Disruption of redox relays in the age-related failure of muscle responses to contractile activity

We propose to study the role that a process called a 'redox relay' plays in the response of skeletal muscles to exercise and how this changes with age. Exercise is important in helping to maintain the health of our skeletal muscles and it is well known that skeletal muscle responds to exercise and to other environmental changes in a positive way to make the muscles stronger and work more efficiently. Muscle does this by increasing or decreasing the content of different proteins that are produced after exercise. Unfortunately as we age, these adaptations following exercise become less efficient, increasingly leading to a failure to maintain muscle mass and function.

As muscle exercises it generates some very reactive molecules, called reactive oxygen species, and it is known that these species can act as signals to initiate some of the positive adaptations that exercise induces in muscle. Unfortunately the precise way in which these reactive species undertake this is unknown, as is how these actions change as we age. One of these reactive species is hydrogen peroxide which is present in muscles at very low amounts and is the most important of these signalling molecules. Our research team has a great deal of experience of studying hydrogen peroxide and other similar species in muscle and we have previously shown in general terms how important these species are to maintaining muscle integrity following exercise and we also demonstrated that this process appears to become disrupted as we age.

Recent studies have shown that a small number of proteins present in our muscles can react with hydrogen peroxide and can transmit an oxidising signal from hydrogen peroxide to other key proteins with which hydrogen peroxide cannot react directly. The process that these intermediary proteins undertake is called a "redox relay" and the proposed project will examine the way that these "redox relay" proteins can transfer the oxidising signal from hydrogen peroxide. It will identify which proteins are able to do this, which other proteins they then react with to transfer the signal and which then go on to stimulate positive adaptations in the muscle. Finally we will determine how this process is modified by ageing. The initial part of the project will involve detailed molecular studies that require us to modify proteins in muscles in order to understand their actions and this will be undertaken in mice. Once we have identified the pathways involved we will then examine key aspects of these that can be measured in muscle biopsy samples that we will obtain from healthy young and older human subjects undertaking exercise.

On completion of the project we should understand how these signals are transmitted in muscle following exercise to stimulate positive adaptations in the muscle and how they are changed by ageing and their relevance to the poor responses elderly subjects have to exercise. This information will facilitate the design of logical and targeted interventions to correct the ageing changes with potential benefits of maintaining muscle strength in the elderly.

Hydrogen peroxide is a key signalling molecule that is generated by contractions and stimulates adaptations in skeletal muscle fibres, but current theories for the mechanisms involved cannot be sustained in the light of the absolute levels of hydrogen peroxide found in muscle fibres. Recent data have shown that some peroxidases, including peroxiredoxins (Prx) and thioredoxins (Trx) are orders of magnitude more reactive with hydrogen peroxide than other proteins and can transmit oxidising equivalents to other less reactive proteins by disulphide exchange. The proposed project will examine the possibility that Prx and/or Trx in muscle mediate the transfer of oxidising equivalents to signalling proteins that regulate multiple adaptations to contractions and that ageing leads to a disruption of these redox-signalling pathways and a reduced ability to respond to contractile activity.

The overall objective of the proposed project is therefore to determine the role of disulphide signalling in the responses of muscle to contractile activity, the role that Prxs and Trxs play in these processes and how they are disrupted during ageing.

State-of-the-art techniques will be used to assess the formation of intermolecular disulphides in Prx and Trx in muscle fibres following contractions together with a "trapping mutant" approach to identify partner proteins that form transient intra-molecular disulphides with Prx or Trx as a mechanism to transmit oxidising equivalents to the partner proteins. Adaptations of muscle to exercise involve activation of numerous pathways including the transcription factors NF-kappaB, AP-1 and FOXO3 and the essentiality of Prx and Trx in this activation will be assessed by deletion of these proteins by shRNA. Finally, we will translate these data through examination of key components of those redox relays identified by analysis of muscle biopsies obtained from groups of exercising young and older healthy volunteers.

Researchers interested in the responses of muscle to exercise and in the area of ageing and frailty and those undertaking research into appropriate lifestyle interventions to prevent age-related disorders will be direct beneficiaries upon completion of this research.

The pharmaceutical and personal care products sectors of industry may also benefit from this research. Data generated throughout the project may provide a resource to guide identification of pharmaceutical or non-pharmaceutical interventions to enhance the training effects of exercise and to reduce age-related loss of muscle mass and function. With the increasing elderly population there is increased demand for anti-ageing products. Anti-ageing products and lifestyle interventions have enormous economic potential for the pharmaceutical and personal care products sector and there is therefore potential for EU and UK economic benefit and improved quality of life as a consequence of their development. Understanding the mechanisms by which muscle responds to exercise, how this is modified by ageing and how loss of muscle mass occurs with increasing age is also relevant in the agriculture and animal husbandry industry, since meat yields decline in older farm animal species.

In the longer term the impact of this research will be on understanding the most effective forms of achieving beneficial effects from exercise training and on the health and quality of life of the elderly. Thus, local and national charities and policy-makers may benefit from regular contact with the research group throughout the project to disseminate information to promote and guide healthy ageing. An example of the approach used to facilitate this will be through attendance at the Institute of Ageing and Chronic Disease annual 'lay' Open Day which is advertised as a "Meet the Scientists" day and held at the World Museum, Liverpool. In 2014 it was attended by 1500 people over 2 days and included interested parties such and local and national charities and local MPs. This will provide a forum for briefing such groups in both general terms and with specific information related to this project. In addition, local schoolchildren and teachers are invited to attend the Open Day where we hold specific interactive discussions to inform both schoolchildren and teachers. This will demonstrate the specific need for this research, inform about outcomes and provide an educational forum for increasing awareness of musculoskeletal biology. This approach also helps identify areas which school outreach parties can focus on when small groups of active research staff (at all career levels), including the applicants and postdoctoral scientist funded on this project visit local schools.

The timescale of development of appropriate interventions deriving from the current work may be several years. However, if successful, the optimisation of adaptations resulting from exercise and prevention of age-related loss of skeletal muscle mass and function would lead to an improvement in quality of life for elderly individuals with a major economic impact on UK health and social care costs.