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Novel approaches to elucidate the molecular basis of muscle contraction: FRET-FLIM imaging applied to single muscle fibres

Reference	BB/I019448/1
Principal Investigator / Supervisor	Professor Michael Ferenczi
Co-Investigators / Co-Supervisors
Institution	Imperial College London
Department	National Heart and Lung Institute
Funding type	Research
Value (£)	371,765
Status	Completed
Type	Research Grant
Start date	01/08/2011
End date	31/07/2014
Duration	36 months

Abstract

The myosin and actomyosin ATPase mechanisms have been extensively studied; however, the specific conformational changes that take place and their link to ADP, Pi release, production of mechanical impulse and the consequent muscle contraction remain unclear. In this work we will take advantage of FRET-imaging (Forster Resonance Energy Transfer) to measure nanometre-scale distances at the cross-bridge level with the aim of detecting the conformational changes the myosin head undergoes during contraction. Our working hypothesis is that the myosin lever arm rotation is dependent on strain. To this end several FRET pairs will be located in strategic positions within the myosin head: we focus our attention on the essential light chain, ELC, as this proximal part of the lever arm forms an interface with the catalytic domain. Four mutations of recombinant ELC, each containing a single cysteine residue in a different position (ELC-127, 142, 160 and 180) will be exchanged with the native ELC of the fibre and their interactions with SH1 helix (labelling Cys707) will be analyzed. Dynamic measurements will be performed on rigor fibres by applying small stretch/release cycles to alter the interaction distance between the ELC and SH1. In this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction, directly testing the lever arm hypothesis.

Summary

Movement in living cells results from the coordinated behaviour of proteins. This energy-requiring process is driven by the breakdown of ATP (adenosine triphosphate), the cells' molecular fuel. Muscle cells specialise in converting this chemical energy into movement and mechanical work with their precisely dimensioned and aligned filaments to maximise power output, speed, fatigue resistance and efficiency of movement. At the molecular level, the movement is brought about by a cyclical change in conformation in one of the main protein, myosin. The details of the changes in protein conformation and their link to the steps in the ATP breakdown process are not known. In this project, we intend to develop advanced fluorescence microscopy techniques to measure changes in protein conformation during force production. To do this, we intend to attach fluorescent molecules to specific regions on the myosin molecule and make use of an energy transfer process to measure distances in the nm range. The process of energy transfer implies that the fluorescence emission of one molecule is affected by the fluorescence mechanism of the other, in a distance-specific manner. The regular arrangement of filaments into sarcomeres allows spatial averaging over dozens of functionally equivalent segments, thus giving rise to an improvement in the signal/noise ratio that is not available in other living cells. With this novel method, we plan to investigate changes in the distance between the fluorescent molecules specifically attached on myosin during force development and during perturbations of the muscle length. The result will be a map of the movement of these parts of the myosin molecule, and the determination of the time-course of these movements.

Impact Summary

The importance of studying muscle contraction is evident, since muscle tissue is one of the most abundant tissues in the human body and regulates major physiological processes such as blood circulation, heart function, force production, temperature regulation and motion. Many aspects of muscle contraction have been elucidated in the last decades, but in order to successfully target muscle and heart disease, the mechanism of muscle contraction need to be studied at a molecular level. As such, the scope of the work extends beyond our goals of improving our understanding of muscle contraction. From the point of view of human health, the cross-bridge mechanism in skeletal muscle is relevant to understanding of cardiac muscle contraction. Indeed, understanding of muscle contraction at the molecular level may provide key information for the development of therapies in a number of areas such as heart disease, hypertension, age-related muscle weakening, etc. The importance of studying cardiac muscle is highlighted by the rising incidents of heart disease around the world whereas the information about smooth muscle, a basic component of blood flow regulation is still limited. By carefully optimizing the methodology here proposed, applications for various other experimental fields could be developed.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	Technology Development for the Biosciences
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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