Mutations in the central part of α Tropomyosin molecule alter Ca2+ sensitivity and tension relaxation of skeletal muscle myofibrils after Troponin-Tropomyosin removal and reconstitution

Tropomyosin (Tm) is a α-helical coiled-coil actin-binding protein regulating the cooperative activation of muscle contraction. Previous observations reported the presence in the central part of Tm of highly conserved non canonical residues Asp-137 and Gly-126 modulating Tm conformational mobility . Replacement of these residues by canonical ones (Leu and Arg, respectively) stabilizes the middle part of Tm decreases molecular flexibility and modulates the calcium sensitivity of in vitro regulated acto-myosin systems (Sumida et al., 2008; Nevzorov et al., 2011). Here, we investigated the functional impact of recombinant α Tm mutants previously characterized in vitro and carrying one (D137L) or both stabilizing substitutions (D137LG126R) on the mechanical behaviour of skeletal myofibrils. Endogenous Tm and Tn were replaced into rabbit psoas myofibrils with purified Tn (sk Tn) and recombinant α Tm (WT, D137L and D137LG126R) Force recordings from myofibrils showed that at saturating [Ca2+], maximal isometric tension and the rates of force activation (kACT) and force redevelopment (kTR) were slightly or not significantly affected by Tm mutations. Interestingly, a clear effect was observed on force relaxation parameters: D137LG126R reconstituted myofibrils showed prolonged duration of the slow phase of relaxation and a decreased rate of the fast phase compared to WT. This effect was smaller in single D137L myofibrils. Tm mutations also strongly decrease slack sarcomere length (s.l.) at sub-activating [Ca2+] as well as they increase the steepness s.l.- passive tension relation. These effects were reversed by 10 mM BDM, supporting that single and double Tm mutations compromise the full inhibition of acto-myosin interactions in the absence of Ca2+. These data support the hypothesis that flexibility of the Tm coiled-coiled structure imparted by Asp-137 and Gly-126 critically modulate the turning–on/off of the thin filament system and muscle activation/relaxation dynamics.