Close regulation of peroxiredoxin oxidation is essential for maintenance of muscle mass and function in the elderly.

It is well known that skeletal muscle responds to exercise in a positive way to make the muscle stronger and work more efficiently. Muscle does this by increasing or decreasing the content of different proteins, but we still do not understand the key factors produced by exercising muscle which stimulate the signalling processes leading to changes in protein content. As muscle exercises it generates molecules, called reactive oxygen species, which are potential signals that initiate positive adaptations to exercise. The key reactive oxygen species is hydrogen peroxide and our research team have a great deal of experience of studying this molecule in muscle. We have shown that the very low levels of hydrogen peroxide generated in muscle during contraction can oxidise three highly reactive proteins, called peroxiredoxins 1, 2 and 3. We believe that these proteins act as effectors (or intermediaries) and activate signalling pathways that lead to adaptations to the muscle. We need to understand the roles of these peroxiredoxins, and which adaptations to exercise they mediate, since they may provide novel targets for interventions to help maintain muscle mass and function in situations where subjects cannot exercise and adapt efficiently, such as in ageing and in various disease states.

Our detailed studies will therefore examine muscle cells from humans and muscles from mice in which the different peroxiredoxins are deleted to achieve the following:

To characterise the way that human muscle cells respond to electrically induced contractions and to hydrogen peroxide when individual peroxiredoxins are deleted from the cells.

To determine how a lack of individual peroxiredoxins affects the functional responses to exercise in mice in which specific peroxiredoxins have been deleted by transgenic techniques.

To identify the key regulators of peroxiredoxin oxidation during contractile activity in skeletal muscle in order to find how the oxidation can be controlled as a potential approach to improve the way that muscle responds to exercise in the elderly.

Hydrogen peroxide (H2O2) is a key signalling molecule that is generated by contractions in skeletal muscle fibres and acts to stimulate beneficial adaptations of the muscle to exercise. The mechanisms by which H2O2 generated during contractions induces such adaptive changes have been unclear, but we have demonstrated that three 2-Cys peroxiredoxins (1, 2 and 3) in skeletal muscle fibres are oxidised following contractile activity, but we have not defined whether these oxidised proteins then mediate the adaptive responses to contractile activity.

We will therefore follow an unbiased testing approach to identify the pathways involved including targeted RNAseq and proteomics. The project will determine the responses and adaptations to contractile activity in skeletal muscle which are mediated by specific peroxiredoxins and identify factors that control the extent of Prx oxidation and determine how they are modified by ageing.

This will be achieved through 3 specific Aims:

1. To characterise the oxidation of peroxiredoxins and adaptive responses to H2O2 and contractile activity in immortalised human muscle myotubes in which specific peroxiredoxins are knocked out.

2. To determine functional responses to contractile activity in vivo in mice lacking specific peroxiredoxins (Prx1, 2 or 3 KO mice).

3. To identify key redox regulators of Prx oxidation in order to determine potential approaches that may be exploited to enhance the efficacy of the Prx signalling system.