Denervation of small numbers of fibres during ageing leads to dysregulation of ROS and functional changes in whole skeletal muscles

Lead Research Organisation: University of Liverpool
Department Name: Institute of Ageing and Chronic Disease

Abstract

As we age our muscle become smaller and weaker and the reduced muscle function can eventually contribute to frailty and poor quality of life. These changes occur partly because we lose some of the muscle cells (called muscle fibres) as we age, but also the muscle we retain is weak, does not respond well to exercise or training and releases pro-inflammatory compounds that make it more sensitive to damage. It is currently unknown how muscle fibres are lost during ageing, but when these fibres die, the motor nerves that normally control their activity are also lost. We believe that as the muscle fibres and motor nerves die, they release a highly reactive molecule (hydrogen peroxide) that affects the function of the neighbouring muscle fibres and makes them weak, prevents them from responding well to exercise or training and causes them to release pro-inflammatory molecules that make them more sensitive to damage.

This possibility will be tested using a mouse model in which a branch of one nerve that innervates a small portion of a muscle in the mouse limb will be cut under anaesthetic to induce denervation of a small number of muscle fibres embedded within a large number of intact innervated fibres. This will not significantly affect muscle function and mobility in the mouse, but will allow us to look at the effects of denervation of a small number of fibres on the neighbouring innervated fibres as a mimic of what occurs during ageing. The effect of this denervation of a small number of fibres on the force generation by the adjacent intact fibres, their ability to adapt following contractions and their release of pro-inflammatory compounds will be examined. The possibility that these changes are mediated by hydrogen peroxide released from the denervated fibres will also be examined.

Completion of these studies will provide further information on the mechanisms underlying muscle weakness during ageing and potentially provide indicators of the type of therapeutic interventions that may help preserve muscle function during ageing.

Technical Summary

Ageing causes loss of fibres from skeletal muscle and the fibres that remain show changes that limit muscle function. These include reduced specific force generation, attenuation of adaptive responses to contractions and increased generation of pro-inflammatory cytokines. The loss of fibres is associated with loss of whole motor units that occurs following cycles of denervation and re-innervation of fibres. Whether this fibre denervation affects the function of adjacent fibres has not been examined, but denervation of whole muscles causes large increases in ROS (hydrogen peroxide) generation by mitochondria. The increase is such that if only 1 in 100 fibres were denervated, this could increase apparent hydrogen peroxide generation from mitochondria isolated from whole muscle to the extent which has been claimed to mediate loss of muscle function during ageing. This project will use a novel partial denervation model to examine whether denervation of small numbers of fibres in a muscle leads to age-related changes in function in adjacent innervated fibres through increased mitochondrial ROS generation in the denervated fibres.

The project will examine transection of the superior muscular nerve as a model to denervate a defined group of fibres in the mouse medial gastrocnemius. Mitochondrial ROS generation in small bundles of permeablised denervated muscle fibres will be compared with permeblised innervated fibres from this muscle and microdialysis techniques will be used to determine whether partial denervation increases interstitial ROS in the innervated part. Effects of partial denervation on the function of adjacent innervated fibres will be examined by assessing their specific force generation and adaptive responses to an isometric contraction protocol. Finally, whether effects of denervation are caused by hydrogen peroxide (rather than other ROS or cytokines) will be examined by pre-treatment of mice with polyethylene glycol-tagged catalase.

Planned Impact

Researchers in the area of ageing and frailty will be direct beneficiaries upon completion of this research in addition to those studying other neuromuscular disorders associated with denervation and muscle atrophy such as motor neurone disease (ALS), diabetes, incontinence.

The pharmaceutical and personal care products sectors of industry will also benefit from this research. Data generated throughout the project may provide a resource to guide identification of pharmaceutical or non-pharmaceutical interventions 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 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 the development of anti-ageing products. Understanding the mechanisms by which 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 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.

The timescale of development of appropriate interventions deriving from the current work may be several years. However, if successful, the 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.
 
Description The key findings associated with this award are that:
1. Denervation of a muscle lead to loss of pre-synaptic axons within 1-3 days with no significant morphological changes in post-synaptic structures up to 10 days post-surgery, a time when decreased muscle mass and fiber size were apparent.
2. Mitochondria in the denervated muscle had increased peroxide generation that was apparent by 3 days post-transection. Use of electron transport chain (ETC) substrates and inhibitors of specific pathways indicated that the ETC did not contribute to increased ROS generation, but monoamine oxidase B, NADPH oxidase and phospholipase enzymes were implicated.
3. Partial denervation of muscle through transection of one branch of the motor nerve caused denervation of some muscle fibers while others retained innervation, but increased mitochondrial peroxide generation was found to occur in both denervated and innervated fibers and was associated with decreased muscle fiber sizes similar to that in the fully denervated muscle.
4. Thus the presence of recently denervated fibers in skeletal muscle leads to increased reactive oxygen species generation by mitochondria in neighbouring innervated fibres providing a novel explanation for the increased mitochondrial oxidative stress and damage seen with aging in skeletal muscles.
Exploitation Route Publication describing these key findings has been published and are reported here. We are developing translational aspects of this work through new MRC-funded studies and also in studies funded by the US National Institute on Aging (NIH).
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description MRC project grant (Jackson,, Barratt-Jolley, McArdle, Vasilaki)
Amount £648,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2015 
End 01/2018
 
Description NIH (Jackson, McArdle, Vasilaki, McDonagh)
Amount $1,300,000 (USD)
Organisation National Institute on Aging 
Sector Public
Country United States
Start  
 
Description Dissemination event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact Invitation to present research programmes to business/industry
Year(s) Of Engagement Activity 2014,2015
 
Description Radio interview 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Interviews on Radio Merseyside relating to ageing and frailty
Year(s) Of Engagement Activity 2014,2016