Understanding the mechanisms underlying myosinopathies

Lead Research Organisation: King's College London
Department Name: Ctr of Human & Aerospace Physiolog Sci

Abstract

Over 40% of the human body is made up of skeletal muscle, which are essential for breathing, and for movement. Skeletal muscles are composed of numerous cells called muscle fibres. Within the muscle fibres, two main proteins called myosin and actin are organised into thick and thin filaments respectively, and these filaments have highly precise, lengths. These filaments are organised into muscle sarcomeres, which are in turn organised in long linear arrays from one end of the muscle fibre to the other. The interaction of myosin with actin in each muscle sarcomere drives a small shortening of each sarcomere (known as a contraction), which is summed along the muscle fibre, to drive muscle shortening, which in turn drives movement.

Genetic alterations to the genes that encode part of the myosin molecule mean that a faulty (or mutant) protein is made, and this leads to severe muscle weakness in patients, in a type of disease known as myosinopathies. We do not understand how these faulty (or mutant) myosin proteins cause skeletal muscle weakness.
Our research will help us to obtain a new understanding of how faulty myosin proteins cause myosinopathies. We will be able to test how mutations affect the molecular structure of the myosin, how this affects its ability to form precisely built filaments, and how this then results in changes to muscle structure, leading to muscle weakness. Our approaches will range from investigating individual fragments of myosin to investigating the organisation and properties of myosin in intact human patient samples to enable us to obtain a deep understanding. This new knowledge will not only greatly advance our understanding of myosinopathies, but, most importantly, suggest pharmacological targets that may be exploited for effective therapeutic interventions.

Technical Summary

A large number of mutations located in human myosin heavy chains are associated with skeletal myopathies termed myosinopathies. However, their molecular and cellular determinants remain unclear. In this new project, we aim to identify the structural, biochemical and kinetic properties of a commonly occuring human mutations in two skeletal myosin isoforms, MYH7 and MYH2. Our analysis shows that most myosinopathy-causing mutations in these myosins are located in the light meromyosin (LMM) region of myosin. This region of myosin is formed of coiled coil, and known to be important for filament formation. We have identified 10 human LMM mutations known to be pathogenic and leading to muscle phenotypes (weakness) and myosinopathies that we wish to focus on. Based on our preliminary data, we hypothesise that these mutations will compromise filament structure with dramatic consequences on the relaxed and active states of myosin molecules in vivo. We further speculate that these LMM defects will result in sarcomeric and myofibre contractile dysregulation contributing to muscle phenotypes (weakness). To test this, we will define how these mutations specifically alter the various biochemical myosin states by using a combination of Mant-ATP experiments, in vitro motility assays and X-ray diffraction and uncover how these molecular functional alterations affect sarcomere integrity, dynamics and cellular contractility (King's College London). In complimentary studies at the University of Leeds, we will determine how each of these 10 mutations affect the structure and filament forming ability, using circular dichroism, electron microscopy and advanced light microscopy. By achieving these aims, our proposal will link the fundamental impairments to the clinical phenotypes. It will also reveal potential mechanisms necessary for the design of novel treatments.

Planned Impact

The benefits of our proposed research are multiple:
1. Impact on basic research and academia: The present research programme combining state-of-the-art methods and unique technologies has the potential to take the broader muscle disease field into new directions and as well increasing our understanding of human muscle biology in general. We thus therefore predict that the outcomes of the proposed project will have impact on both the basic biomedical science and clinical communities.
2. Impact on the pharmaceutical industry: We believe that a number of pharmaceutical companies (following on from the interest stemming from our previous work) would be highly attracted by the outcomes of these studies, which are likely to have applicability not just in myosinopathies, but across many skeletal muscle conditions where myosin function is impaired. There are thus potential implications for the development of therapeutic interventions by identifying the major processes. The applicants have pre-existing links to the pharmaceutical industry.
3. Impact on patients: The muscle weakness resulting from LMM mutations is a long term debilitating condition with dramatic implications on quality of life impacting upon ability to live, work and perform daily tasks of life without difficulty/ assistance. Understanding the mechanisms by which this occurs is crucial if we are to develop novel therapeutic interventions to mitigate this and improve long-term patient outcomes.
4. Impact on the economy: The economic consequences of muscle weakness are extremely large (e.g. large medical costs) and the impact on family members and carers can be enormous. The ultimate development of effective therapeutic interventions for patients with LMM mutations would have important economic implications, not only in the savings to the NHS, but in terms of the increased ability of affected patients (and carers) to live and work.
5. Impact by training: We are concerned that young scientists who come for training should be given as wide a scope as possible in order to further develop their scientific careers. This programme will allow the development of the appointed individuals. They will acquire a large range of unique and state of the art skills in human muscle biology, in study design and delivery, through data processing, leading through to good communication skills by presenting their work to the scientific and medical communities, as well as to general audience.
6. Impact on society via engagement of the public in science: The applicants of this application are members of various scientific societies and take active roles in trying to engage interested members of the public, particularly younger people, in science and our engagement for this project would be no different.

Publications

10 25 50
 
Description Unveiling the secrets of the enigmatic myosin-binding protein C (MyBP-C)
Amount kr 5,000,000 (DKK)
Organisation Carlsberg Foundation 
Sector Charity/Non Profit
Country Denmark
Start 02/2021 
End 01/2024
 
Description Molecular Dynamics 
Organisation Queen Mary University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution My team has provided mutations information to Dr Arianna Fornili. Drugs unravelled by her group will be further tested with our unique molecular an cellular assays.
Collaborator Contribution Dr Arianna Fornili and her group are using computational studies on our mutations to target pharmacological drugs to be used in patients
Impact n/a
Start Year 2019
 
Description Mouse model design 
Organisation University of Colorado Boulder
Country United States 
Sector Academic/University 
PI Contribution Prof Leslie Leinwand has provided muscles from her newly developed mouse model of MYH7 related diseases. We test the muscles using our unique molecular and cellular assays.
Collaborator Contribution Prof Leslie Leinwand has designed a unique mouse model of the disease and is writing a R01 NIH grant application where our team is listed as a main collaborator.
Impact n/a
Start Year 2019
 
Description Myomaker in health and disease 
Organisation University of Cincinnati
Country United States 
Sector Academic/University 
PI Contribution We are testing the muscle samples given by our collaborator.
Collaborator Contribution Dr Doug Millay is giving us mouse muscle samples to test. He is also designing a new mouse model relevant to human diseases due to mutations in MYMK (myomaker).
Impact Paper published in Nature communication. PMID: 33293533
Start Year 2020
 
Description Myosin in the ICU 
Organisation Free University of Amsterdam
Country Netherlands 
Sector Academic/University 
PI Contribution We test the muscles given by our collaborator using our unique molecular and cellular assays.
Collaborator Contribution Prof Coen Ottenheijm has provided muscles from ICU patients.
Impact Outputs and outcomes are still to come.
Start Year 2021