Activating mitochondrial biogenesis in skeletal muscle through repression of the acetyltransferase GCN5.

Skeletal muscle in a healthy adult accounts for approximately 50% of total body mass. In addition to its primary tasks of maintaining posture, breathing and locomotion, skeletal muscle also represents a vital nutrient store and metabolic regulator. During ageing, approximately 30% of an individual's muscle mass is lost by the age of 80. This decline is critical as it dramatically increases the prevalence of 'diseases of ageing', such as type 2 diabetes and sarcopenia (age-related muscle atrophy). Further, mitochondrial function is directly linked to physical activity levels, as inactivity coupled with poor habitual diet is associated with function in skeletal muscle. A fundamental cellular process, central in the aetiology of sarcopenia, is the function and maintenance of mitochondria (the principal energy producing organelles of the cell). Mitochondrial content and function declines during ageing and is thought to lead to impaired glucose and lipid utilisation, insulin resistance and increased adiposity. Thus, therapies aimed at maintaining or boosting mitochondrial function hold tremendous therapeutic potential for combating sarcopenia.

The best countermeasure currently identified to increase mitochondrial content in skeletal muscle is exercise; however how this adaptation occurs at the cellular level is poorly understood. This application will test the role of a protein called GCN5 in the regulation of mitochondria in skeletal muscle. Preliminary data suggests that GCN5 regulates mitochondrial adaptive responses to exercise such that reducing GCN5 activity in skeletal muscle will increase mitochondrial biogenesis. Therefore, to examine the specific role of GCN5 in skeletal muscle we will study mice in which GCN5 has been specifically deleted from skeletal muscle (GCN5 mKO). GCN5 mKO mice will undergo exercise training and we will examine how mitochondrial biogenesis is altered with the loss of GCN5. Further, we will then test whether a small compound inhibitor of GCN5 called CPTH2 can increase mitochondrial adaptation to exercise in healthy mice undergoing exercise training. Finally we will use a cell-based approach to try and identify how exercise regulates GCN5 activity. Using a small electrical field to stimulate contraction in muscle cells, we will use a proteomic approach to study and identify the process by which exercise leads to GCN5 repression. Overall we aim to identify a new biological pathway that is critical to how skeletal muscle adapts to exercise.

This proposal will identify fundamental bioscience and improve our understanding of how signals generated in muscle act to maintain and improve our health in response to physical (in)activity. It will also identify the suitability of inhibiting GCN5 in skeletal muscle, thus testing the potential for using drugs and nutritional supplements to target GCN5 and benefit our overall health.

Reversible lysine acetylation has emerged as a widespread post-translational modification, now thought comparable to phosphorylation in the hierarchical control of metabolic function. 80% of all proteins involved in skeletal muscle contraction undergo acetylation, with mitochondria demonstrating the greatest sub-cellular acetylation 'pool' in rodent and human skeletal muscle. Thus understanding the role and regulation of acetylation in skeletal muscle appears fundamentally important for determining the cellular regulation of skeletal muscle mitochondrial biogenesis. This application will manipulate acetylation signatures in skeletal muscle via targeted repression of the acetyltransferase GCN5. To achieve this I will utilize a GCN5 muscle specific knockout mouse and a compound inhibitor of GCN5 to study whether loss of GCN5 activity leads to greater mitochondrial biogenesis in basal and exercise-trained skeletal muscle. Further, I will then use an in vitro cell-contraction model to examine how exercise modifies GCN5 activity. Specifically I will test the role of the AMP-activated protein kinase AMPK in GCN5 regulation following exercise.

Aims and methods:
Utilizing a muscle-specific GCN5 knockout mouse (GCN5 mKO) and the GCN5 pharmacological inhibitor CPTH2, I will:
(i) Use targeted proteomics to determine the exercise dependent GCN5 acetylome in skeletal muscle
(ii) Examine whether GCN5 deletion increases mitochondrial biogenesis (mitochondrial protein synthesis, enzyme activity, mitochondrial content and gene expression) following chronic exercise training.

Utilizing a cell based contraction model I will examine how exercise modifies GCN5 activity. Specifically I will:
(i) Use contraction, compound activators, inhibitors and siRNA approaches to manipulate the activity of AMPK in vitro.
(ii) Examine whether AMPK directly phosphorylates GCN5 in vitro.
(iii) Use targeted proteomics to study how exercise alters post-translational modification of GCN5.

This project will investigate an important aspect of muscle exercise physiology focussing on preventing age-associated decline in skeletal muscle. As such it is likely to impact numerous beneficiaries:

Academic circle - There is a wide national and international academic circle of users who will derive immediate and long-term benefit from this research. These include basic bio scientists, exercise and muscle physiologists, and technologists whose research is directed towards muscle metabolic physiology and how to assess it.

Commercial sector - There is potential for long term interest from the commercial and private sector who could use the pathways involved and the data generated to think about small molecules drugs and nutritional supplements that have exercise mimetic properties through inhibition of GCN5 (e.g. CPTH2 administration). Policy makers, government agencies or regulators could benefit through an improved knowledge base to prepare policy driving a health economy.

Public sector - Clinical practitioners, dieticians and associated health professionals will benefit from improved knowledge of the impact of nutrition and exercise on skeletal muscle adaptation and related health benefits. Healthcare decision makers may also benefit through improved knowledge of muscle metabolic physiology and how it may pertain to disease in an aging population. This would add synergy in situations where GPs and or clinical academics, dietician and policy makers have greater information on which to decide benefit options of diet and exercise in at risk populations.

The public and third sector - Will derive potentially long term benefit through the activities of the identified beneficiaries making more informed policy and decision through to the end user, the general public. They will also benefit through direct information and advocacy through charities and the media to improve health through using newly identified biological mechanisms as the basis.

We will ensure results are highlighted to beneficiaries using the appropriate media. For academics we will publish and present the results at local, national and international meetings, and data will be published in high impact factor journals. To reach public sector and related academic beneficiaries, I will engage local clinical collaborators and service providers through the MRC-ARUK centre for musculoskeletal aging, highlighting the relevance of our investigations and outcomes. The University of Birmingham has very effective groups which oversee the commercialisation of research, and whom will be involved in the ongoing evaluation of this project. Within the College, the dedicated Technology Transfer Team regularly assesses IP and potential commercial development strategies. The College has its own External Commercialisation Board made up of top industry experts to advise on and support this process. This is then linked to the University's own dedicated commercialisation company, Alta Innovations, who support the patent process and drive licensing and spin-out opportunities. We will also seek, through ongoing education programmes and outreach to continue propagating good science.

Exercise is a bonafide countermeasure to offset declining health, thus enhancing lifespan. Through education and knowledge transfer, research such as that proposed herein has the capacity for long lasting impact that can directly influence human health. Exploitation of this data using the correct public forums may eventually lead to synergistic improvements in nutritional, pharmacological and exercise countermeasures, which could be realised in the next decade.