CCL5 Attenuates Palmitate-Induced Myotube Atrophy through Cytoskeletal Stabilization Rather than Proteostasis Regulation
Article information
Abstract
PURPOSE
Sarcopenia is characterized by progressive skeletal muscle atrophy associated with impaired proteostasis, mitochondrial dysfunction, and chronic metabolic stress. Through transcriptomic analysis of aged skeletal muscle, CCL5 was identified as a stress-responsive immune-related factor consistently upregulated under sarcopenic conditions. This study aimed to investigate the role of CCL5 in a palmitate (PA)-induced in vitro model of sarcopenia.
METHODS
Fully differentiated C2C12 myotubes were treated with PA to induce metabolic stress–associated atrophy. Morphological changes, myogenic signaling, protein turnover pathways, mitochondrial function, and apoptosis-related markers were analyzed. Candidate genes identified from transcriptomic analysis of aged skeletal muscle were validated in the PA-induced atrophy model, and the effects of exogenous recombinant CCL5 treatment were examined.
RESULTS
PA treatment recapitulated key pathological features of muscle atrophy, including reduced myotube diameter, suppressed myogenic signaling, inhibition of the AKT–mTOR pathway, increased proteolysis, mitochondrial dysfunction, and activation of apoptosis. Among the candidate genes identified through transcriptomic analysis of aged skeletal muscle in our previous study, Ccl5 was markedly upregulated in PA-treated myotubes. Notably, exogenous CCL5 treatment partially restored myotube diameter under PA-induced stress. However, CCL5 did not significantly alter canonical regulatory pathways of muscle atrophy, including protein synthesis, protein degradation, or mitochondrial biogenesis. Instead, CCL5 selectively restored the expression of desmin, an intermediate filament protein critical for myofiber structural integrity.
CONCLUSIONS
CCL5 may attenuate metabolic stress–induced myotube atrophy by preserving cytoskeletal stability rather than regulating classical proteostasis pathways, highlighting a potential CCL5–CCR5–mediated mechanism for maintaining skeletal muscle structural integrity.
INTRODUCTION
Sarcopenia is a systemic muscle disorder characterized by the progressive loss of skeletal muscle mass, strength, and physical function with aging. Traditionally, it was considered a natural consequence of aging [1,2]. However, accumulating evidence indicates that sarcopenia is strongly associated with an increased risk of falls, fractures, diabetes, and cardiovascular diseases, and is ultimately linked to increased mortality [3]. Despite its growing clinical importance, no pharmacological treatment for sarcopenia has yet been approved by the U.S. Food and Drug Administration (FDA), and current management strategies largely rely on nutritional support and exercise interventions [4-9]. Therefore, identifying novel molecular targets based on the underlying pathophysiology of sarcopenia remains an important research priority.
The development of sarcopenia involves multiple interconnected pathological mechanisms [10]. One key mechanism is impaired proteostasis, characterized by reduced protein synthesis through suppression of the PI3K/AKT/mTOR signaling pathway and increased protein degradation mediated by FoxO-dependent E3 ubiquitin ligases, including MuRF1 and MAFbx, as well as activation of autophagy pathways [11-13]. Another major contributor is mitochondrial dysfunction, which includes reduced mitochondrial biogenesis associated with decreased PGC-1α expression, imbalanced mitochondrial dynamics, and excessive accumulation of reactive oxygen species (ROS) [14,15]. In addition, chronic low-grade inflammation and apoptosis are frequently observed in aged skeletal muscle [16-19]. With aging, impaired lipid metabolism can lead to abnormal accumulation of fatty acids in skeletal muscle. Palmitate (PA), the most abundant saturated fatty acid in the human body, is known to accumulate in skeletal muscle under conditions such as aging and obesity [20]. In this context PA-treated C2C12 myotubes are widely used as an in vitro model of metabolic stress–induced muscle atrophy [21,22].
