Introduction: Gross disuse atrophy (DA) of skeletal muscle results from a lack of contractile activity in both health and disease. However, DA rates of individual muscles exhibit marked heterogeneity; some muscles display atrophy resistance (e.g., tibialis anterior (TA)) whilst others appear (e.g., medial gastrocnemius (MG)) susceptible (Belavý et al. 2009; Bass et al. 2021). The mechanistic basis of the heterogeneity in DA susceptibility is not well understood.

Aims: We investigated the transcriptomic profiles of human MG and TA across 15-days of unilateral immobilization, hypothesizing muscle-specific programming.

Methods: Twelve[BP1]  healthy males aged 18-40y were recruited. Muscle mass was assessed using dual x-ray absorptiometry (DXA) and ultrasonography (Hardy et al. 2024). Biopsies were obtained from the MG and TA of the control and contralateral immobilized leg. Total RNA was extracted and underwent RNA-sequencing (RNA-seq). After QC of raw data, pseudo-alignment counts were generated with salmon. The limma-voom procedure implemented in R 4.4.1 was used to assess differential gene expression. Assessment of up-/down-regulation of genes in Broad Institute Hallmark gene categories (Liberzon et al. 2015) and muscle specific gene categories (Malatras, Duguez, and Duddy 2019) was carried out using the camera method. Potential upstream transcriptional control mechanisms were examined using the ChEA3 tool.

Results: Volume and mass were significantly reduced in the MG (p=0.0125 and p=0.0002 respectively) but not the TA (p=0.065 and p=0.18 respectively). 1087 genes were differentially regulated in the MG (255 up and 832 down) at a false-discovery rate (FDR) of 5%. No genes were differentially regulated in the TA (FDR <5%; 394 up and 366 down with raw p-value <0.05). Category enrichment suggested the up-/downregulation of 12 Hallmark genesets (FDR < 0.05) in the MG indicating reduced energy metabolism, muscle differentiation and upregulated inflammatory processes. Muscle specific genesets suggested immobilisation induced a gene signature ‘similar’ to ageing/illness in the MG (52 genesets regulated at FDR <5%). In contrast, we found that no Hallmark genesets were regulated in the TA (FDR <5%) and only 13 muscle-specific genesets were regulated (FDR <5%); the latter also suggesting a profile of ageing/illness, albeit subtler than that seen in the MG. To examine potential upstream mechanisms, we extracted genes with raw p-value <0.05 from each dataset into the ChEA3 transcription factor (TF) ranking tool. We then took the top 20 ranked TFs forward for further analysis. We found that signatures for the muscle specific TFs MYF6 & MYOD1 were enriched in MG and TA but in different directions (downregulated in MG and upregulated in TA) suggesting a possible role for these TFs in the divergent DA response.

Conclusions: These data substantiate our prior findings on TA and MG susceptibility to DA (Bass et al. 2021). Dampened molecular responses and muscle specific transcription factors MYF6 and MYOD1 may play a role in the transcriptomic control of the divergent DA response and suggest therapeutic targets.

Ethical statement: All studies had local ethical approval (University of Nottingham Faculty of Medicine and Health Sciences Research Ethics Committee: 103-1809) and conformed to the Declaration of Helsinki.