Mutations in the central part of a Tropomyosin molecule alter Ca2+ sensitivity and tension relaxation of skeletal muscle myofibrils after troponin–tropomyosin removal and reconstitution

Tropomyosin (Tm) is a a-helical coiled-coil actin-binding protein regulating muscle contraction. Previous observations suggested that the highly conserved non canonical residues Asp-137 and Gly-126 in the central part of Tm confer instability. Replacement of these residues by canonical ones (Leu and Arg, respectively) decreases molecular flexibility and modulates the Ca2+ sensitivity of in vitro regulated actomyosin systems (Sumida et al. J Biol Chem 283:6728–6734, 2008; Nevzorov et al. J Biol Chem 286:15766–15772, 2011). Here, we investigated the functional impact of recombinant a Tm carrying one (D137L) or both (D137L/G126R) stabilizing substitutions on the mechanical behavior of skeletal myofibrils. Endogenous Tm and Tn were replaced into rabbit psoas myofibrils (Scellini et al. Adv Exp Med Biol 682:163–174, 2010) with purified Tn and recombinant a Tm (WT,D137L and D137L/G126R). Force recordings from myofibrils (15 C) showed the increase in Ca2+ sensitivity expected from in vitro studies. At saturating [Ca2+] maximal isometric tension and the rates of force activation (kACT) and redevelopment (kTR) were not significantly affected by Tm mutations. Interestingly, a clear effect was observed on force relaxation: D137L/G126R 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 D137L myofibrils. Tm mutations also strongly decreased slack sarcomere length (SL) at sub-activating [Ca2+] as well as they increased the steepness of the SL—passive tension relation. These effects were reversed by 10 mM BDM, suggesting 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 critically modulates the turning off of the thin filament system and muscle relaxation dynamics. Supported by PRIN 2010–2011 of the Ministero Universita` e Ricerca (MIUR).