In our previous study, we demonstrated that gene sets associated with immune function were upregulated in the skeletal muscle of 21-monthold aged rats compared to young rats, based on transcriptomic analysis. Among the genes commonly upregulated in both muscles, Ccl5 (RANTES) was identified as a potential sarcopenia-related candidate gene. CCL5 is a CC chemokine that binds primarily to the CCR5 receptor and is well known for regulating immune cell recruitment and inflammatory responses [23]. Interestingly, recent studies suggest that CCL5 is also expressed in skeletal muscle cells and satellite cells and may participate in muscle differentiation, tissue homeostasis, and cytoskeletal remodeling [24,25]. Recent studies suggest that immune-related signaling molecules contribute to skeletal muscle adaptation and stress responses under aging and metabolic dysfunction [26,27]. Among these factors, chemokines have emerged as potential regulators of tissue remodeling and cellular homeostasis in skeletal muscle. Conversely, under chronic metabolic stress conditions such as aging and obesity, excessive CCL5 expression has been reported to enhance inflammatory responses and exacerbate muscle atrophy [28-30]. Therefore, whether CCL5 exerts protective or detrimental effects in skeletal muscle under metabolic stress conditions remains unclear.
Therefore, the purpose of this study was to investigate the functional role of CCL5 in a PA–induced skeletal muscle atrophy model. Specifically, we examined whether the increased expression of Ccl5 observed in aged skeletal muscle is also reproduced in PA-treated C2C12 myotubes. In addition, we evaluated the effects of exogenous CCL5 treatment on myotube diameter, protein synthesis and degradation signaling pathways, cytoskeletal stability, and mitochondrial function. Through these approaches, we aimed to clarify the functional role of the CCL5 axis in skeletal muscle and explore its potential as a novel molecular target for the treatment of sarcopenia.
METHODS
1. Experimental design
Fully differentiated C2C12 myotubes were treated with PA to induce metabolic stress–associated atrophy, and the effects of exogenous CCL5 treatment were examined. Cells were divided into three groups: vehicle-treated control (Control), PA-treated (PA), and PA plus CCL5-treated (PA+CCL5). In the CCL5 treatment group, recombinant CCL5 was administered 30 mintues prior to PA exposure, followed by co-treatment with PA for 24 hours. After treatment, morphological changes in myotubes and molecular markers related to protein synthesis and degradation, cytoskeletal stability, mitochondrial function, MAPK signaling, and apoptosis were analyzed.
2. Cell culture and differentiation
The mouse skeletal muscle cell line C2C12 (ATCC, CRL-1722) was cultured in a humidified incubator at 37°C with 5% CO₂. Cells were maintained in growth medium consisting of high-glucose Dulbecco’s Modified Eagle Medium (DMEM, 25 mM glucose) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. When cells reached approximately 100% confluence, differentiation was induced by replacing the growth medium with differentiation medium containing 2% horse serum. Cells were allowed to differentiate for 72–120 hours, and the medium was replaced every 24 hours to allow the formation of multinucleated myotubes.
3. Palmitate (PA) treatment
PA was conjugated with fatty acid–free bovine serum albumin (FAFBSA) prior to treatment. Fully differentiated C2C12 myotubes were treated with PA at a final concentration of 0.75 mM to induce metabolic stress–related muscle atrophy. PA was dissolved in serum-free medium containing 2% FAF-BSA and applied for 24 hours. Control cells received vehicle medium containing the same concentration of FAF-BSA.
4. CCL5 treatment
To evaluate the effects of exogenous CCL5, recombinant mouse CCL5 (BioLegend, USA) was diluted in serum-free medium containing 1% FAF-BSA to a final concentration of 200 ng/mL. In the PA+CCL5 group, CCL5 was administered 30 min prior to PA treatment and co-incubated with PA for 24 hours.
5. Cell viability assay
Cell viability was assessed using an MTT assay. After treatment, cells were incubated with MTT solution (0.5 mg/mL) for 2 hours at 37°C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader (PowerWaveX; Bio-Tek Instruments, VT, USA).
6. Immunofluorescence staining and myotube diameter analysis
To evaluate morphological changes in myotubes, immunofluorescence staining was performed using an antibody against myosin heavy chain fast isoform (MHC(f)). After treatment, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton X-100. Cells were then blocked with 5% bovine serum albumin (BSA) for 1 hour and incubated with primary MHC(f) antibody (1:500) overnight at 4°C. After washing, cells were incubated with Alexa Fluor 488–conjugated secondary antibody (1:500) and counterstained with DAPI to visualize nuclei. Fluorescence images were captured using an Olympus IX83 fluorescence microscope (Olympus, Tokyo, Japan). For myotube diameter analysis, three random fields were captured per well, and 50 multinucleated myotubes containing at least three nuclei were selected. Myotube diameter was quantified using cell-Sens imaging software (Olympus, Japan).
