Sarcomere proteostasis in titinopathies

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy


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 protein turnover are emerging to originate at the M-band and the Z-disk. These structures contain proteins that can sense mechanical stress and control the activity of the protein degradation machinery. 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, severe muscle diseases are the result. This study will shed light on the compositions and regulation of the M-band, its role as a regulator of proteostasis, and why mutations in two of the giant proteins that are involved in its assembly, titin and obscurin, can lead to muscle disease.
Inherited defects in the giant muscle protein titin, the largest in the human body, are increasingly identified as common causes of a broad range of muscle diseases. Many of these mutations cause defective proteins that the muscle cell would need to prevent from behaving abnormally by clumping together and interfering with normal function, which may be a major disease mechanism. We will study the impact of code-changing "missense" mutations in titin on the ability of the cell to cope with defective proteins, called protein quality control. The findings will help us to understand the basic mechanisms of how sarcomeres regulate sarcomere quality control, and how this fundamental mechanism is perturbed in severe inherited myopathies affecting mainly children.

Technical Summary

This project will use a cross-disciplinary approach using to understand the interplay between the sarcomeric M-band and muscle protein quality control pathways controlling the turnover of myofibrils, and how mutations in the major sarcomeric protein titin affect this interplay of cellular structure, mechanics and signalling. Biochemical studies will measure the impact of missense mutations on titin domains for paediatric and adult myopathies. We will use cell and animal models to inform on the impact of pathogenic titin mutations on the basic biological mechanism of sarcomere proteostasis and to discover cellular pathways that lead to the tissue-specific impact of these mutations. Cardiomyocyte cultures from neonatal and adult rodents, iPSC- derived myocytes and skeletal myocyte cultures will be used as easily manipulated models for studying cellular responses to physiological and pathological effects on proteostasis stimuli. This will be complemented by super- resolution confocal microscopy and advanced image analysis to visualize with resolution approaching the dimension of individual M-band components the spatial relations of these events, and the changes induced by mechanical activity or genetic disruptions in animal 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. Structural analysis by X-ray crystallography, small-angle X-ray scattering and NMR 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 with components of the protein turnover machinery, but reveal the mechanisms of pathogenic mutations and guide the development of small molecule compounds that could target such interactions 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- with particular relevance to the pathomechanisms of hereditary 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. We are aware that output from our research on titin kinase has led to drug-discovery programme by a clinical-stage drug development company that discovers and develops novel, small- molecule drugs for the treatment of muscle disorders. We expect that new pathways and deeper insight into known pathways emerging during our proposed programme will benefit development- oriented companies in implementing new drug development programmes, and we remain in active communication to achieve and facilitate this. For example, we make develop reagents and protocols and make these available for testing biological activities of M-band signalling proteins that can be implemented in high-throughput screens.
We also generate new monoclonal antibodies against sarcomeric proteostasis components that will be of considerable value in the diagnostic analysis of titinopathies, 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.
Patients and patient support groups. We are aware 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. We are actively engaging with patient organisations to understand their needs and communicate our results. These impacts can be realised within the tenure of the grant or shortly afterwards.
Description European Research Council Synergy Grant
Amount € 12,000,000 (EUR)
Funding ID 856118 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 01/2020 
End 02/2026
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 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 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 cryo-electron tomography/microscopy of sarcomeres 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Physiology
Country Germany 
Sector Academic/University 
PI Contribution Co-wrote the research programme, provided preliminary data, provide samples and guidance
Collaborator Contribution Provide crucial expertise in muscle structure, cryo-electron microscopy, cryo-focussed ion beam milling, nanobody generation.
Impact multi-disciplinary
Start Year 2015
Description Outreach to titinopathy Patient Organisation Team Titin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact Presented our research on titinopathies to patient organisation in lay terms, outlining new developments and potential avenues for future therapeutic developments
Year(s) Of Engagement Activity 2020