ECM components are essential for proper in vitro myogenesis

Ugur Akpulat

doi:10.58600/eurjther2260

Authors

Ugur Akpulat Department of Medical Biology, Faculty of Medicine, Kastamonu University, Kastamonu, Türkiye https://orcid.org/0000-0001-8126-8209

DOI:

https://doi.org/10.58600/eurjther2260

Keywords:

Myogenesis, fibronectin, laminin, matrigel, gelatin, poly-l-lysine

Abstract

Objective: In vitro models of skeletal muscle often utilize primary myoblast cells or myoblast cell lines. Myoblasts require adhesion to the extracellular matrix (ECM) to grow, proliferate, migrate, and differentiate in their natural environments in vivo. To meet the adhesion needs of adhesive cells under in vitro conditions, culture surfaces are coated with various biological or synthetic compounds. Within the scope of the study, the differentiation potential of H2K myoblasts, a cell line resembling primary myoblasts, were comparatively evaluated through morphological analysis on culture surfaces coated with various ECM and synthetic materials.

Methods: The culture surfaces were coated with fibronectin and laminin, the major adhesion proteins of ECM; gelatin, a molecular derivative of collagen; matrigel, an ECM extract; and PLL, a synthetic poly-amino acid. Cells were allowed to differentiate in each culture medium for 4 days and their capacity to adhere to the surface and differentiation rates from myoblast to myotube were evaluated by morphological analysis.

Results: In the uncoated culture environment, cells could only attach to 30-50% of the culture surface and myotube development was limited and not aligned with each other. On surfaces coated with PLL, no myotube development was observed and cells could only attach to 30-40% of the culture surface. Myotube development and alignment were similar on all surfaces coated with ECM components. On surfaces coated with ECM components laminin, fibronectin and matrigel, cells covered the entire culture surface and exhibited similar myotube development. However, on surfaces coated with gelatin, both cell adhesion to the surface and myotube development were limited compared to other ECM components. The mean myotube diameters of fibronectin, laminin, matrigel, PLL+laminin and gelatin were 49.71µm (±16.3µm), 52.31µm (±15.7µm), 51.9µm (±15.3µm), 53.06µm (±14.2µm) and 35.25µm (±11.4µm), respectively.

Conclusion: Within the scope of the study, it was revealed that coating the culture surface with only a cationic material such as PLL does not support myogenesis and ECM components are needed for cell viability and differentiation.

Metrics

Metrics Loading ...

References

Brooks SV, Guzman SD, Ruiz LP (2023) Skeletal muscle structure, physiology, and function. Handb Clin Neurol 195:3-16. https://doi.org/10.1016/B978-0-323-98818-6.00013-3

Frontera WR, Ochala J (2015) Skeletal muscle: a brief review of structure and function. Calcif Tissue Int 96:183-195. https://doi.org/10.1007/s00223-014-9915-y

Lieber RL, Friden J (2000) Functional and clinical significance of skeletal muscle architecture. Muscle Nerve 23:1647-1666. https://doi.org/10.1002/1097-4598(200011)23:11<1647::aid-mus1>3.0.co;2-m

Mukund K, Subramaniam S (2020) Skeletal muscle: A review of molecular structure and function, in health and disease. Wiley Interdiscip Rev Syst Biol Med 12:e1462. https://doi.org/10.1002/wsbm.1462

Argiles JM, Campos N, Lopez-Pedrosa JM, Rueda R, Rodriguez-Manas L (2016) Skeletal Muscle Regulates Metabolism via Interorgan Crosstalk: Roles in Health and Disease. J Am Med Dir Assoc 17:789-796. https://doi.org/10.1016/j.jamda.2016.04.019

Westerblad H, Bruton JD, Katz A (2010) Skeletal muscle: energy metabolism, fiber types, fatigue and adaptability. Exp Cell Res 316:3093-3099. https://doi.org/10.1016/j.yexcr.2010.05.019

Lieber RL, Ward SR (2011) Skeletal muscle design to meet functional demands. Philos Trans R Soc Lond B Biol Sci 366:1466-1476. https://doi.org/10.1098/rstb.2010.0316

Chang M, Cai Y, Gao Z, Chen X, Liu B, Zhang C, Yu W, Cao Q, Shen Y, Yao X, Chen X, Sun H (2023) Duchenne muscular dystrophy: pathogenesis and promising therapies. J Neurol 270:3733-3749. https://doi.org/10.1007/s00415-023-11796-x

Shefner JM, Musaro A, Ngo ST, Lunetta C, Steyn FJ, Robitaille R, De Carvalho M, Rutkove S, Ludolph AC, Dupuis L (2023) Skeletal muscle in amyotrophic lateral sclerosis. Brain 146:4425-4436. https://doi.org/10.1093/brain/awad202

Cruz-Jentoft AJ, Bahat G, Bauer J, Boirie Y, Bruyere O, Cederholm T, Cooper C, Landi F, Rolland Y, Sayer AA, Schneider SM, Sieber CC, Topinkova E, Vandewoude M, Visser M, Zamboni M, Writing Group for the European Working Group on Sarcopenia in Older P, the Extended Group for E (2019) Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing 48:16-31. https://doi.org/10.1093/ageing/afy169

Martin A, Gallot YS, Freyssenet D (2023) Molecular mechanisms of cancer cachexia-related loss of skeletal muscle mass: data analysis from preclinical and clinical studies. J Cachexia Sarcopenia Muscle 14:1150-1167. https://doi.org/10.1002/jcsm.13073

