Characterisation of sestrin proteins in aged skeletal muscle

Ageing is associated with a progressive loss of skeletal muscle mass, known as sarcopenia, which can have detrimental effects on health and quality of life in the elderly population. Sestrins (sestrin 1-3) are a group of highly conserved stress responsive proteins that exhibit antioxidant, nutrient sensing and cytoprotective properties and may therefore be important in the maintenance of skeletal muscle health across the life-course. However, little is known about sestrins in skeletal muscle. In this thesis, we aimed to understand more about sestrin proteins in skeletal muscle ageing through the use of cell lines, primary muscle cell cultures and human muscle tissue. Specifically, we aimed to (i) determine how sestrins respond to cellular stresses in skeletal muscle cells, (ii) explore the sestrin content in human skeletal muscle tissue of young and older individuals, and determine the impact of chronic exercise on their content, (iii) investigate how sestrin protein levels change in primary human skeletal muscle cells during myogenesis, and the impact of ageing on this, and (iv) determine if sestrin 2 is required for amino acid-induced mTORC1 activation in skeletal muscle.

In Chapter 3, C2C12 myotubes were exposed to three chemically induced stresses that are known to occur in ageing and age-related diseases; oxidative stress induced by hydrogen peroxide (H2O2), endoplasmic reticulum (ER) stress induced by tunicamycin and inflammation induced by the addition of Lipopolysaccharide (LPS). We observed that sestrin 1 was unresponsive to any stress and sestrin 3 levels increased only in response to high levels of LPS. Sestrin 2 expression specifically increased with both H2O2 and tunicamycin treatment, which was closely associated with elevations in Ddit3 mRNA expression and ATF4 protein levels. The increase in sestrin 2 under stress conditions was sensitive to 4-phenylbutyrate (4PBA), which is a chemical chaperone resulting in attenuated ER stress. Thus, for the first time in skeletal muscle it was established that sestrin 2 is specifically upregulated by H2O2 and tunicamycin treatment and is closely related to ER stress.

In Chapter 4, we established an experimental model using C2C12 myotubes to test for the role of sestrin 2 in the activation of the mechanistic target of rapamycin complex 1 (mTORC1) by amino acids. We found that 6-hour amino acid deprivation was associated with a more than 50% reduction in mTORC1 signalling, which can be re-activated by 30 min incubation with 5 mM of leucine. This experimental model was then implemented to test for the role of sestrin 2 in mTORC1 sensitivity to amino acids. This was achieved by siRNA mediated knockdown of sestrin 2 in C2C12 myotubes. The results point towards a role for sestrin 2 as an important regulator of mTORC1 activation in response to leucine, but there were limitations in the experimental methods which make the results somewhat difficult to interpret and further work in this area is required.

In the final chapter, we assessed the effects of ageing on sestrin content in human muscle tissues and in primary muscle cells derived from human muscle biopsies throughout myogenesis as a foundation to understand the role of sestrins in ageing muscle. As a confounding factor, physical activity levels were accounted for by including a group of physically active elderly men in our analysis. We measured the protein content of sestrins in the skeletal muscle of young males (18-25 years), and older (65-80 years) inactive and active males. The relationships between physical activity and muscle fiber size were also analysed. There were no statistical differences in the content of any of the three sestrin proteins between the young and old individuals, although large effect sizes were observed, and our sample size was restricted due to the Covid-19 pandemic and the closures of research laboratories during data collection. When data points from all groups were analysed together, there were also negative correlations between sestrin 2 and muscle fibre crosssectional area and sestrins 1 and 3 and insulin signalling following amino acid feeding. In primary human muscle cells, the content of sestrins 1-3 were explored in the cells isolated from young (18-25 years) and old (65-80 years) participants throughout myogenesis. Whilst all sestrin isoforms were higher in myotube cultures compared to sub-confluent cultures, this pattern was altered for sestrin 2 and 3 in cells obtained from older individuals, and ATF4 protein levels displayed similar agerelated changes.

In conclusion, the experiments in this thesis show that sestrin 2 is responsive to age related cellular stresses and might be important for amino acid (leucine) induced mTORC1 activation in C2C12 myotubes. Whilst larger sample sizes are required, it appears that sestrins are differentially regulated in ageing in muscle tissue and myoblasts and future work should aim to fully discern the functions and importance of sestrin isoforms in human muscle health.