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Bleomycin-treated myoblasts undergo p21-associated cellular senescence and have severely impaired differentiation

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Abstract

As we age, the ability to regenerate and repair skeletal muscle damage declines, partially due to increasing dysfunction of muscle resident stem cells—satellite cells (SC). Recent evidence implicates cellular senescence, which is the irreversible arrest of proliferation, as a potentiator of SC impairment during aging. However, little is known about the role of senescence in SC, and there is a large discrepancy in senescence classification within skeletal muscle. The purpose of this study was to develop a model of senescence in skeletal muscle myoblasts and identify how common senescence-associated biomarkers respond. Low-passage C2C12 myoblasts were treated with bleomycin or vehicle and then evaluated for cytological and molecular senescence markers, proliferation status, cell cycle kinetics, and differentiation potential. Bleomycin treatment caused double-stranded DNA breaks, which upregulated p21 mRNA and protein, potentially through NF-κB and senescence-associated super enhancer (SASE) signaling (p < 0.01). Consequently, cell proliferation was abruptly halted due to G2/M-phase arrest (p < 0.01). Bleomycin-treated myoblasts displayed greater senescence-associated β-galactosidase staining (p < 0.01), which increased over several days. These myoblasts remained senescent following 6 days of differentiation and had significant impairments in myotube formation (p < 0.01). Furthermore, our results show that senescence can be maintained despite the lack of p16 gene expression in C2C12 myoblasts. In conclusion, bleomycin treatment provides a valid model of damage-induced senescence that was associated with elevated p21, reduced myoblast proliferation, and aberrant cell cycle kinetics, while confirming that a multi-marker approach is needed for the accurate classification of senescence within skeletal muscle.

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Acknowledgements

The authors would like to thank Todd Prior and Linda May (McMaster University) for their technical assistance during laboratory analysis. Figure graphics were created with BioRender.com.

Funding

M.K. was supported in part by the Natural Sciences and Engineering Research Council (NSERC) of Canada post-graduate scholarship. G.P was supported by the Natural Sciences and Engineering Research Council (NSERC) Discovery award.

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  1. Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, ON, Canada

    Michael Kamal, Sophie Joanisse & Gianni Parise

  2. Department of Sport and Exercise Sciences, Musculoskeletal Science and Sport Medicine Research Centre, Institute of Sport, Manchester Metropolitan University, Manchester, UK

    Sophie Joanisse

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MK, SJ, and GP were involved in the conception and design of the study. MK conducted data acquisition and analysis. MK, SJ, and GP were involved in data interpretation. MK drafted the manuscript. All authors read and approved the final version.

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Kamal, M., Joanisse, S. & Parise, G. Bleomycin-treated myoblasts undergo p21-associated cellular senescence and have severely impaired differentiation. GeroScience 46, 1843–1859 (2024). https://doi.org/10.1007/s11357-023-00929-9

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