7. Western blot analysis
Cells were lysed using RIPA buffer to extract total proteins. Protein concentrations were determined using a bicinchoninic acid (BCA) assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies. After incubation with HRP-conjugated secondary antibodies, protein bands were detected using enhanced chemiluminescence (ECL). Proteins analyzed included markers related to myogenesis and cytoskeletal stability (MHC, Myogenin, MyoD, Desmin), protein synthesis (p-AKT, p-mTOR, p-Raptor, p-p70S6K, p-4E-BP1), protein degradation and autophagy (p-FoxO1/3a, MuRF1, MAFbx, Ubiquitin, p62, LC3), mitochondrial function and dynamics (p-AMPK, SIRT1, PGC-1α, MFN1/2, OPA1, DRP1, FIS1, PINK1, Parkin), MAPK signaling (p-p38, p-ERK1/2, p-JNK), and apoptosis-related proteins (cleaved Caspase-3, cleaved PARP). Detailed information for all primary and secondary antibodies, including manufacturer, catalog number, and dilution conditions, is provided in Table 1.
List of antibodies used in western blot
8. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted using TRIzol reagent. RNA concentration and purity were determined using a NanoDrop spectrophotometer. Complementary DNA (cDNA) was synthesized using reverse transcription reagents. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed using SYBR Green chemistry to assess mRNA expression levels of Ccl5, Ccl11, Tmem158, and muscle-related genes. Gene expression levels were normalized to Gapdh, and relative expression was calculated using the 2–ΔΔCT method. Primer sequences used for qRT-PCR are listed in Table 2.
Primer sequences used for quantitative real-time reverse transcription polymerase chain reaction
9. Mitochondrial function analysis
Mitochondrial membrane potential was assessed using MitoTrackerTM Red CMXRos. Cells were incubated with MitoTrackerTM Red CMXRos (100 nM) for 30 minutes prior to imaging, followed by fluorescence microscopy and quantification of fluorescence intensity. Intracellular ATP levels were measured using a commercial ATP assay kit according to the manufacturer’s instructions.
10. Statistical analysis
All data are presented as mean±standard deviation (SD). Because of the relatively small sample size and variability inherent to biological replicate measurements in cell-based experiments, nonparametric statistical analyses were applied to minimize potential violations of normality assumptions. Differences between two groups were analyzed using the Mann–Whitney U test, whereas comparisons among three or more groups were performed using the Kruskal–Wallis test. The one-sample t-test was used only when normalized values were compared against reference control value. Statistical analyses were conducted using GraphPad Prism (version 10.1.0). Statistical significance was indicated as follows: *p<.05, **p<.01, and ***p<.001.
RESULTS
1. Palmitate (PA) induces myotube atrophy in C2C12 cells
To establish experimental conditions for PA-induced muscle atrophy, C2C12 myotubes were treated with increasing concentrations of PA, and cell viability was assessed using an MTT assay. PA treatment at 0.75 mM for 24 hours induced metabolic stress without causing significant cytotoxicity and was therefore selected for subsequent experiments (Fig. 1A).
Validation and molecular characterization of palmitate-induced myotube atrophy in C2C12 cells. (A) Fully differentiated C2C12 myotubes were treated with increasing concentrations of palmitate (PA; 0–1 mM) for 24 h, and cell viability was assessed using an MTT assay (n=4). (B) Representative immunofluorescence images of myosin heavy chain fast isoform (MHC; green) and nuclei (DAPI; blue) following PA treatment (0.75 mM, 24 h). Scale bar=200 μm. (C) Quantification of myotube diameter and relative frequency distribution derived from immunofluorescence images (n=15). (D) Protein expression of myogenic markers (MHC, Myogenin, and MyoD) analyzed by Western blotting (n=6–7). (E) Phosphorylation levels of AKT were evaluated by Western blotting (n=6–8). (F) Phosphorylation of FoxO3a and total ubiquitination levels were analyzed as markers of protein degradation (n=3–7). (G) Apoptotic markers, including cleaved Caspase-3 and cleaved PARP, were examined by Western blotting (n=6). (H) Mitochondrial membrane potential was visualized using MitoTrackerTM Red CMXRos. Representative fluorescence images and quantitative fluorescence intensity analysis are shown. Scale bar=200 μm (n=7). Statistical analysis. Data are presented as mean±SD. Statistical analyses were performed using the Mann–Whitney U test, Kruskal–Wallis test, or one-sample t-test as appropriate. *p<.05, **p<.01, and ***p<.001 versus control.
