title: NF-κB activation in skeletal muscle during ageing: role in development of sarcopenia.
Creators: name: Owen, Euan
Publisher: name: University of Liverpool Repository
Year: 2022

description: Sarcopenia is the loss of muscle mass and function in older age. An increase in pro-inflammatory cytokines is associated with many age-related conditions including sarcopenia. A chronic activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) has been shown in muscles from old wild type (WT) and this has also been shown in a model of accelerated loss of muscle mass and function, mice lacking CuZn superoxide dismutase (SOD1KO mice). The consequence of such a chronic activation of NF-κB is unclear, but muscle is known to be an endocrine organ, with changes in the release of cytokines and chemokines, particularly following contractions where pro inflammatory cytokines can be released. In advancing age there is a notable decrease in muscle mass, with substantial loss of protein, suggesting that the balance between protein synthesis and degradation is net negative. Activation of NF-κB is also associated with activation of protein degradation processes. The aims of is thesis were to: 1) Quantify the changes in mass, structure and function of muscles of old WT and adult SOD1KO mice compared with adult WT mice; 2) Determine the localisation of nuclei with increase activation of the canonical pathway of NF-κB in muscles of old WT and adult SOD1KO compared with adult WT mice; 3) Identify whether any changes in cytokine and chemokine levels in the plasma of old WT and adult SOD1KO mice are associated with increased cytokine production by muscle and 4) to determine whether activation or inhibition of protein turnover pathways are associated with changes in NF-κB activity in muscles of old WT mice when compared with adult WT mice. Morphological and functional characteristics of muscle were examined using the tissue cell geometry plugin on ImageJ and MyoVision. Components of the NF-κB pathway were measured through qPCR and western blotting of lysates from gastrocnemius muscles and by immunohistochemistry analysis of EDL muscles. The levels of cytokines and chemokines were determined in muscle lysates and plasma and in media derived from isolated muscle fibres from adult WT, old WT and adult SOD1KO mice. The association of nuclear localisation of p65 with the regenerative stage of the muscle fibre was also examined in a model of regenerating extensor digitorum longus (EDL) muscles from adult WT, old WT and SOD1KO mice following injury induced via BaCl2 injection into the EDL muscle. Finally, fractional synthesis rates of individual proteins were determined in muscles of adult WT and old WT mice using heavy water SILAM where mice and unlabelled proteomics was also performed on these mice. The datasets from these experiments were compared with an RNA sequencing dataset to determine whether transcription was the driving factor in differences observed in fractional synthesis rates. There was an increase in inflammatory cytokines/chemokines in the plasma of old WT mice and an increase in specific chemokines in muscle homogenates or released from isolated muscle fibres from old WT muscles, suggesting that muscle of old WT mice may be a source of specific cytokines/chemokines, some of which add to the plasma pool and others acting more locally. The levels and patterns of cytokines/chemokines in plasma and muscle of adult SOD1KO mice were very different, suggesting that these mice are a poor model of inflammaging. Higher levels of p65 (the major transcription factor involved with canonical NF-κB activation) were observed in muscle fibres containing a centrally positioned nucleus, seen particularly in muscles of SOD1KO mice and during regeneration following chemically induced muscle damage but it is unlikely that this activation results in a substantial increase in release of cytokines/chemokines by these muscle fibres. Bioinformatic analysis of the unlabelled proteomics dataset revealed there was a clear upregulation of protein degradation pathways and protein misfolding pathways in the muscles of old WT mice. There was little evidence for changes in overall protein synthesis rates suggesting that increased protein degradation is the main driver of muscle protein loss with age. Increased activation of NF-κB may play a role in increased activation of degenerative pathways in muscles of old WT mice, but further analyses including inhibitor studies are required to confirm the role of NF-κB.