Modelling Ageing Skeletal Muscle In Vitro to Determine the Effect of Aged Skeletal Muscle Secretomes on Macrophage Function and Their Role in Sarcopenia Progression

Abstract

[This thesis is in two volumes; both volumes are included in this electronic copy]. Skeletal muscle to immune system crosstalk maintains normal functioning of muscle tissue, however this communication could be altered during age-associated muscle conditions. This body of work aims to better understand this communication and suggests components of an ageing muscle secretome, including extracellular vesicles (EVs) could be responsible for the progression of the age-related muscle wasting disorder, sarcopenia. Most studies in the muscle field employ serum-based methods to generate differentiated skeletal muscle, however the use of serum can interfere with EV-related research. This study is built on the foundation of a serum-free skeletal muscle model for the uncomplicated study of muscle-derived EVs; an approach many muscle EV researchers avoid. Data here show a serum-free murine skeletal muscle system can be used to model sarcopenia and muscle cellular senescence in vitro for the investigation of downstream effects of aged muscle secretomes on both skeletal muscle cells and macrophages, and their involvement in furthering muscle atrophy. Sarcopenic and senescent myotube secretomes propagate an atrophic muscle phenotype in healthy myotubes and therefore facilitate the progression of sarcopenia via the bystander effect. Regarding immunomodulation, sarcopenic and senescent myotube secretomes and their isolated components are shown to have different and at times contradictory effects on macrophage function. Data presented here highlight the complexity of the aged skeletal muscle secretome role in modulating macrophage function, and suggest they induce an intermediate macrophage phenotype, which is responsible for maintaining age related muscle atrophy. It is proposed that aged muscle secretomes can promote anti-inflammatory features in these hybrid macrophages, such as reducing nitric oxide production and therefore limiting an M1 macrophage phenotype, whilst concomitantly encouraging pro-inflammatory functions, including reactive oxygen species release. Moreover, proteomic analyses suggest a role for aged skeletal muscle secretome derived EVs in both directly stimulating sarcopenia in healthy muscle cells and indirectly by stimulating an aberrant macrophage response to ageing skeletal muscle.