Morphological changes in myotubes were examined by immunofluorescence staining for MHC. PA-treated myotubes exhibited visibly thinner fibers compared with the control group (Fig. 1B). Quantitative analysis confirmed a significant reduction in mean myotube diameter, and frequency distribution analysis further demonstrated a shift toward smaller myotube diameters in the PA-treated group (Fig. 1C).
To further characterize the molecular features of PA-induced atrophy, the expression of myogenic markers was analyzed. Western blot analysis revealed a significant reduction in Myogenin expression, while MHC and MyoD also showed a decreasing trend following PA treatment (Fig. 1D).
Next, signaling pathways related to protein metabolism were evaluated. Phosphorylation of AKT, a key regulator of protein synthesis, was significantly decreased in PA-treated myotubes (Fig. 1E). Consistently, phosphorylation of FoxO3a was reduced, accompanied by an increase in total ubiquitination levels, indicating enhanced protein degradation (Fig. 1F). Apoptotic signaling was also examined. The levels of cleaved Caspase-3 and cleaved PARP were significantly increased following PA treatment, suggesting activation of apoptosis (Fig. 1G).
In addition, mitochondrial function was assessed by measuring mitochondrial membrane potential using MitoTrackerTM Red CMXRos. PAtreated myotubes displayed markedly reduced fluorescence intensity compared with control cells, indicating impaired mitochondrial membrane potential (Fig. 1H).
2. PA disrupts proteostasis and mitochondrial regulatory pathways in C2C12 myotubes
To further define the molecular features of PA-induced atrophy, we examined signaling pathways associated with protein synthesis, protein degradation, and mitochondrial regulation in C2C12 myotubes.
Analysis of mTOR signaling components, a pathway related to protein synthesis, phosphorylation of mTOR was decreased following PA treatment, whereas phosphorylation levels of Raptor and p70S6K were significantly increased, with no consistent change observed in 4E-BP1 (Fig. 2A).
Detailed analysis of protein turnover and mitochondrial regulatory pathways following palmitate treatment. (A) Phosphorylation levels of proteins involved in the protein synthesis pathway (mTOR, Raptor, p70S6K, and 4E-BP1) were analyzed by Western blotting. Phosphorylated proteins were normalized to their respective total proteins (n=6–8). (B) Components of the protein degradation pathway, including phosphorylation of FoxO3a and expression of MuRF1 and MAFbx, as well as autophagy markers (p62 and LC3-I/II), were analyzed by Western blotting. Phosphorylated proteins were normalized to total proteins, and other proteins were normalized to α-actinin (FoxO3a: n=3–7; p62/LC3: n=2). (C) Protein expression of mitochondrial dynamics regulators (MFN1, MFN2, OPA1, DRP1, and FIS1) and mitophagy-related proteins (PINK1 and Parkin) was analyzed by Western blotting and normalized to α-actinin. Data are presented as mean±SD. Statistical analyses were performed using the Mann–Whitney U test, Kruskal–Wallis test, or one-sample t-test as appropriate. *p<.05, **p<.01, and ***p<.001 versus control.
PA treatment showed an increasing trend in protein degradation pathways including MuRF1, p62, and LC3-II/I ratio, while MAFbx expression was reduced compared with control (Fig. 2B).
To evaluate mitochondrial quality control mechanisms, we analyzed the expression of genes involved in mitochondrial dynamics and mitophagy (Fig. 2C). Expression levels of MFN1, MFN2, OPA1, DRP1, and Parkin were not significantly altered, whereas PINK1 showed a decreasing trend and FIS1 expression was significantly reduced.
3. Validation of sarcopenia-related genes in the PA-induced atrophy model
To validate sarcopenia-associated candidate genes identified from transcriptomic analysis, we examined the expression levels of selected genes in PA-treated C2C12 myotubes. qRT-PCR analysis confirmed that three genes—Ccl5, Ccl11, and Tmem158—were significantly upregulated following PA treatment compared with control (Fig. 3). These genes were previously identified as being elevated in aged skeletal muscle, indicating a consistent pattern of induction across both in vitro metabolic stress conditions and in vivo aging models.
