Thiol Signalling in Skeletal Muscle Ageing

An age-related loss of muscle mass is associated with increased frailty in the elderly. The effect is felt at both a national scale, with an increased budgetary demand for health services directed towards the ageing population, and by the individual where reduced mobility significantly reduces their quality of life. It is unclear whether all skeletal muscle types are affected in the same manner. This thesis considered how thiol signalling, facilitated through reactive thiol groups on cysteine amino acids, may affect skeletal muscle ageing as it is crucial for normal intracellular function. Several studies have identified reactive oxygen species (ROS) as crucial signalling molecules in healthy muscle and various proteins can detect and respond to changes in their concentration. The cysteines are evolutionarily conserved in functionally important locations and have a direct impact on protein function, affecting either its active site or conformation. In healthy muscle, proteins can quickly and efficiently respond to changes in ROS concentrations via this mechanism whereas in aged muscle these responses appear to be impaired. The quadriceps and soleus muscles were selected because of their differing primary metabolic pathways and physiology, reflecting fast and slow twitch muscle respectively. This enabled determination of age related changes to the redox proteome between two different skeletal muscles. They are hypothesised to age differently and to determine this, adult (12 months) and old (24 months) tissue were subjected to a deep proteomics investigation, elucidating changes to the global proteome of ageing mouse muscle as well as using differential labelling of reduced and reversibly oxidised cysteine residues to identify redox-susceptible locations on individual proteins. Prior to this a proteomics study had not analysed changes to the redox proteome between two skeletal muscle tissues before. Analysis of the quadriceps label free results identified changes to redox protein abundance such as a significant increase in Protein Disulphide Isomerase, crucial to disulphide bond formation and breakage. HSC70, important for protein folding, was significantly decreased with age. Differential labelling of specific cysteine residues demonstrated Cys46 increased in its reduced form with age in PARK7. Furthermore, many changes observed in the label free analysis highlighted cytoskeletal proteins as those primarily affected. The soleus label free results demonstrated significant decreases in abundance of a number of mitochondrial proteins involved in the electron transport chain such as NAD(P)H dehydrogenase and ATP Synthase. One example of differential labelling highlighted ATP Synthase Cys101 as becoming increasingly reduced with age. This increase in a reduced redox state of cysteines was observed across a range of other mitochondrial proteins, possibly indicating a negative impact on energy metabolism in the soleus with age. A successful preliminary study considered the effect of stretching C2C12 mouse skeletal muscle cells in vitro. A protocol for testing the effect of mechanical stretching on C2C12 cells was optimised with a future goal of producing replicable in vitro proteomics data and thereby reducing the requirement for animal tissue. The studies in this thesis identified various redox proteome changes in quadriceps and soleus muscle with age. This data will provide a basis for a targeted analysis of musculoskeletal proteins with a view to a better understanding of musculoskeletal ageing and its impact via the proteome