Endocrinol Metab.  2021 Apr;36(2):455-465. 10.3803/EnM.2020.942.

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

• Kim DA
• Park SJ
• Lee JY
• Kim JH
• Lee S
• Lee E
• Jang IY
• Jung HW
• Park JH
• Kim BJ

• KMID: 2515471
• DOI: http://doi.org/10.3803/EnM.2020.942

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.

Keyword

Chemokine CCL11; Muscle development; Sarcopenia; Aging; Biomarkers

Figure

• 

  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.

Reference

1. Moretto J, Girard C, Demougeot C. The role of arginase in aging: a systematic review. Exp Gerontol. 2019; 116:54–73.

2. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013; 153:1194–217.

3. Hou Y, Dan X, Babbar M, Wei Y, Hasselbalch SG, Croteau DL, et al. Ageing as a risk factor for neurodegenerative disease. Nat Rev Neurol. 2019; 15:565–81.

4. Niccoli T, Partridge L. Ageing as a risk factor for disease. Curr Biol. 2012; 22:R741–52.

5. Schosserer M, Grillari J, Wolfrum C, Scheideler M. Age-induced changes in white, brite, and brown adipose depots: a mini-review. Gerontology. 2018; 64:229–36.

6. Barja G. Towards a unified mechanistic theory of aging. Exp Gerontol. 2019; 124:110627.

7. Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, et al. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science. 2014; 344:630–4.

8. Villeda SA, Luo J, Mosher KI, Zou B, Britschgi M, Bieri G, et al. The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature. 2011; 477:90–4.

9. Laviano A. Young blood. N Engl J Med. 2014; 371:573–5.

10. Villeda SA, Plambeck KE, Middeldorp J, Castellano JM, Mosher KI, Luo J, et al. Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nat Med. 2014; 20:659–63.

11. Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, et al. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 2013; 153:828–39.

12. Rebo J, Mehdipour M, Gathwala R, Causey K, Liu Y, Conboy MJ, et al. A single heterochronic blood exchange reveals rapid inhibition of multiple tissues by old blood. Nat Commun. 2016; 7:13363.

13. Kim BJ, Lee SH, Koh JM. Clinical insights into the kynurenine pathway in age-related diseases. Exp Gerontol. 2020; 130:110793.

14. Choi KM. Sarcopenia and sarcopenic obesity. Endocrinol Metab (Seoul). 2013; 28:86–9.

15. Kim TN, Choi KM. Sarcopenia: definition, epidemiology, and pathophysiology. J Bone Metab. 2013; 20:1–10.

16. Kim G, Kim JH. Impact of skeletal muscle mass on metabolic health. Endocrinol Metab (Seoul). 2020; 35:1–6.

17. Cruz-Jentoft AJ, Bahat G, Bauer J, Boirie Y, Bruyere O, Cederholm T, et al. Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing. 2019; 48:16–31.

18. Cruz-Jentoft AJ, Sayer AA. Sarcopenia. Lancet. 2019; 393:2636–46.

19. Dao T, Green AE, Kim YA, Bae SJ, Ha KT, Gariani K, et al. Sarcopenia and muscle aging: a brief overview. Endocrinol Metab (Seoul). 2020; 35:716–32.

20. Lim HS, Park YH, Suh K, Yoo MH, Park HK, Kim HJ, et al. Association between sarcopenia, sarcopenic obesity, and chronic disease in Korean elderly. J Bone Metab. 2018; 25:187–93.

21. Ponath PD, Qin S, Post TW, Wang J, Wu L, Gerard NP, et al. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J Exp Med. 1996; 183:2437–48.

22. Garcia-Zepeda EA, Rothenberg ME, Ownbey RT, Celestin J, Leder P, Luster AD. Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat Med. 1996; 2:449–56.

23. Jose PJ, Griffiths-Johnson DA, Collins PD, Walsh DT, Moqbel R, Totty NF, et al. Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation. J Exp Med. 1994; 179:881–7.