Validation of sarcopenia-related candidate genes in the PA-induced atrophy model. mRNA expression levels of selected candidate genes (Ccl5, Ccl11, and Tmem158) were measured in PA-treated C2C12 myotubes (0.75 mM, 24 h) using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR; n=12). Data are presented as mean±SD. Statistical significance was determined using the Mann–Whitney U test, Kruskal–Wallis test, or one-sample t-test as appropriate. **p<.01, and ***p<.001 versus control.
Among these candidates, Ccl5 exhibited the most pronounced increase. In our previous transcriptomic analysis of aged skeletal muscle, Ccl5 expression was markedly elevated, showing approximately 4.5-fold upregulation in the soleus and 14.5-fold upregulation in the white gastrocnemius compared with young muscle [31]. A comparable increase in Ccl5 expression was also observed in PA-treated C2C12 myotubes in the present study.
4. CCL5 attenuates PA-induced myotube atrophy through restoration of desmin expression
To evaluate the functional role of CCL5 under atrophic conditions, recombinant mouse CCL5 (rCCL5) was applied to PA-treated C2C12 myotubes. MTT assays confirmed that rCCL5 (0 to 400 ng/mL) was not cytotoxic, and 200 ng/mL was selected as the optimal concentration for subsequent experiments (Fig. 4A).
CCL5 co-treatment partially attenuates PA-induced myotube atrophy. (A) Cell viability following treatment with recombinant mouse CCL5 (rCCL5; 0–400 ng/mL, 24 h) was assessed using an MTT assay (n=4). (B) Representative immunofluorescence images of C2C12 myotubes stained for myosin heavy chain (MHC; green) and nuclei (DAPI; blue) under control (NC), PA (0.75 mM, 24 h), and PA+CCL5 (rCCL5 200 ng/mL+PA 0.75 mM, 24 h) conditions. Scale bar=200 μm. (C) Quantification of myotube diameter and frequency distribution analysis based on MHC-stained images. (D) Desmin protein expression under PA treatment and PA+CCL5 co-treatment conditions was analyzed by Western blotting and normalized to GAPDH (n=2). Data are presented as mean±SD. Statistical significance was determined using the Mann–Whitney U test, Kruskal–Wallis test, or one-sample t-test as appropriate. **p<.01 versus control.
rCCL5 significantly attenuated PA-induced myotube atrophy (Fig. 4B). Histogram analysis of myotube diameter distribution further demonstrated that the left-shifted distribution induced by PA treatment was partially restored toward the control distribution following rCCL5 cotreatment (Fig. 4C).
Given that the protective effects of CCL5 were not explained by classical proteostasis pathways, we next investigated cytoskeletal stability. Desmin, a muscle-specific intermediate filament protein, plays a critical role in maintaining structural integrity by linking myofibrils to the sarcolemma [32,33]. Desmin protein expression was markedly reduced following PA treatment. Notably, rCCL5 co-treatment partially restored desmin protein levels (Fig. 4D).
5. CCL5 does not restore proteostasis signaling pathways or inflammatory responses in PA-treated myotubes
To determine whether the morphological recovery induced by rCCL5 was linked to restoration of canonical proteostatic signaling, we examined pathways involved in protein synthesis, degradation, autophagy, inflammation, and apoptosis in PA-treated C2C12 myotubes.
Despite its effect on myotube morphology, rCCL5 co-treatment did not restore the expression of key myogenic markers at either the protein or transcript level, including MHC, Myogenin, and MyoD (Fig. 5A, B). In parallel, major signaling pathways regulating protein synthesis remained largely unaltered, as phosphorylation levels of AKT–mTOR axis components showed no meaningful recovery in response to rCCL5 (Fig. 5C).
