A myopathy-linked tropomyosin mutation severely alters thin filament conformational changes during activation

Abstract

Human point mutations in β-and γ-tropomyosin induce contractile deregulation, skeletal muscle weakness, and congenital myopathies. The aim of the present study was to elucidate the hitherto unknown underlying molecular mechanisms. Hence, we recorded and analyzed the X-ray diffraction patterns of human membranepermeabilized muscle cells expressing a particular β-tropomyosin mutation (R133W) associated with a loss in cell force production, in vivo muscle weakness, and distal arthrogryposis. Upon addition of calcium, we notably observed less intensified changes, compared with controls, (i) in the second (1/19 nm ) actin layer lines of cells set at a sarcomere length, allowing an optimal thin-thick filament overlap; and (ii) in the second actin layer line of overstretched cells. Collectively, these results directly prove that during activation, switching of a positive to a neutral charge at position 133 in the protein partially hinders both calcium-and myosin-induced tropomyosin movement over the thin filament, blocking actin conformational changes and consequently decreasing the number of cross-bridges and subsequent force production.actin | cross-bridge | single-muscle fiber | X-ray diffraction T ropomyosin is expressed in multiple isoforms in most mammalian cell types (1, 2). The isoform diversity is related to alternative splicing, alternative promoters, and differential RNA processing, resulting in specific striated muscle, smooth muscle, and nonmuscle isoforms (2). In skeletal muscle there are three major isoforms, α, β, and γ, which are encoded by the TPM1, TPM2, and TPM3 genes, respectively (2). A number of missense defects in these TPM2 and TPM3 genes cause single amino acid changes in β-and γ-tropomyosin, skeletal muscle weakness, and myopathies (3). The molecular mechanisms by which such subtle defects result in muscle contractile dysfunction remain unclear and need to be elucidated with the aim of targeting potential therapies.A unique β-tropomyosin mutation (R133W) resulting from a defect in the TPM2 gene was recently identified in patients with distal arthrogryposis and an autosomal dominant congenital myopathy (4). These patients suffered from generalized weakness in both proximal and distal muscles. Biopsy specimens did not reveal any signs of atrophy or other histopathological abnormalities (4). Nevertheless, in normal-sized fibers, cell-physiological experiments disclosed a large decrease in maximal isometric force production ( Fig. 1) as well as changes in kinetics: that is, a slower rate of force development and a faster maximum unloaded shortening velocity (5), implying a slower rate of motor protein myosin attachment and a faster rate of detachment from actin monomers. This finding suggests a reduced number of cross-bridges in the strong binding state, resulting in overall weakness in the patients (5). How does the R133W mutation induce such dysfunction? To date, at the molecular level no experimentally-based explanation exists.In skeletal muscle, tropomyosin is an integral component of the sar...

X-ray recordings reveal how a human disease-linked skeletal muscle α-actin mutation leads to contractile dysfunction

“…1 and Table 2). This specific D286G mutation is not directly located in a region where myosin head residues bind (Ochala et al, 2012(Ochala et al, , 2008(Ochala et al, , 2011. Hence, the observed reduction in the intensification of the first MM reflection together with the reduced I 1,1 to I 1,0 ratio (À23% , Table 1) -suggesting a limited proper strong myosin Fig.…”

“…Arrays of approximately 30 fibers were prepared (Iwamoto, 2009;Iwamoto et al, 2001Iwamoto et al, , 2002Iwamoto et al, , 2003Ochala et al, 2012Ochala et al, , 2008Ochala et al, , 2011. For each myofiber, both ends were clamped to half-split gold meshes for electron microscopy (width, 3 mm), which had been glued to precision-machined ceramic chips (width, 3 mm) designed to fit into a specimen chamber.…”

Myopathies associated with β-tropomyosin mutations

“…The physiological mechanism for muscle weakness was further studied in detail in muscle cells by applying X-ray diffraction analysis where the movement of tropomyosin during the activation of muscle contraction can be determined. The results from this study demonstrated that the movement of tropomyosin over actin was impaired by the R133W mutation, which may directly explain the weakness, as this will cause a reduced number of actin-myosin crossbridges [59]. The E41K mutation associated with cap/nemaline myopathy was also studied in vitro on single muscle fibres from patients.…”

A myopathy-related actin mutation increases contractile function

“…Fibre mechanics and X-ray recordings were performed using established methodologies [9,14,15]. Full details are presented in the supplementary file.…”