The mechanism of stretch activation in muscle: a multidisciplinary approach

Understanding how muscle works is important, both for our in depth comprehension of physiology, and to allow us to design new therapies to cure muscle diseases. In particular, understanding how cardiac muscle, works is essential for the prevention and treatment of all cardiomyopathies. Regulation of muscle contraction is now known to involve two different mechanisms, one triggered by calcium, the second initiated by mechanical stretch. While the first is relatively well studied, very little is known about the second.
The goal of this application is to understand the mechanism of stretch activation in all muscles. We plan to use insect flight muscle, because in these muscles, stretch activation plays a predominant role, and can thus be studied in the absence of the masking effects of the otherwise predominant calcium-activation mechanism. We hope to find out what aspects of regulation are common to all striated muscles. We will use a giant Asiatic waterbug, since its flight muscles are particularly suitable for physiological studies, thanks to their appreciable size. There have been ground breaking studies on the flight muscle of the giant waterbug, some of which were carried out by members of this team. The flight muscle has the most ordered structure of any muscle, which makes it ideal for following structural changes when it contracts.

We will exploit the expertise of two teams, one in London and the other in York, both of which have previous long experience of working on muscle proteins using different but complementary analytical approaches to tackle this challenging and fascinating problem. Both teams have state-of-the-art facilities in their own discipline, and both have many years experience in developing analytical methods for studying biomolecules, and biomolecular interactions and pathways. The two teams have also been working in collaboration before, producing published results that have increased our understanding of muscle functioning.

The project holds enormous promise, not only for understanding the mechanism of stretch activation, but also for providing new tools for the future development of compounds able to intervene in muscle activation. Both cardiac muscle and insect flight muscle contract rhythmically and are activated by stretch. Thus, our research will help us to understand how the regulation of contraction has evolved; we will gain more insight into the way cardiac and other muscles work. This has ultimately the potential to increase our understanding of the causes of human cardiomyopathies.

We aim to advance the field of muscle research by gaining an understanding of the molecular basis of stretch activation, one of the two mechanisms that allow muscles to contract.

We intend to combine two teams with complementary expertise in developing different analytical, structural and functional methods, to understand the regulation of muscle contraction through protein-protein interactions. We propose to map in atomic detail the changes in troponin structure that are involved in stretch activation, and to suggest how these could lead to muscle contraction. Despite being debated for several decades, and the increasing interest in the effects of mechanical stress on biological systems, stretch activation is poorly understood. The results will add to our knowledge of the physiology of all muscles, and the way in which regulatory mechanisms have evolved; importantly, the proteins responsible for regulating contraction will be described at atomic resolution.

The project is challenging from a structural viewpoint, in that it involves proteins of >70 kDa in size that are filamentous, and in many cases, intrinsically unstructured, which assemble into large complexes. It is also challenging because some components are prone to denaturation and aggregation. We plan to use a combination of state-of-the-art NMR methods (to provide atomic resolution information about protein binding sites) and small angle scattering (to obtain the overall shape), supported by other biophysical approaches (ITC, SEC-MALS, MST). We also plan to substitute mutated proteins into muscle fibres and to make mechanical measurements.

Our results will significantly advance our understanding of stretch activation and increase our knowledge of the mechanisms that allow muscle to contract.

Scientific impact: Insects are the most diverse of any group of living organism: there are more than a million documented species, which make up 80% of all species. Knowledge of how insects fly will benefit our species, both for the potential of controlling insect-borne disease and for ensuring the survival of beneficial insects. Stretch activation of flight muscle is the mechanism that enables insects to fly.

The mechanical basis of stretch activation has been studied for more than 50 years but little is known about the structural changes that occur in the contractile proteins. Our study of stretch activation has both biochemical and medical applications, since well functioning muscle is important for health and wellbeing, especially in the elderly. The results will interest scientists, pharmacists and medical researchers in both academia and industry. Muscles researchers will benefit, as will clinicians involved in myopathies. The results may be exploited by scientific centres associated with our institutions: KCl has a strong muscle community who will be particularly interested in the output of this project.

Supporting knowledge: Pastore and Bullard regularly present their work at major national and international scientific conferences in their disciplines. When appropriate, press releases will be issued by the University of York, King's College and BBSRC, which may disseminate the results to the general public. While at NIMR, AP organized several press releases to spread information on her research and she intends to continue to do it at KCL. We will ensure that the research staff involved will participate in the promotion and dissemination of the research results by attending scientific meetings and publishing the results in high profile, peer-reviewed journals.

Industrial impact and technology translation: It is envisaged that the long term findings of the proposed research could be exploited in translational research for developing strategies for structure-based design of new drugs and hence, may result in social and economic benefit to the UK, and the community dedicated to improving human health. Intellectual properties at KCl and University of York are protected by their central offices. One of the teams has produced a panel of monoclonal antibodies to flight muscle proteins that are sold by the BBSRC Babraham Institute and Abcam. Other antibodies could potentially be marketed.

Delivering highly skilled people: The proposed project will train two PDRFs in new technologies; they will develop skills in advanced methods of structural molecular biology, nano-science and biochemistry. This will benefit their future careers, whether in academia or industry. Over the past few years, skilled post-graduate and post-doctoral fellows from the laboratories of both applicants have gone on to post-doctoral fellowships and lectureships in other universities, and positions in industry.

Public engagement: It is important that the public is involved in understanding and valuing scientific research, as this is the basis for the support and development of research. It will be important to find the most effective ways to communicate and disseminate the results of the proposed research to the non-scientific community. AP and BB have been committed to giving seminars to students at different levels, and to a general audience. The Wohl Institute of KCl is also committed to creating a specific interface with the public, and to involving it in their research. The Biology Department at York has Open Days for school children, and encourages their participation in research projects.