CCL5 co-treatment does not restore proteostasis, MAPK signaling, inflammatory responses, or apoptosis pathways. (A) Protein expression of myogenic markers (MHC, Myogenin, and MyoD) was analyzed by Western blotting (MHC, MyoD: n=8; Myogenin: n=12). (B) mRNA expression of myogenic genes (Myh7, Myh4, Myh1, Myog, and Myod1) was measured by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR; n=4). Gene expression was normalized to Gapdh and calculated using the 2–ΔΔCT method. (C) Phosphorylation levels of proteins in the protein synthesis pathway (AKT, mTOR, Raptor, p70S6K, and 4E-BP1) were analyzed by Western blotting (n=6–8). (D) Phosphorylation of FoxO3a and expression of protein degradation markers (MuRF1, MAFbx, and ubiquitin) were analyzed by Western blotting. Phosphorylated proteins were normalized to total proteins, and other proteins were normalized to α-actinin (FoxO3a: n=6; ubiquitin/MuRF1: n=4; MAFbx: n=2). (E) mRNA expression of proteolysis-related genes (Trim63, Fbxo32, and Mstn) was analyzed by qRT-PCR (n=4). Gene expression was normalized to Gapdh and calculated using the 2–ΔΔCT method. (F) Autophagy markers p62 and LC3-I/II were analyzed by Western blotting. p62 was normalized to α-actinin, and LC3-II was normalized to LC3-I (n=4). (G) Phosphorylation levels of MAPK signaling proteins (p38 MAPK, ERK1/2, and JNK) were analyzed by Western blotting. Phosphorylated proteins were normalized to total proteins (ERK: n=2; p38: n=12; JNK: n=4). (H) mRNA expression of inflammatory cytokines (Il6, Tnf, and Il1b) was analyzed by qRT-PCR (n=4). Gene expression was normalized to Gapdh. (I) Apoptotic markers, including cleaved Caspase-3 and cleaved PARP, were analyzed by Western blotting. Cleaved proteins were normalized to their respective total proteins (n=10). Data are presented as mean±SD. Statistical analyses were performed using the Mann–Whitney U test, Kruskal–Wallis test, or one-sample t-test as appropriate. *p<.05, **p<.01, and ***p<.001 were considered statistically significant.
Likewise, markers associated with protein degradation were not substantially affected by rCCL5 treatment. The phosphorylation status of FoxO3a, as well as the expression of MuRF1, MAFbx, and ubiquitin, remained comparable to those observed under PA treatment alone (Fig. 5D, E). Consistent with this, autophagy-related markers, including p62 and LC3-II/I ratio, also showed no appreciable changes following rCCL5 co-treatment (Fig. 5F).
Furthermore, rCCL5 did not modulate MAPK signaling pathways or inflammatory responses, as phosphorylation levels of p38, ERK1/2, and JNK, along with the expression of pro-inflammatory cytokines (Il6, Tnf, and Il1b), remained largely unchanged (Fig. 5G, H). Similarly, apoptotic markers, including cleaved Caspase-3 and PARP, were not significantly altered by rCCL5 treatment (Fig. 5I).
6. CCL5 does not restore mitochondrial homeostasis under PA-induced stress
To determine whether CCL5-mediated effects extend to mitochondrial regulation, we examined key pathways involved in mitochondrial biogenesis, dynamics, redox balance, and bioenergetics under PA-induced stress in C2C12 myotubes.
rCCL5 co-treatment did not restore the AMPK–SIRT1–PGC-1α signaling axis, as phosphorylation of AMPK and expression levels of SIRT1 and PGC-1α remained largely unchanged compared with PA-treated cells (Fig. 6A). Consistently, protein levels of mitochondrial dynamics regulators (MFN1, MFN2, OPA1, DRP1, and FIS1) and mitophagy-related proteins (PINK1 and Parkin) were not substantially altered by rCCL5 (Fig. 6B).
Effects of CCL5 co-treatment on mitochondrial homeostasis under PA-induced stress. (A) Proteins involved in the AMPK–SIRT1–PGC-1α signaling pathway were analyzed by Western blotting. Phosphorylated proteins were normalized to total proteins, and other proteins were normalized to α-actinin (AMPK: n=8; SIRT1: n=6; PGC-1α: n=4). (B) Protein expression of mitochondrial dynamics regulators (MFN1, MFN2, OPA1, DRP1, and FIS1) and mitophagy- related proteins (PINK1 and Parkin) was analyzed by Western blotting and normalized to α-actinin (MFN1: n=6; OPA1/DRP1/FIS1: n=4; MFN2: n=2; PINK1/Parkin: n=8). (C) mRNA expression of redox and inflammation-related genes (Nfe2l2, Sod1, Sod2, Cat, Txn1, Cybb, Ptgs2, and Nos2) was analyzed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression was normalized to Gapdh (Sod1, Sod2, Cat: n=4; Nfe2l2, Txn1, Cybb, Ptgs2, Nos2: n=2). (D) mRNA expression of mitochondrial biogenesis-related genes (Ppargc1a, Nrf1, Tfam, Sco1, and Vdac1) was analyzed by qRT-PCR (n=2). Gene expression was normalized to Gapdh. (E) Mitochondrial membrane potential was visualized using MitoTrackerTM Red CMXRos staining. Representative fluorescence images and quantitative fluorescence intensity measured using Olympus cellSens software are shown. Scale bar=500 μm (n=12). (F) Intracellular ATP levels were measured using an ATP assay kit (n=4). Data are presented as mean±SD. Statistical analyses were performed using the Mann–Whitney U test, Kruskal–Wallis test, or one-sample t-test as appropriate. *p<.05, ***p<.001 were considered statistically significant.
