Congenital myopathy-causing tropomyosin mutations induce thin filament dysfunction via distinct physiological mechanisms

Abstract

In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit thin filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative thin filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in thin filament length. (ii) In contrast, the TPM2-E181K mutation increased thin filament activation, cross-bridge binding and force generation. In the former mechanism, modulating thin filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the thin filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.

Figure 1.

MyHC isoform composition. A predominance of type I fibers was observed for TPM3-R167H and TPM2-E181K, whereas TPM2-null had a similar MyHC isoform distribution as CTL. In the figure, examples of electrophoretic separations are displayed. Lanes 1–3 and 5–9 are single fibers, whereas lane 4 is a muscle homogenate showing the three different MyHC isoforms (I, IIa and IIx, from bottom to top).

Figure 2.

Maximal fiber force production. Maximal isometric steady-state force generation was not significantly modified by the various tropomyosin mutations at saturating [Ca²⁺] (pCa 4.50) and optimal sarcomere length (between 2.60 and 2.80 µm).

Figure 3.

Force generation as a function of sarcomere length. The mean relative force–sarcomere length relationships of fibers expressing the type I MyHC isoform (pCa 4.50) clearly demonstrates a lower force generation at 3.20 µm for TPM3-R167H when compared with other groups. In the figure, asterisk indicates a significant difference when compared with CTL (P < 0.05).

Figure 4.

Fiber force production and stiffness at non-saturating [Ca²⁺]. The various typical curves for fibers expressing the type I MyHC isoform highlight the decrease (TPM3-R167H and TPM2-null) or increase (TPM2-E181K) in Ca²⁺ sensitivities of force and stiffness when compared with CTL.

Figure 5.

Rate of force development. In fibers expressing the type I MyHC isoform, at saturating [Ca²⁺] (pCa 4.50) (A), k_tr was significantly lower for TPM3-R167H and TPM2-E181K when compared with TPM2-null and CTL while at submaximal [Ca²⁺] (pCa 6.30 and 5.90) (B), relative k_tr was significantly higher for TPM3-R167H and TPM2-null when compared with TPM2-E181K and CTL. In the presence of 3 µm of NEM-S1 (C), the elevated relative k_tr returned to normal values. In the figure, asterisk indicates a significant difference when compared with CTL (P < 0.05).

Figure 6.

Relative myosin, actin and tropomyosin content. The relative contents of myosin, actin and tropomyosin were not different between TPM2-null, TPM3-R167H, TPM2-E181K and CTL, except for a tropomyosin isoform switch in type I [C] and IIa [D] fibers of TPM2-null. In [C], a typical electrophoretic separation of actin, α-, β- and γ-tropomyosin is shown for a single fiber from CTL1 expressing the type I MyHC isoform.

Figure 7.

Immunofluorescence microscopy of skeletal muscle thin filament components. (A) Longitudinal cryosections of skeletal muscle fiber bundles from CTL subjects and TPM2-null, TPM3-R167H and TPM2-E181K patients were phalloidin-stained for F-actin, immunostained for Tmod4 to visualize thin filament pointed ends and immunostained for α-actinin to visualize Z-lines. (B) Higher-magnification views of CTL and TPM3-R167H bundles qualitatively suggest that modest thin filament shortening in TPM3-R167H bundles, as evidenced by slightly wider F-actin-free gaps (H-zones) spanning Tmod4 doublets even when sarcomere length and as determined by the distance between successive α-actinin striations, is held constant. (C) Immunostaining for the nebulin M1M2M3 domain reveals no overt changes in the position of the nebulin M1M2M3 domain in TPM3-R167H fiber bundles. Both thin filament length and the position of the nebulin M1M2M3 domain were quantified using DDecon (Table 2). Bars, 3 μm. Yellow brackets indicate thin filament arrays between F-actin-free H-zones.