂‐derived myotubes were cultured in normal growth medium (open bar), with the presence of control macrophage‐conditioned medium (hatched bar), activated macrophage‐conditioned medium (dotted bar), or LPS and IFN‐γ (solid bar). Values are mean ± SD of three independent experiments. Scale bar, 100 µm. Significant difference between the values obtained from control cells (incubated in growth medium) and those incubated in other media was set at *P < 0·05; **P < 0·01.

Release of TNF‐α and IL‐6 from activated macrophage cultures

Supernatants from RAW 264·7 cultures stimulated with LPS and IFN‐γ for 24 hours were collected to measure the concentrations of TNF‐α and IL‐6. Activated macrophage‐conditioned medium contained 35 ± 2 pg/ml of TNF‐α and 9 ± 1 pg/ml of IL‐6. In the scratch wound closure assay, the culture medium contained about 6 pg/ml of TNF‐α and 1·5 pg/ml of IL‐6. The concentrations of TNF‐α and IL‐6 in control macrophage‐conditioned medium were below the detection limits.

Reduced migration in myoblasts and myotubes treated by TNF‐α or IL‐6

To investigate whether TNF‐α or IL‐6 secreted by activated macrophages affect cell migration, after scraping, myoblasts and/or myotubes were cultured in growth medium in the presence of 2, 1, 0·5 and 0 pg/ml of TNF‐α or IL‐6. Figure 3 shows that 2 pg/ml of TNF‐α or IL‐6 significantly reduced myoblast migration at 4, 8 and 12 hours after scraping (P < 0·01) (Figure 3). Myotube migration was also significantly reduced by 1 pg/ml of TNF‐α (P < 0·01) or 0·5 pg/ml of IL‐6 (P < 0·05) at 30 hours after scraping (Figure 4). These phenomena were dose‐dependent.

Figure 3.

Myoblast migration was reduced by TNF‐α and IL‐6. After scraping, C₂C₁₂ myoblasts were cultured in normal growth medium in the presence of 2 (hatched bars), 1 (dotted bars), 0·5 (solid bar) or 0 (open bars) pg/ml of TNF‐α (A) or IL‐6 (B), respectively. The extent of wound closure was measured and calculated as (Distance of original wound gap − Distance of remaining wound gap)/Distance of original wound gap × 100%. Values are mean ± SD of three independent experiments. Significant difference between the values obtained from control cells (incubated in growth medium) and those incubated in other media was set at *P < 0·05; **P < 0·01.

Figure 4.

Myotube migration was reduced by TNF‐α and IL‐6. After scraping, C₂C₁₂‐derived myotubes were cultured in normal growth medium in the presence of 2 (hatched bars), 1 (dotted bars), 0·5 (solid bar) or 0 (open bars) pg/ml of TNF‐α (A) or IL‐6 (B), respectively. The extent of wound closure was measured and calculated as (Distance of original wound gap − Distance of remaining wound gap)/Distance of original wound gap × 100%. Values are mean ± SD of three independent experiments. Significant difference between the values obtained from control cells (incubated in growth medium) and those incubated in other media was set at *P < 0·05; **P < 0·01.

Discussion

This study provides evidence indicating that both myoblast and myotube migrations are reduced by activated macrophage‐conditioned medium. Secondly, potential factors that inhibit migration include TNF‐α and IL‐6. This study has also found that myoblasts migrate at a faster rate than myotubes.

Previous studies have shown that when myoblasts were located a distance from the injury site, macrophage‐secreted cytokines act as chemoattractants, enhancing myoblast migration across a gelatin‐coated polycarbonate filter or endothelium monolayer 26, 30. These apparently contradictory observations can be explained by dissimilar study designs and experimental approaches. The experiments conducted by Torrente et al. 27 and Corti et al. 26 refer to situations when myoblasts were distant from the injury site. However, the in vitro migration experiments were designed to simulate a situation in which myoblasts are present at the injury site where activated macrophages aggregate. In a similar experimental design as in this study, Merly et al. reported in an ex vivo coculture experiment that macrophages stimulate the proliferation and inhibit the differentiation of muscle satellite cells 15. The results of this study and those of Merly et al. suggest that when myogenic precursors are located at the injury site, their migration capability is inhibited to retain these cells in the area of injury 15. High concentrations of TNF‐α were associated with reduced wound closure that might be explained by a higher percentage of activated macrophages involved in muscle inflammation. Additionally, in order to generate more myoblasts for muscle repair at the injury site, myogenic precursors are stimulated to undergo proliferation while their differentiation is delayed. These results are in good agreement with previous reports that during the degeneration/inflammation phase, myogenic precursors migrate to the site of injury where they proliferate 9, 10.

