How does genotype define phenotype? Microphysiology of a tropomyosin mutation in situ shows the limitations of reductionism

Abstract

Tropomyosin is one of the components of muscle thin filaments. In concert with the troponin complex it forms the regulatory Ca2+ switch of striated muscles. The tropomyosin molecule is a parallel coiled-coil dimer of 274 amino acids which is almost 100%α-helix. Two strands of tropomyosin molecules joined end-to-end are wound around the actin double helix to form the core of the thin filament. Traditionally tropomyosin has been regarded as a passive player in muscle regulation, whose function is merely to transmit Ca2+ activation signals from troponin along the filament. However, in recent years tropomyosin mutations have been found to cause a number of clinically significant myopathies including hypertrophic cardiomyopathy, dilated cardiomyopathy, distal arthrogryposis, nemaline myopathy and congenital fibre-type disproportion. In the same way that disease-causing mutations in myosin heavy chain, myosin binding protein C and troponin T have sparked off renewed investigations of apparently well-known proteins, the discovery of mutations in tropomyosin has stimulated many new structural and biochemical studies of phenotype–genotype relationships. The emerging story is that tropomyosin is a much more interesting molecule than previously thought and it plays a very fundamental role in muscle regulation. From structural investigations it is now evident that tropomyosin is not a pure coiled-coil α-helix (Brown et al. 2001). Breaks in the heptad sequence alter the shape of tropomyosin so that it bends round the actin helix and there are also points with increased flexibility that are essential for tropomyosin to change its conformation on actin between the active and relaxed states. Binding sites for troponin T and for actin in the active and relaxed states have been identified. Thus, it should be possible to predict the effect of mutations from their location and nature. In practice, studies on the functional effect of disease-causing mutations in tropomyosin by in vitro methods do not correlate well with predictions from structure since almost any aspect of Ca2+ regulation could be altered by mutations. This should not be surprising since it is well understood that the thin filament is an integrated cooperative–allosteric system so that perturbations in any location will have a global effect on structure and function (Perry, 2003). Thus the complexity of the thin filament at present defies reductionist explanation (see, for instance, our recent study on the E40K and E54K mutations in α-tropomyosin (Mirza et al. 2007)). The proper way to study genetic disease is to study the physiology of the diseased tissue itself. This is a formidable challenge to physiologists but a recent paper in The Journal of Physiology from Ochala et al. shows that it can be done (Ochala et al. 2008). In this study, the authors obtained fresh biopsies of skeletal muscle from a patient with skeletal myopathy due to the mutation of glutamic acid 41 to lysine in β-tropomyosin and performed a comprehensive series of mechanical measurements, in vitro motility and even determined the effect of a Ca2+ sensitiser as a possible therapy. In order to do this they needed to select individual type I fibres for assay based on myosin heavy chain isoform analysis. The key findings are that crossbridge turnover speed and Ca2+ sensitivity are reduced but the relationship between force and stiffness is not affected. The interpretation of these results is that crossbridge properties are not changed but the probability of activation at intermediate Ca2+ concentrations is reduced. It is interesting to note that the observed 2- to 3-fold increase in EC50 is of the same order as that found with mutations that cause dilated cardiomyopathy; indeed a very similar mutation was found at the neighbouring amino acid (E40K in TPM1; Mirza et al. 2005). In both of these cases the mutation is remote from the putative sites where troponin binds to tropomyosin, thus emphasizing the allosteric nature of muscle regulation. Another interesting feature that could only be determined in tissue samples is that the β-tropomyosin content relative to α-tropomyosin in mutant muscle is increased from 68% to 81% and that muscle fibres are small – 28% of control fibre diameter. It is by no means clear how this multiplicity of changes is caused by the initiating mutation nor which changes are significant in the disease phenotype. Nevertheless microphysiology of this kind is most valuable in showing us the overall state of a diseased muscle, which can be quite different from that predicted from the molecular properties of tropomyosin on its own.