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

Abstract

Human point mutations in beta- and gamma-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 membrane-permeabilized muscle cells expressing a particular beta-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(-1)), sixth (1/5.9 nm(-1)), and seventh (1/5.1 nm(-1)) 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.

Fig. 1.

Specific force of membrane-permeabilized muscle cells from controls (CTL) and patients (R133W), corresponding to maximal isometric force production normalized to cell cross-sectional area. Values of fibers expressing the type I and IIa myosin heavy-chain isoforms are pooled together, as they did not differ significantly. These values have already been published elsewhere (5) and are given here as means ± SEs.

Fig. 2.

X-ray diffraction patterns from human control (A–C) and mutated (D–F) cells set at an optimal sarcomere length, in preactivating (A and D) and activating (B and E) solutions. There are differences in intensity profiles (C and F); red and blue colors indicate the enhanced and weakened areas, respectively, after addition of calcium. ALL, actin layer line.

Fig. 3.

X-ray diffraction patterns from human control (A–C) and mutated (D–F) overstretched fibers, in low-EGTA rigor (A and D) and calcium-rigor (B and E) solutions. There are differences in intensity profiles (C and F); red and blue colors indicate the enhanced and weakened areas, respectively, after addition of calcium.

Fig. 4.

Representative plots of slack-test procedure in a control fiber (○) and a mutated cell (●). Lengths were 1,620 and 1,700 μm, respectively.

Fig. 5.

Location (arrows) of the mutation in the heptad repeat and along the seven pseudorepeats of tropomyosin.