Myosin-linked mechanisms for the regulation of muscle contraction

Muscles make us mobile, and mobility is a major factor determining our quality of life. Unfortunately our muscles get weaker as we get older, and everyday tasks like walking, climbing stairs or even getting up out of a chair get more difficult. Many diseases also lead to severe forms of muscle weakness, and some of these affect young children. In general there is no effective treatment for muscle weakness. Using muscles- exercising them- does make them stronger, but this is not always possible, particularly for those suffering from diseases that affect the muscles. This proposal seeks to discover what controls the strength of healthy muscles because, if we understood that, we might be able to design a drug to boost the strength of weak muscles.

Muscles are built from long strings of a basic microscopic building block called a sarcomere. Each sarcomere contains an array of two types of filament, one wider than the other, and these are called thick and thin filaments. The relative sliding of these two sets of filaments is responsible for muscle shortening. When a signal from the brain travels along a nerve and reaches a muscle, it triggers release of calcium from stores inside the muscle cell. The calcium binds to the thin filaments, causing a change in their structure that allows the filaments to slide, and the muscle contracts. This change in thin filament structure is quite well understood, but it works like an OFF/ON switch- it controls when the muscle contracts, but not how strongly. Recently it has become apparent that there is another type of muscle control that works by changing the structure of the thick filaments, and this can alter both the ON and the OFF states. In other words, these changes in thick filament structure can control the strength of a muscle, but also how much energy a muscle uses when it is resting. Since about a third of the weight of our bodies is muscle, understanding how to control the OFF state of muscle might be useful in combatting a very different health problem, allowing us to use muscles to burn off some unwanted calories.

At present we don't know how these OFF and ON states of muscle are controlled- we don't know exactly how the structure of the thick filament changes, and what controls those changes. We do know which proteins are involved, though, and roughly where they are in the sarcomere. In this project we will apply a new method that we developed to tag the thick filament proteins with small fluorescent molecules that report changes in protein structure from inside muscle cells. Our preliminary tests with this method shows that it is capable of answering some key questions about how changes in in thick filament structure control muscle performance: What is the difference between the OFF and ON structures of the thick filaments at the molecular level? How do these structural changes in the thick filament control the muscle strength? How is thick filament structure itself controlled? How is that control related to the calcium switch in the thin filaments?

The answers to these questions will allow us to develop a detailed picture of how the OFF and ON states are controlled by changes in thick filament structure in healthy muscle. This in turn will enable us to suggest ways in which these states might be controlled by drugs, and to develop ways to assess the value of potential new drugs. Finally, since very similar changes in thick filament structure occur in heart muscle using the same protein components, we expect that the results of this project will also be useful in guiding an analogous approach to controlling the strength of heart muscle, and therefore in developing potential new treatments for heart disease.

This project aims to elucidate the molecular mechanisms of thick filament-based regulation in skeletal muscle. These mechanisms supplement the well-known thin filament-mediated pathways to control the strength and duration of contraction in response to muscle length, load and previous activity, making them attractive targets for therapeutic intervention in diseases of both skeletal and cardiac muscle. Using bifunctional fluorescent probes, we will determine the in situ conformation of the regulatory light chain (RLC) region of myosin in the OFF state of the thick filament. We will test the hypothesis that the RLC N-terminal lobe is primarily a regulatory filament-docking domain rather than, as in current models, an extension of the lever arm of the myosin motor. We will characterize the changes in RLC conformation associated with the OFF-ON transition of the thick filament, and investigate how that transition is controlled by the nucleotide state of myosin and the inter-filament spacing. We will determine how RLC conformation is controlled by calcium, both in the steady state at intermediate calcium concentrations and kinetically following a calcium jump, and thus elucidate the relationship between calcium activation of the thin filament and the OFF-ON transition in the thick filament. We will characterise the changes in RLC conformation produced by RLC phosphorylation, and relate them to the increased force output associated with previous muscle activity. We will determine the dependence of RLC conformation on the static and dynamic stress in the thick filament in order to elucidate the role of mechano-sensing in muscle regulation. Using N-terminal fragments of myosin binding protein C, we will determine the role of this protein in signalling between thin and thick filaments. Combining these results, we will produce a molecular model of the physiological regulation of contractility of skeletal muscle that integrates thin and thick filament-based mechanisms.

Who will benefit from this research, and how?

1. The elderly. There are more than 10 million people over normal retirement age in the UK, and worldwide there are more than 600 million people aged over 60, many of whose lives are affected by weakness of their skeletal muscles, as a normal part of the ageing process. This research aims to open up new approaches to the treatment of muscle weakness through a better understanding of the normal mechanisms by which muscle strength is controlled.

2. Those suffering from muscle weakness due to disease. This would include patients suffering from congenital and adult-onset neuromuscular and neurodegenerative disorders, obesity and its associated disorders, and cachexia. Again the potential benefits would come from new approaches to the treatment of muscle weakness through a better understanding of the normal mechanisms by which muscle strength is controlled.

3. Those suffering from heart failure. Over three-quarters of a million people in the UK alone suffer from heart failure, and this is another important factor influencing quality of life for many older people. Because of the close similarity between the regulatory pathways under study in this proposal in skeletal and heart muscle, we expect that the results of the proposal will guide the development and testing of potential new drugs that could improve contractility in the heart, as a means to counteract heart failure.

4. Those suffering from diabetes and obesity. More than three million people have been diagnosed with diabetes in the UK alone, and worldwide over 400 million people were obese in 2008. The potential impact of the present proposal for this group relates to the dominant impact of skeletal muscle metabolism on blood glucose levels and body mass, and the possibility of long-term control of these factors by modulation of the rate of ATP utilization by resting muscle.

5. Commercial organisations. The long-term beneficiaries within the private sector are likely to be companies involved in the further development of small-molecule therapeutics arising from the results of the proposal. We have held a series of meetings with a company specializing in small molecules that modulate the contractility of skeletal and heart muscle, as described in Pathways to Impact, and this would be our preferred initial route for exploring the commercialization of potential new targets arising from the present proposal.

6. Academic beneficiaries. These include researchers in muscle physiology, muscle development, sports and exercise physiology, ageing, skeletal muscle disease, heart disease and obesity, and clinicians working in the corresponding specialities, as described in the section Academic Beneficiaries.

7. Staff employed on the project. The employed staff will benefit from enhanced research, technical and planning skills. The project will be a key step enabling the PDRA to be ready to undertake an independent research career. More widely, the project will help to maintain the shrinking pool of researchers with the necessary skill-base to perform demanding holistic or systems-level approaches to elucidate physiological mechanisms in intact muscle.

Most of the above potential benefits are long-term, and the likelihood of their realization would be assessed during year 3 of the project in order to prioritise future work between the potential targets listed above.