Contractile Proteins

Actin filaments, labelled with rhodamine phalloidin, moving over a bed of myosin in the presence of 1mM ATP (3mb video clip)

Professor Steve Marston, Principal Investigator

Thin filament regulation of the contractile apparatus in cardiac and smooth muscle
We study the contractile and regulatory proteins of cardiac and smooth muscle.

The heart is a muscular organ in which contraction of striated muscle cells pumps blood around the body.  The walls of blood vessels contain a different sort of muscle- smooth muscle- whose contraction is responsible for the control of blood pressure.  In both cases contractility is achieved by a molecular motor made up of interdigitating actin and myosin filaments.  The motor is switched by regulatory proteins, which respond to changes in intracellular Ca²⁺ levels. We are interested in the mechanism of regulation of contraction by Ca²⁺ and how it is altered in muscle diseases.

Our main interest is in the regulatory mechanism of the thin, actin based filaments. In cardiac muscle this involves troponin and tropomyosin whilst in smooth muscle it involves tropomyosin and caldesmon - a protein which we have studied in great detail. We use a wide variety of structural, biochemical and physiological techniques to investigate these systems.

A large part of our work concerns the Ca²⁺ switch incorporated into actin filaments that regulates cardiac muscle contraction.  We have demonstrated that then protein complex tropomyosin-troponinT-troponinI-troponin C is functionally altered in failing hearts and is also functionally altered in genetic diseases hypertrophic cardiomyopathy and dilated cardiomyopathy which can be caused by mutations in actin, tropomyosin, troponin T and troponin I.  We are investigating how changes in protein structure affect function and also whether these changes trigger heart disease.

For these studies our basic technique is the in vitro motility assay.  We study single thin filaments interacting with immobilised myosin and other actin binding proteins.  By image analysis of filament movement we can determine the filament speed (measure of cross bridge turnover rate), the fraction moving (measure of the equilibrium between on and off state of thin filament), the density of bound filaments (measure of weak binding affinity) and relative isometric force. A technique for determining the absolute force velocity relationship of single filaments is under development.

We use the assay to determine the functional consequences of mutations in TnI, TnT, tropomyosin and actin and to relate these to disease phenotype (Hugh Watkins and Charles Redwood, Oxford).  The emerging pattern is that HCM mutations increase sliding speed, increase Ca2+-sensitivity and/or impair relaxation i.e. a hypercontractile phenotype.  DCM mutations decrease sliding speed and Ca2+-sensitivity.

Parallel tests on troponin extracted from normal and failing human hearts show altered function: slower sliding speed and increased Ca2+-sensitivity.  Currently we are investigating the biochemical changes that underlie the functional differences.  An alteration in PKC-catalysed phosphorylation is the present most likely explanation. More detailed time series experiments to try to establish cause/effect relations are planned in an animal model.

Smooth Muscle
Smooth muscle studies are centred on the thin filaments of vascular smooth muscle and the key regulatory proteins tropomyosin, caldesmon and CaBP.

We are engaged in extensive structure/function studies of caldesmon regulatory domain by site directed mutagenesis and protein NMR (with Dr Barry Levine, Birmingham).

We are also studying mechanism of regulation in smooth muscle thin filaments, which involves cooperative and allosteric interactions between the motor protein, myosin and the regulator caldesmon/CaBP through actin and tropomyosin.  We have recently started transient kinetic investigations of this system to dissect the regulated actin-myosin ATPase pathway in detail. A particular interest is the differing regulatory functions of the multiple tissue specific isoforms of tropomyosin and the effects of mutations, which are associated with HCM, DCM, essential hypertension and nemaline myopathy.

Through an INTAS network with laboratories in Moscow (Vorotnikov), St Petersburg, Lund and Koln we are studying the physiology, functional and structural consequences of phosphorylation of caldesmon by cell signalling kinases (MAPK, PAK) which modulates its regulatory properties.