At the transcriptional level, genes associated with redox regulation and inflammation, including Nfe2l2, Sod1, Sod2, Cat, Txn1, Cybb, Ptgs2, and Nos2, showed no consistent changes following rCCL5 co-treatment (Fig. 6C). Similarly, expression of mitochondrial biogenesis-related genes such as Ppargc1a, Nrf1, Tfam, Sco1, and Vdac1 remained largely unaffected (Fig. 6D).
Functionally, mitochondrial membrane potential, assessed by MitoTrackerTM staining, remained reduced in PA-treated myotubes and was not significantly recovered by rCCL5 co-treatment (Fig. 6E). In line with this, intracellular ATP levels also showed no appreciable restoration following rCCL5 treatment (Fig. 6F).
DISCUSSION
The present study investigated the functional role of the chemokine CCL5 in a PA-induced skeletal muscle atrophy model. The main findings are as follows. First, PA treatment reproduced key pathological features of sarcopenia in C2C12 myotubes, including suppression of myogenic signaling, inhibition of AKT–mTOR signaling, enhanced protein catabolism, mitochondrial dysfunction, and activation of apoptosis. Second, Ccl5 expression was consistently upregulated in both aged skeletal muscle and the PA-induced atrophy model, identifying it as a potential stress-responsive candidate gene associated with metabolic stress–related muscle remodeling. Third, exogenous CCL5 treatment partially attenuated myotube diameter reduction and restored the expression of the cytoskeletal protein desmin, despite having minimal effects on classical pathways regulating protein synthesis, degradation, or mitochondrial biogenesis. Together, these findings suggest that CCL5 may exert a protective effect against metabolic stress–induced muscle atrophy by maintaining cytoskeletal integrity rather than directly regulating proteostasis.
PA-induced lipotoxicity is widely used as an experimental model that mimics metabolic stress conditions associated with aging and obesity that are targets of exercise intervention. Previous studies have reported that PA treatment suppresses AKT signaling, activates FoxO-dependent E3 ubiquitin ligases, impairs mitochondrial function, and promotes inflammatory responses, ultimately leading to an atrophic phenotype [34, 35]. Consistent with these reports, the present study observed decreased AKT phosphorylation, increased ubiquitin expression, reduced mitochondrial membrane potential, and activation of apoptotic signaling following PA treatment. These findings support the validity of the PA-induced myotube model as an in vitro system for investigating mechanisms of metabolic stress–related muscle atrophy by maintaining cytoskeletal integrity rather than directly regulating classical anabolic and catabolic pathways.
Through integrative analysis of aged skeletal muscle transcriptomes and the PA-induced cellular model, CCL5 was identified as a gene consistently upregulated under both conditions. CCL5 is a CC chemokine that primarily interacts with the CCR5 receptor and is well known for its role in immune cell recruitment and inflammatory regulation. However, emerging evidence suggests that CCL5 is also expressed in skeletal muscle and may participate in muscle regeneration, tissue remodeling, and cellular stress responses. The increased expression of Ccl5 observed in aged muscle and PA-treated myotubes may therefore represent a compensatory response to metabolic or structural stress conditions rather than a direct regulator of anabolic signaling.
Interestingly, exogenous CCL5 treatment partially restored myotube diameter but did not significantly alter canonical signaling pathways associated with muscle atrophy. This negative finding suggests that the protective effects of CCL5 may occur independently of traditional proteostasis- regulating mechanisms. An important observation of the present study is that CCL5-mediated protection was primarily associated with restoration of desmin expression, rather than recovery of protein synthesis pathways, mitochondrial biogenesis, or inflammatory signaling.
Desmin is a muscle-specific intermediate filament protein that plays a critical role in maintaining the structural integrity of muscle fibers by linking sarcomeres to the sarcolemma [33]. Genetic deletion of desmin in mice results in severe disruption of myofibrillar organization and impaired muscle function [32]. In the present study, PA treatment markedly reduced desmin expression, whereas CCL5 co-treatment partially restored its levels. These findings suggest that preservation of desmin expression may contribute to maintenance of cytoskeletal integrity under lipotoxic stress conditions.
