Sarcomeric signalling by giant muscle M-band proteins in health and disease

Lead Research Organisation: King's College London
Department Name: Cardiovascular

Abstract

The function of striated muscles, so called because of their highly regular striation pattern when viewed in a microscope, is crucial for the movement of our body and heart muscles. These stripes are formed from the repetitive arrangements of molecular machines, called sarcomeres that generate force and movement. In the sarcomere, three systems of molecular filaments are working together: actin filaments, which are held together at the Z-disk, myosin filaments, held together at the M-band, and the giant protein filament titin, which links the actin and myosin filaments. Muscle responds rapidly to changes in use, with disuse leading to muscle loss (called atrophy) and exercise leading to muscle growth (called hypertrophy). These processes need to be constantly balanced, and are linked in a coordinated way to those controlling muscle repair by making new proteins for sarcomere repair and replacement of other unwanted or damaged components of the cell. Signals controlling muscle growth, atrophy and repair signals are emerging to originate at the M-band and the Z-disk. These structures contain proteins that can sense mechanical stress, the most important factor controlling muscle growth and atrophy (as seen by the rapid loss of more than half the muscle mass in two weeks when muscles are immobilised in a plaster cast). Many of these proteins, however, remain enigmatic or haven't even been discovered, and often even their most fundamental functions have not been elucidated. Yet, when the integration of the M-band as a machinery combining structural, mechanical and communication functions is disrupted by genetic defects or extreme muscle inactivity (for instance in intensive care unit patients), severe muscle diseases are the result. This study will shed light on the compositions and regulation of the M-band, its role as a sensor for mechanical stress, and why mutations in two of the giant proteins that are involved in its assembly, titin and obscurin, can lead to muscle disease.

Technical Summary

This project will use a cross-disciplinary approach using to understand the links of the sarcomeric M-band to protein kinase signalling pathways controlling the turnover of myofibrils, and how mutations in the major sarcomeric protein titin affect this interplay of cellular structure, mechanics and signalling. It will use animal models to inform us on basic biological parameters of titin kinase as well as to allow us studying models of human disease. Cardiomyocyte cultures from neonatal and adult rodents will be used as easily manipulated models for studying cellular responses to mechanical stimuli, as well as for their particular suitability for live imaging approaches, which will inform us on the temporal dynamics and interactions of the M-band components studied, and the impact of defined mutations thereon. This will be complemented by super-resolution confocal microscopy and advanced image analysis (stress maps) to visualize with resolution approaching the dimension of individual M-band components the spatial relations of these, and the changes induced by mechanical activity or genetic disruptions in anmal models or patient biopsies. An underpinning activity will be the protein biochemistry necessary to generate the enzymes and structural proteins required for functional and kinetic studies, and for the nanoscale force measurements by single-molecule force spectroscopy and optical tweezers. Similarly, the structural analysis by X-ray crystallography, small-angle X-ray scattering and electron microscopy with single-particle reconstruction will be highly dependent on our proven ability to generate highly functional proteins for these types of studies, which will not only show basic molecular principles of M-band protein interactions or enzyme structure, but reveal the mechanisms of pathogenic mutations and guide the development of small molecule compounds that could target such interaction interfaces or active sites.

Planned Impact

Our research addresses both fundamental questions of striated muscle biology- the assembly of sarcomeric structures, the identification of novel components thereof, and the mechanisms governing their controlled turnover- as well as the pathomechanisms of hereditary and acquired muscle disease. Research from our laboratory has been, and will be, of benefit for the diagnosis and treatment of acquired and hereditary myopathies.

Our research will foster economic performance by benefiting commercial private companies interested in new physiological targets for muscle diseases. Currently, we are aware that output from our research on titin kinase has already spawned a new drug-discovery programme by a clinical-stage drug development company that discovers and develops novel, small-molecule drugs for the treatment of muscle disorders (Rigel Pharmaceuticals, San Francisco, USA; www.rigel.com). Rigel collaborates with e.g. AstraZeneca, Pfizer, Merck, Johnson & Johnson and Novartis in these activities. We expect that new pathways and deeper insight into known pathways emerging during our proposed programme will benefit development-oriented companies like Rigel in implementing new drug development programmes, and we remain in active communication to achieve and facilitate this. For example, we make reagents and protocols available for testing biological activities of M-band signalling proteins that can be implemented in high-throughput screens.
We also propose here to generate new monoclonal antibodies against M-band titin that will be of considerable value in the diagnostic of M-band myopathies, where deletion of parts of the titin C-terminus emerges as a common feature and an unmet need for standardised reagents in diagnostic application arises. We will collaborate with UK partners in the generation and eventual commercialisation of these reagents. These impacts can be realised within the tenure of the grant or shortly afterwards.

Patients and patient support groups.
We predict that the proposed research will be of benefit to patients and patient support organizations (e.g. international and national muscular dystrophy, and heart associations), by raising awareness of titin mutations as causes of hereditary muscle diseases, and their diagnostic and prognostic implications. These impacts can be realised within the tenure of the grant or shortly afterwards.

Publications

10 25 50
 
Guideline Title 219th ENMC International Workshop Titinopathies International database of titin mutations and phenotypes, Heemskerk, The Netherlands, 29 April-1 May 2016
Description Contribution to diagnostic guidelines for titinopathies
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in clinical guidelines
Impact Contributions led to establishment of evidence-based criteria how to define pathogenic TTN mutations, which parameters should be included in a titinopathy database, and which clinical parameters should be preferentially monitored in affected patients for best health outcomes and improved diagnosis.
 
