Unravelling the transcriptomic symphony of muscle ageing: key pathways and hub genes altered by ageing and caloric restriction in rat muscle revealed by RNA sequencing.

Age-related muscle wasting, sarcopenia is an extensive loss of muscle mass and strength with age and a major cause of disability and accidents in the elderly. Mechanisms purported to be involved in muscle ageing and sarcopenia are numerous but poorly understood, necessitating deeper study. Hence, we employed high-throughput RNA sequencing to survey the global changes in protein-coding gene expression occurring in skeletal muscle with age. Caloric restriction (CR) is a known prophylactic intervention against sarcopenia. Therefore, total RNA was isolated from the muscle tissue of both rats fed ad libitum and CR rats. RNA-seq data were subjected to Gene Ontology, pathway, co-expression, and interaction network analyses. This revealed the functional pathways most activated by both ageing and CR, as well as the key "hub" proteins involved in their activation.RNA-seq revealed 442 protein-coding genes to be upregulated and 377 to be downregulated in aged muscle, compared to young muscle. Upregulated genes were commonly involved in protein folding and immune responses; meanwhile, downregulated genes were often related to developmental biology. CR was found to suppress 69.7% and rescue 57.8% of the genes found to be upregulated and downregulated in aged muscle, respectively. In addition, CR uniquely upregulated 291 and downregulated 304 protein-coding genes. Hub genes implicated in both ageing and CR included Gc, Plg, Irf7, Ifit3, Usp18, Rsad2, Blm and RT1-A2, whilst those exclusively implicated in CR responses included Alb, Apoa1, Ambp, F2, Apoh, Orm1, Mx1, Oasl2 and Rtp4. Hub genes involved in ageing but unaffected by CR included Fgg, Fga, Fgb and Serpinc1. In conclusion, this comprehensive RNA sequencing study highlights gene expression patterns, hub genes and signalling pathways most affected by ageing in skeletal muscle. This data may provide the initial evidence for several targets for potential future therapeutic interventions against sarcopenia.

Declarations. Ethics approval and consent to participate: The animal study from which samples were collected [25] received ethical approval from the University of Liverpool’s Animal Welfare Committee and adhered to the Animal (Scientific Procedures) Act 1986 and its amendments. The research proposal underwent an ethical review and scoring process before grant submission, followed by additional review from the funding body. After funding approval, a Project Licence (PPL 40/2533), classified as "Mild," was obtained from the Home Office Animal Inspectorate, which involved detailed scrutiny and compliance with the principles of Replace, Reduce, and Refine. The Home Office conducted unannounced visits to ensure adherence to protocols, and animals were humanely euthanised by cervical dislocation after stunning, as required by law. The process was overseen by licensed officials to maintain high standards of animal welfare and ethical compliance. Consent for publication: Not applicable. Competing interests: JPM is CSO of YouthBio Therapeutics, an advisor/consultant for the BOLD Longevity Growth Fund, 199 Biotechnologies, and NOVOS, and the founder of Magellan Science Ltd, a company providing consulting services in longevity science. There are no competing interests declared by any other authors. There are no competing interests declared by any other authors.