Early studies of muscle inflammation implicated M1 macrophages in the early stages of muscle injury 31, 32. It is known that Th1 cytokine IFN‐γ drives the classical activation of macrophages to an M1 phenotype that is proinflammatory. M2 macrophages are activated by Th2 cytokines, such as IL‐4 and IL‐10, and play an important role in anti‐inflammation 33. In a previous study, muscle damage induced by eccentric contraction resulted in significantly increased TNF‐α level on the second day but increased IL‐10 concentrations after the fourth day 34. Therefore, in this study, we used LPS/IFN‐γ to activate macrophages to an M1 phenotype and secrete proinflammatory cytokines, such as TNF‐α and IL‐6. The results of this study suggest that reduced migration is associated with 6 pg/ml of TNF‐α and 1·5 pg/ml of IL‐6, in the activated macrophage (RAW 264·7 cell)‐conditioned medium. As it has been shown that the effects of TNF‐α on myogenic cells are dependent on TNF‐α concentration 25, we infer that the effects on myogenic migration may also be influenced by TNF‐α concentration. For instance, in a chemotactic study performed by Torrente et al., the authors reported that the ability of TNF‐α to attract distantly located myogenic cells was markedly reduced at the highest concentration (400 U/ml) compared with lower TNF‐α concentrations (100 or 200 U/ml) 27. The migration inhibition of TNF‐α on myogenic cells was found in this study and the chemotaxis stimulation shown by Torrente et al. may be caused by different concentrations of TNF‐α or/and their different culture model 27. In addition, a previous study indicated that 0·05 ng/ml TNF‐α stimulated myogenesis, whereas 0·5 or 5 ng/ml TNF‐α inhibit myogenesis 35. The effects of TNF‐α have been shown to be dependent on the differentiation status of myogenic cells 24. This study found that the inflammatory stimuli applied have distinct effects on myoblasts and myotubes. It is possible that the reduced migration in myogenic cells in this study is related to the differentiation status. A number of studies have reported that IL‐6 is involved in the process of myogenic proliferation 19 and differentiation 18. Here, we further demonstrated that IL‐6 exerts similar effects as those of TNF‐α on myoblast and myotube migrations. The exact mechanisms underlying the differential effects of TNF‐α on migration and proliferation are still elusive and, therefore, future studies are needed to determine this.

To the best of our knowledge, no previous studies have compared the migration rate of C₂C₁₂‐derived myotubes with that of C₂C₁₂ myoblasts. Results of this study show that myotubes migrate at a much slower rate than myoblasts with or without the treatment of inflammatory mediators. Although myoblasts and myotubes were cultured in growth medium with different percentages of serum, we found no significant difference in the migration percentage of myoblast between DMEM medium with 10% and 2% fetal calf serum (data not shown). This indicates that the migration capacity of myotubes is significantly impaired following differentiation. This observation is plausible as it is known that during normal physiological process of skeletal muscle repair, differentiated myotubes do not migrate. However, the physiology of myotubes may be affected by muscle injury. For instance, Alvarez et al. reported that macrophage‐secreted cytokines, such as TNF‐α, affect the protein contents of myotubes 25. However, the limitation of this study was the isolation of the affected cells next to the scratch wound that were associated with scratch stress and wound closure. The results presented here indicate that the greatly weakened migration capacity of myotubes was further reduced by inflammatory mediators from activated macrophage‐conditioned medium, TNF‐α and IL‐6. Whether the expression levels of adhesion molecules, chemotactic receptors and chemotactic factors are influenced by different concentrations of TNF‐α and IL‐6 requires further study.

In summary, this study has shown that myoblast and myotube migrations were significantly reduced in the presence of activated macrophage‐conditioned medium. In addition, C₂C₁₂‐derived myotubes migrate at a much slower rate than C₂C₁₂ myoblasts. The results of this study also demonstrate that inflammatory mediators, TNF‐α and IL‐6, play important key roles in regulating myoblast and myotube migrations. Although detailed mechanisms underlying these observations need further investigation, this may provide novel insights into the interactions of the immune and muscle systems during the degeneration/inflammation phase of skeletal muscle repair.