Description citation in clinical reviews in the neurological area
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in clinical reviews
Impact improved genetic diagnostics of early-onset myopathies
 
Description 'Next Generation Optical Microscopy' - co-funded by MRC and EPSRC
Amount £1,647,764 (GBP)
Funding ID MR/K015664/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 02/2013 
End 01/2018
 
Description BHF Professorship-renewal
Amount £1,511,811 (GBP)
Funding ID CH/08/001 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2018
 
Description BHF Programme Grant
Amount £1,169,309 (GBP)
Funding ID RG/15/8/31480 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2015 
End 08/2020
 
Description Wellcome Trust Collaborative Award in Sciences
Amount £1,164,059 (GBP)
Funding ID 201543/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 09/2020
 
Title reagents and diagnostic criteria for Human Myopathy with Early Respiratory Failure 
Description antibodies against Nbr1, MURF2 for immunohistochemistry and Western blotting 
Type Of Material Antibody 
Year Produced 2006 
Provided To Others? Yes  
Impact Research Material used for differential diagnosis for Human Myopathy with Early Respiratory Failure 
 
Title reagents and diagnostic criteria for Salih Myopathy and LGMD2J 
Description antibodies against titin and obscurin, obscurin-like 1 used in immunohistochemistry and Western blotting of patient material 
Type Of Material Antibody 
Year Produced 2008 
Provided To Others? Yes  
Impact Research Material used for differential diagnosis for Human Myopathy with Early Respiratory Failure, Salih Myopathy, tibial muscular dystrophy (TMD) and LGMD2J 
 
Description Biophysics of cytoskeletal proteins 
Organisation Technical University of Munich
Department Department of Biophysics
Country Germany 
Sector Academic/University 
PI Contribution Concept of research project, generation of recombinant proteins and cell biological validation
Collaborator Contribution technical and conceptual advances on single-molecule biophysics
Impact One joint paper: Pernigo et al., Proc. Natl. Acad. Sci. USA, (2010). 107(7): p. 2908-2913. Multi-disciplinary collaboration between the physics team in Munich with expertise in optical trap measurements of protein-protein complexes, and our input in muscle protein biochemistry, analysis of sarcomeric protein interactions, and muscle cell biology.
Start Year 2008
 
Description Congenital autophagy disorders 
Organisation Guy's and St Thomas' NHS Foundation Trust
Department Paediatric Neurology
Country United Kingdom 
Sector Hospitals 
PI Contribution Analyse the impairment of the autophagy/lysosomal pathway and muscle ubiquitin ligases in hereditary paediatric disorders with myopathy/cardiomyopathy.
Collaborator Contribution Identifies patients and candidate disease genes
Impact myotubular myopathy trust award
Start Year 2011
 
Description Critical illness myopathy 
Organisation Umea University
Department Clinical Neurophysiology Unit
Country Sweden 
Sector Academic/University 
PI Contribution We analyse muscle samples from a unique rat model for critical illness myopathy developed in Umea for biochemical and cellular changes in sarcomeric mechanosignalling complexes
Collaborator Contribution We receive samples from a unique rat model of critical illness myopathy to assess changes in mechanosignalling complexes in the pathogenesis of this condition
Impact One joint paper: Ochala et al., J. Physiol., (2011). 589(8): p. 2007-26. A multi-disciplinary collaboration that combines clinical expertise in critical illness myopathy in Umea with our expertise in muscle mechanosignalling.
Start Year 2006
 
Description Early-onset Myopathies 
Organisation Pierre and Marie Curie University - Paris 6
Department UMR 787 (Institute of Myology)
Country France 
Sector Academic/University 
PI Contribution We analyse sarcomeric protein mutations leading to early-onset (paediatric) myopathies on the biochemical, biophysical and cell-biological level.
Collaborator Contribution Has provided valuable insight into the human biology of titin kinase and M-band titin by identifying crucial regions affected in early-onset myopathies.
Impact Two joint publications: Carmignac et al., Ann. Neurol., (2007). 61(4): p. 340 - 351. Fukuzawa et al., J. Cell Sci., (2008). 121(11): p. 1841-1851. A multi-disciplinary, translational collaboration that elucidates the disease mechanisms of novel early-onset myopathies.
Start Year 2006
 
Description Mechanoenzymatics 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution We expanded our research on mechanoenzymatic kinase signalling in collaboration with the Gaub team at the LMU. We identified new target proteins, expressed those and designed new studies.
Collaborator Contribution publications, shared personnel
Impact Two 2008 publications (Puchner et al., Proc. Natl. Acad. Sci. USA, (2008). 105(36): p. 13385-13390. Puchner et al., Biophys. J., (2008). 95(1): p. 426-434.), and one recent paper (Stahl et al., Biophys. J., (2011). 101(8): p. 1978-86). Ongoing multi-disciplinary work with the Munich physics team, and our expertise and conceptual background in muscle biochemistry and cell biology.
Start Year 2006
 
Description BHF donor lab visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 12 donors attended this visit, which included lab tours, practical demonstrations and short presentations.

A cheque over £10000 was issued on the spot.
Year(s) Of Engagement Activity 2012
 
Description BHF lab visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact Visit of BHF fundraising and outreach teams to DR. Yin-Biao Sun's team. Introduced the Randall Division and presented cardiovascular research activities in cell and molecular biophysics.
Year(s) Of Engagement Activity 2017