Espino-Gonzalez E, Dalbram E, Mounier R, Gondin J, Farup J, Jessen N, Treebak JT (2024) Impaired skeletal muscle regeneration in diabetes: From cellular and molecular mechanisms to novel treatments. Cell Metab 36:1204-1236. https://doi.org/10.1016/j.cmet.2024.02.014

Romagnoli C, Iantomasi T, Brandi ML (2021) Available In Vitro Models for Human Satellite Cells from Skeletal Muscle. Int J Mol Sci 22. https://doi.org/10.3390/ijms222413221

Carter S, Solomon TPJ (2019) In vitro experimental models for examining the skeletal muscle cell biology of exercise: the possibilities, challenges and future developments. Pflugers Arch 471:413-429. https://doi.org/10.1007/s00424-018-2210-4

Chal J, Pourquie O (2017) Making muscle: skeletal myogenesis in vivo and in vitro. Development 144:2104-2122. https://doi.org/10.1242/dev.151035

Taylor L, Wankell M, Saxena P, McFarlane C, Hebbard L (2022) Cell adhesion an important determinant of myogenesis and satellite cell activity. Biochim Biophys Acta Mol Cell Res 1869:119170. https://doi.org/10.1016/j.bbamcr.2021.119170

Burkin DJ, Kaufman SJ (1999) The alpha7beta1 integrin in muscle development and disease. Cell Tissue Res 296:183-190. https://doi.org/10.1007/s004410051279

Cooke MJ, Phillips SR, Shah DS, Athey D, Lakey JH, Przyborski SA (2008) Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins. Cytotechnology 56:71-79. https://doi.org/10.1007/s10616-007-9119-7

Wysotzki P, Gimsa J (2019) Surface Coatings Modulate the Differences in the Adhesion Forces of Eukaryotic and Prokaryotic Cells as Detected by Single Cell Force Microscopy. Int J Biomater 2019:7024259. https://doi.org/10.1155/2019/7024259

Morgan JE, Beauchamp JR, Pagel CN, Peckham M, Ataliotis P, Jat PS, Noble MD, Farmer K, Partridge TA (1994) Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines. Dev Biol 162:486-498. https://doi.org/10.1006/dbio.1994.1103

Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, Kioussis D (1991) Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci U S A 88:5096-5100. https://doi.org/10.1073/pnas.88.12.5096

Thorsteinsdottir S, Deries M, Cachaco AS, Bajanca F (2011) The extracellular matrix dimension of skeletal muscle development. Dev Biol 354:191-207. https://doi.org/10.1016/j.ydbio.2011.03.015

Mayer U (2003) Integrins: redundant or important players in skeletal muscle? J Biol Chem 278:14587-14590. https://doi.org/10.1074/jbc.R200022200

Boppart MD, Mahmassani ZS (2019) Integrin signaling: linking mechanical stimulation to skeletal muscle hypertrophy. Am J Physiol Cell Physiol 317:C629-C641. https://doi.org/10.1152/ajpcell.00009.2019

Crawley S, Farrell EM, Wang W, Gu M, Huang HY, Huynh V, Hodges BL, Cooper DN, Kaufman SJ (1997) The alpha7beta1 integrin mediates adhesion and migration of skeletal myoblasts on laminin. Exp Cell Res 235:274-286. https://doi.org/10.1006/excr.1997.3671

Cachaco AS, Pereira CS, Pardal RG, Bajanca F, Thorsteinsdottir S (2005) Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse. Dev Dyn 232:1069-1078. https://doi.org/10.1002/dvdy.20280

Gullberg D, Tiger CF, Velling T (1999) Laminins during muscle development and in muscular dystrophies. Cell Mol Life Sci 56:442-460. https://doi.org/10.1007/pl00000616

Shibuya S, Wakayama Y, Inoue M, Kojima H, Oniki H (2003) Merosin (laminin-2) localization in basal lamina of normal skeletal muscle fibers and changes in plasma membrane of merosin-deficient skeletal muscle fibers. Med Electron Microsc 36:213-220. https://doi.org/10.1007/s00795-003-0227-y

Yurchenco PD, McKee KK, Reinhard JR, Ruegg MA (2018) Laminin-deficient muscular dystrophy: Molecular pathogenesis and structural repair strategies. Matrix Biol 71-72:174-187. https://doi.org/10.1016/j.matbio.2017.11.009

Snow CJ, Peterson MT, Khalil A, Henry CA (2008) Muscle development is disrupted in zebrafish embryos deficient for fibronectin. Dev Dyn 237:2542-2553. https://doi.org/10.1002/dvdy.21670

Hughes CS, Postovit LM, Lajoie GA (2010) Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 10:1886-1890. https://doi.org/10.1002/pmic.200900758

Wohlgemuth RP, Brashear SE, Smith LR (2023) Alignment, cross linking, and beyond: a collagen architect's guide to the skeletal muscle extracellular matrix. Am J Physiol Cell Physiol 325:C1017-C1030. https://doi.org/10.1152/ajpcell.00287.2023

Kiss AA, Somlyai-Popovics N, Kiss M, Boldogkoi Z, Csiszar K, Mink M (2019) Type IV Collagen Is Essential for Proper Function of Integrin-Mediated Adhesion in Drosophila Muscle Fibers. Int J Mol Sci 20. https://doi.org/10.3390/ijms20205124