Effect of CCL11 on In Vitro Myogenesis and Its Clinical Relevance for Sarcopenia in Older Adults

Abstract

Background: The C-C motif chemokine ligand 11 (CCL11) has been receiving attention as a potential pro-aging factor. Accordingly, it may be involved in muscle metabolism and sarcopenia, a key component of aging phenotypes. To clarify this potential, we investigated the effects of CCL11 on in vitro muscle biology and its clinical relevance for sarcopenia parameters in older adults.

Methods: Myogenesis was induced in mouse C2C12 myoblasts with 2% horse serum. Human blood samples were collected from 79 participants who underwent a functional assessment. Thereafter, CCL11 level was measured using a quantikine ELISA kit. Sarcopenia was defined using the Asian-specific guideline.

Results: Recombinant CCL11 treatment significantly stimulated myogenesis in a dose-dependent manner, and consistently increased the expression of myogenic differentiation markers. Among the C-C chemokine receptors (CCRs), CCR5, not CCR2 and CCR3, was predominantly expressed in muscle cells. Further, the CCR5 inhibitor blocked recombinant CCL11-stimulated myogenesis. In a clinical study, serum CCL11 level was not significantly different according to the status of sarcopenia, low muscle mass, weak muscle strength, and poor physical performance, and was not associated with skeletal muscle index, grip strength, short physical performance battery score, gait speed, and time to complete 5 chair stands, after adjusting for sex, age, and body mass index.

Conclusion: Contrary to expectations, CCL11 exerted beneficial effects on muscle metabolism at least in vitro system. However, its impact on human muscle health was not evident, suggesting that circulating CCL11 may not be a useful biomarker for sarcopenia risk assessment in older adults.

Keywords: Aging; Biomarkers; Chemokine CCL11; Muscle development; Sarcopenia.

Fig. 1

Recombinant C-C motif chemokine ligand 11 (CCL11) stimulates in vitro myogenesis. (A) Mouse C2C12 myoblasts (MBs) were differentiated into myotubes (MTs) with 2% horse serum after exposure to the indicated concentrations of recombinant CCL11 for 4 days. MTs were stained with the anti-myosin heavy chain (MyHC) antibody while the nuclei were counterstained with 4,6-diamidino-2-phenyindole (DAPI). Quantitative results per field are presented. Scale bars, 100 μm. (B) Western blot and (C) quantitative reverse-transcription polymerase chain reaction analyses of myogenin and MyHC in C2C12 cells without or with 2% horse serum in the presence of the indicated concentrations of recombinant CCL11 for 3 days, respectively. Data are expressed as mean±standard error of mean. ^aP<0.05 vs. control or MB; ^bP<0.05 vs. CCL11 1 nM or untreated MT.

Fig. 2

C-C chemokine receptor 5 (CCR5) is the major receptor for C-C motif chemokine ligand 11 (CCL11) in muscle cells. (A) Mouse C2C12 myoblasts (MBs) were differentiated into myotubes with 2% horse serum for the indicated days. Western blot analysis was performed to determine the expression of myogenin, myosin heavy chain (MyHC), and CCR5. (B) Interaction between CCL11 and CCR5 in C2C12 MBs using binding enzyme-linked immunosorbent assay (ELISA). Varying amounts of cell lysates were incubated in CCL11- or bovine serum albumin (BSA)-coated wells. Thereafter, the level of CCR5 was determined by ELISA. (C) Mouse C2C12 MBs were differentiated into myotubes with 2% horse serum in the presence or absence of recombinant CCL11 and/or CCR5 inhibitor for 4 days. Myotubes were stained with the anti-MyHC antibody while the nuclei were counterstained with 4,6-diamidino-2-phenyindole (DAPI). Quantitative results per field are presented. Scale bars, 100 μm. Data are expressed as mean±standard error of mean. OD, optical density. ^aP<0.05 vs. control.

Fig. 3

Differences in serum C-C motif chemokine ligand 11 (CCL11) levels based on the status of sarcopenia and related parameters (A) before and (B) after adjusting for sex, age, and body mass index (BMI). Analysis of covariance was used to generate and compare the estimated means with 95% confidence intervals.

Fig. 4

Differences in the sarcopenia parameters based on the serum C-C motif chemokine ligand 11 (CCL11) quartiles (A) before and (B) after adjusting for sex, age, and body mass index (BMI). Analysis of covariance was used to generate and compare the estimated means with 95% confidence intervals. Serum CCL11 quartiles: Q1, 46–115 pg/mL; Q2, 116–155 pg/mL; Q3, 156–195 pg/mL; and Q4, 196–327 pg/mL. SMI, skeletal muscle mass index; SPPB, short physical performance battery.