24. Kindstedt E, Holm CK, Sulniute R, Martinez-Carrasco I, Lundmark R, Lundberg P. CCL11, a novel mediator of inflammatory bone resorption. Sci Rep. 2017; 7:5334.

25. Lee JY, Park SJ, Kim DA, Lee SH, Koh JM, Kim BJ. Muscle-derived lumican stimulates bone formation via integrin α2β1 and the downstream ERK signal. Front Cell Dev Biol. 2020; 8:565826.

26. Kim BJ, Lee YS, Lee SY, Baek WY, Choi YJ, Moon SA, et al. Osteoclast-secreted SLIT3 coordinates bone resorption and formation. J Clin Invest. 2018; 128:1429–41.

27. Park SJ, Lee JY, Lee SH, Koh JM, Kim BJ. SLIT2 inhibits osteoclastogenesis and bone resorption by suppression of Cdc42 activity. Biochem Biophys Res Commun. 2019; 514:868–74.

28. Jang IY, Lee S, Kim JH, Lee E, Lee JY, Park SJ, et al. Lack of association between circulating apelin level and frailty-related functional parameters in older adults: a cross-sectional study. BMC Geriatr. 2020; 20:420.

29. Roberts HC, Denison HJ, Martin HJ, Patel HP, Syddall H, Cooper C, et al. A review of the measurement of grip strength in clinical and epidemiological studies: towards a standardised approach. Age Ageing. 2011; 40:423–9.

30. Peel NM, Kuys SS, Klein K. Gait speed as a measure in geriatric assessment in clinical settings: a systematic review. J Gerontol A Biol Sci Med Sci. 2013; 68:39–46.

31. Jung HW, Roh H, Cho Y, Jeong J, Shin YS, Lim JY, et al. Validation of a multi-sensor-based kiosk for short physical performance battery. J Am Geriatr Soc. 2019; 67:2605–9.

32. Oh JH, Song S, Rhee H, Lee SH, Kim DY, Choe JC, et al. Normal reference plots for the bioelectrical impedance vector in healthy Korean adults. J Korean Med Sci. 2019; 34:e198.

33. Jang IY, Jung HW, Lee CK, Yu SS, Lee YS, Lee E. Comparisons of predictive values of sarcopenia with different muscle mass indices in Korean rural older adults: a longitudinal analysis of the Aging Study of PyeongChang Rural Area. Clin Interv Aging. 2018; 13:91–9.

34. Chen LK, Woo J, Assantachai P, Auyeung TW, Chou MY, Iijima K, et al. Asian working group for sarcopenia: 2019 consensus update on sarcopenia diagnosis and treatment. J Am Med Dir Assoc. 2020; 21:300–7.

35. Kitaura M, Nakajima T, Imai T, Harada S, Combadiere C, Tiffany HL, et al. Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J Biol Chem. 1996; 271:7725–30.

36. Ogilvie P, Bardi G, Clark-Lewis I, Baggiolini M, Uguccioni M. Eotaxin is a natural antagonist for CCR2 and an agonist for CCR5. Blood. 2001; 97:1920–4.

37. Boyle PA, Buchman AS, Wilson RS, Leurgans SE, Bennett DA. Association of muscle strength with the risk of Alzheimer disease and the rate of cognitive decline in community-dwelling older persons. Arch Neurol. 2009; 66:1339–44.

38. Gray SL, Anderson ML, Hubbard RA, LaCroix A, Crane PK, McCormick W, et al. Frailty and incident dementia. J Gerontol A Biol Sci Med Sci. 2013; 68:1083–90.

39. Loskutova N, Honea RA, Vidoni ED, Brooks WM, Burns JM. Bone density and brain atrophy in early Alzheimer’s disease. J Alzheimers Dis. 2009; 18:777–85.

40. Ye J, Kohli LL, Stone MJ. Characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor CCR3. J Biol Chem. 2000; 275:27250–7.

41. Alblowi J, Tian C, Siqueira MF, Kayal RA, McKenzie E, Behl Y, et al. Chemokine expression is upregulated in chondrocytes in diabetic fracture healing. Bone. 2013; 53:294–300.