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

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.

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.

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.