Importantly, the present findings indicate that maintenance of cytoskeletal architecture may represent an independent protective axis in skeletal muscle under metabolic stress. Unlike classical anti-atrophy mechanisms that primarily target protein synthesis or degradation pathways, preservation of intermediate filament organization may delay structural destabilization and maintain myofiber integrity during prolonged lipotoxic stress.
Although CCL5 treatment did not restore the expression of contractile proteins such as MHC or Myogenin, preservation of cytoskeletal integrity may still provide important physiological benefits. Maintenance of the structural framework of myofibers may delay morphological deterioration and preserve the physical scaffold required for subsequent functional recovery once metabolic stress is relieved. In this context, CCL5 may function as a structural protective factor rather than a direct anabolic regulator in skeletal muscle.
The mitochondrial analyses performed in this study further support the notion that the protective effects of CCL5 are largely independent of mitochondrial recovery pathways. Although CCL5 treatment showed a modest tendency to preserve mitochondrial membrane potential, it did not significantly increase total intracellular ATP levels. One possible explanation is that CCL5 may preserve mitochondrial structural integrity and delay membrane depolarization, but does not restore oxidative phosphorylation once it has been severely impaired by PA-induced lipotoxicity [36]. Nevertheless, partial preservation of mitochondrial membrane potential may still help limit excessive activation of mitochondria-mediated apoptotic pathways.
Taken together, CCL5 appears to function as a context-dependent regulator in skeletal muscle, with its role diverging between physiological adaptation and chronic pathological stress. Rather than acting as a simple pro-inflammatory mediator, CCL5 is increasingly recognized as part of a dynamic immune signaling network associated with metabolic stress and tissue remodeling. In this context, sustained upregulation of Ccl5 under conditions such as lipotoxicity, sedentary aging, or metabolic dysfunction may reflect an initially compensatory response aimed at preserving muscle integrity, but one that becomes maladaptive when persistently activated. This duality suggests that the biological significance of CCL5 cannot be defined solely by its expression level, but rather by the temporal and physiological context in which it is regulated, highlighting its potential role as a key mediator linking inflammatory signaling, muscle adaptation, and sarcopenic progression [39,40].
This study has several limitations. First, the present findings are based on in vitro experiments and therefore require validation in aged animal models. Second, the direct role of endogenous CCL5 was not evaluated under CCL5-deficient conditions. Third, whether the restoration of desmin expression is mediated specifically through CCR5 signaling remains to be determined. Future studies combining in vivo validation with pharmacological inhibition of CCR5 or Rho/ROCK signaling will help clarify the mechanistic role of CCL5 in skeletal muscle structural maintenance under metabolic stress conditions.
CONCLUSION
In summary, this study establishes a PA-induced lipotoxic myotube atrophy model and identifies Ccl5 as a stress-responsive factor consistently upregulated in both aged skeletal muscle and metabolically stressed myotubes. Functional analyses revealed that exogenous CCL5 partially attenuates myotube atrophy without restoring canonical proteostasis pathways. Instead, CCL5 preserved cytoskeletal integrity through restoration of the intermediate filament protein desmin and modest stabilization of mitochondrial membrane potential. These findings suggest that CCL5 functions as a structural protective factor that delays morphological muscle atrophy by maintaining cytoskeletal stability under lipotoxic stress. However, further in vivo validation and mechanistic studies are required to support this previously unrecognized role of CCL5 in skeletal muscle homeostasis and cytoskeletal stabilization.
Notes
ACKNOWLEDGMENTS
This work was supported by the Ministry of Education of the Republic of Korea and National Research Foundation of Korea (NRF-2022S1A5C2A03092407).
CONFLICT OF INTEREST
It is stated that no financial or any other support has been received from any organization in the writing of this paper, and that there are no conflicts of interest.
AUTHOR CONTRIBUTIONS
Conceptualization: So CY, S Kim; Data curation: S Moon, KEK Mantik; Formal analysis: S Moon, Pali DV; Funding acquisition: JH Kang, Kwak HB; Methodology: S Moon, KEK Mantik; Project administration: JH Kang, DH Park; Visualization: So CY, S Kim; Writing - original draft: So CY; Writing - review & editing: JH Kang, S Kim. All authors have read and approved the final manuscript.