Mechanical loading of bioengineered skeletal muscle in-vitro to mimic the transcriptional and epigenetic regulation of skeletal muscle in-vivo

Turner, Daniel Colin

Abstract

Skeletal muscle (SkM) is an extremely abundant and mechano-sensitive tissue, demonstrating increases in mass and function following mechanical loading/resistance exercise (RE). Over the past 3 decades, there has been considerable progress in the development of bioengineered SkM models for elucidating the cellular and molecular mechanisms underpinning load-induced SkM adaptation in vitro. However, the majority of studies often employ loading regimes which lack resemblance of intermittent eccentric-lengthening contraction experienced during RE in-vivo. As the field of SkM epigenetics has only more recently begun to emerge, there is a paucity of data surrounding the epigenetic regulation of mechanical loading in bioengineered SkM. The work conducted herein therefore intermittently loaded bioengineered fibrin SkM in-vitro to determine whether mechanical loading alone recapitulates both the transcriptional and epigenetic responses in SkM following loading/RE in-vivo. The initial experiments (chapter 3) first characterised the use of a novel bioreactor system (i.e. which has never previously been used to bioengineered SkM) for loading bioengineered SkM which demonstrated a comparable mechano-transcriptional response of candidate genes that were differentially regulated after loading in previous well-characterised bioreactors. In order to identify appropriate genes to analyse in response to loading in bioengineered SkM, the most frequently regulated genes across both the human transcriptome and methylome after acute RE in-vivo were determined in chapter 4. Indeed, extensive bioinformatics analysis revealed a number of transcriptionally and epigenetically regulated genes in human SkM that were subsequently analysed at the mRNA and DNA methylation level following acute mechanical loading in fibrin engineered SkM (chapter 5). Despite few changes observed in DNA methylation, mechanical loading alone induced similar changes in gene expression compared to loading/RE in human and rodent SkM in-vivo. Amongst the array of genes analysed, UBR5 demonstrated the largest increase in mRNA expression across all models of loading. Therefore, the final experimental chapter of this thesis (chapter 6) wished to elucidate the mechanistic role of UBR5 after mechanical loading in human myotubes. Most interestingly, mechanical loading was able to rescue the siRNA-induced reduction in UBR5 gene expression, further suggesting a pivotal role in load-induced adaptation.
Overall, the present thesis suggests that mechanical loading of C2C12 bioengineered fibrin muscle is a useful in-vitro model for investigating the transcriptional response to loading in-vivo. Therefore, providing a representative model for investigating the molecular mechanisms underpinning load-induce SkM adaptation. However, the application of repeated/chronic loading, with or without electrical stimulation may be necessary to evoke epigenetic alterations that are comparable to those observed following loading/RE in-vivo.

Citation

Turner, D. C. Mechanical loading of bioengineered skeletal muscle in-vitro to mimic the transcriptional and epigenetic regulation of skeletal muscle in-vivo. (Thesis). Keele University. https://keele-repository.worktribe.com/output/417183

• Thesis Type: Thesis
• Publicly Available Date: May 26, 2023
• Public URL: https://keele-repository.worktribe.com/output/417183
• Award Date: 2020-06