Abstract
Disuse atrophy is a secondary complication that often exacerbates the aetiology of injury and chronic disease. Identifying changes in mechanisms that control muscle mass is necessary to characterise atrophy and maintain a healthy functional capacity. This thesis aimed to use transcriptome- and epigenome-wide analysis to study gene expression and its regulation in human skeletal muscle following short-term limb immobilisation. In addition, transcriptome-wide analysis was used to characterise gene expression in a bi-directional, selectively bred rat model for high responders (HRT) and low responders (LRT) to aerobic exercise.In the rat study female LRT and HRT rats underwent a 3-day hindlimb casting protocol, with matched line controls. Following hindlimb unloading plantaris and soleus muscles were excised, and RNA extracted for transcriptome-wide RNA sequencing. LRT rats have previously been shown to be susceptible to metabolic disease, while HRT rats show a similar risk profile to other rat strains. After immobilisation, plantaris muscle mass was reduced in both LRT and HRT, however the change was only statistically significant in LRT. Soleus muscle mass was also reduced in LRT but failed to reach statistical significance. Transcriptomic differences between the two lines were analysed, with differences observed between well-established biological processes such as protein ubiquitination, and newly identified processes including extracellular matrix organisation, chromatin remodeling and DNA replication. Analysis of individual genes revealed the atrogenes, Trim63 and Fbxo32, were upregulated in plantaris muscle in both LRT and HRT, but unchanged in soleus muscle. The results presented support the hypothesis that a reduced metabolic state, like the one seen in the LRT line, negatively impacts cellular ability to regulate muscle mass during periods of unloading.
The human study employed twenty-one healthy male participants (20-45 years) who completed 4 weeks of standardised physical activity prior to a 14-day limb (left leg) immobilisation period with dietary control. Skeletal muscle biopsies were collected from the m. vastus lateralis before and after 3 days and 14 days of immobilisation. Skeletal muscle RNA and DNA were isolated and analysed using Illumina RNA sequencing and DNA methylation 850K EPIC BeadChips, respectively. Strength testing, DEXA and MRI were also performed pre- and post-immobilisation. Strength (-16.4%), lean mass (-3.2%) and quadricep cross-sectional area (-7.6%) were all significantly reduced after 14 days.
RNA sequencing analysis (17,034 genes) revealed positive enrichment of protein ubiquitination biological processes after 3 days, which returned to baseline after 14 days. Translation processes were also positively enriched after 3 days, however this appeared to be a transient response as they were negatively enriched after 14 days. Other important biological processes identified included autophagy, ion transport, rRNA processing, and chromatin remodelling. Cell deconvolution analysis was used to estimate cell type composition and proportions in the skeletal muscle samples. At Day 3 there was an increase in cell signatures associated with satellite cells and a decrease in signatures associated with pericyte cells. After 14 days there was a significant decrease in signatures related to Type IIa muscle fiber proportions, whilst Type I muscle fiber signatures were unchanged. Differential methylation analysis (750,000 CpG probes) assessed genomic (relative to CpG island) and genic (promoter, gene body etc.) location to infer their biological significance. This characterisation identified differential methylation at CpG rich sites (islands) and changes in promoter methylation at both time points. Further analysis focused solely on promoter methylation (18,740 genes), due to the well-known association with gene repression. More significant biological processes were identified after 3 days compared to 14 days, which indicates promoter methylation may be only a short-term regulator of gene expression associated with muscle unloading.
Finally, integration of transcriptomic and epigenomic data in humans aimed to identify key regulatory pathways controlled by promoter DNA methylation that are responsible for skeletal muscle disuse atrophy. After 3 days there were upregulated biological processes corresponding to cell signaling, Wnt signaling, positive regulation of transcription, and protein ubiquitination, and downregulated biological processes relating to metabolic processes. After 14 days biological processes were predominantly downregulated and corresponded to translation, ribosome biogenesis, mitochondrial respiratory chain, and metabolic pathways. Several genes including HDAC4, CHRNA1, GADD45A, TRIM55, and EIF3F were also identified as central regulators. Altogether these results show that promoter methylation and corresponding gene expression is involved in regulating a number of key genes associated with protein ubiquitination, as an early response to unloading, and translation, as an intermediate response. This research is the first extensive transcriptomic and epigenomic study of short-term disuse atrophy in human skeletal muscle and contributes to understanding of the regulatory processes associated with and progressing the loss of skeletal muscle in response to limb immobilisation.
| Date of Award | 15 Jun 2022 |
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| Original language | English |
| Supervisor | Kevin Ashton (Supervisor) & Vernon Coffey (Supervisor) |