Tropomyosin D137L of reduced flexibility increases submaximal calcium-activated tension in skeletal muscle myofibrils after troponin-tropomyosin removal and reconstitution

Tropomyosin (Tm) is a coiled-coil alpha-helix regulating the cooperative activation of muscle contraction by the thin filament. Tm forms strands along actin filaments which azimuthally move between switched-on/off locations under the influence of Ca2þ -troponin (Tn) and strong crossbridge formation. The flexibility of Tm strongly influences its movement so that the relaxation-activation mechanism within the thin filament may be critically modulated. Here we study the mechanical consequences of the presence of chicken recombinant alpha tropomyosin, D137L, in rabbit skeletal muscle myofibrils, previously characterized in vitro (Sumida et al., 2008). Endogenous Tm and Tn are replaced into rabbit skeletal muscle myofibrils (Siththanandan et al., 2009) with recombinant rabbit Tn (WT) and chicken alphaTm (WT and D137L). SDSPAGE analyses of samples of myofibrils show that Tm-Tn replacement was about 90%. Force recordings from small bundles of myofibrils (Tesi et al., 2002) show that at saturating [Ca2þ ] (pCa 4.5), maximal tension was not affected by Tm flexibility nor were the rates of force activation (kACT) and force redevelopment (kTR). However, at submaximal [Ca2þ ] (pCa 5.9), the presence of D137L Tm significantly increases (about 15-20%) both force and kinetics of force generation. Consistently, force-pCa curves obtained from myofibrils replaced with chicken D137L Tm showed a 0.15 increase in pCa50 compared to WT. This result supports the hypothesis (Sumida et al., 2008) that the increased flexibility imparted to the Tm coiled-coiled structure by Asp at 137 avoids excessive turning-on of the system at the high physiological myosin concentrations. Supported by Telethon-Italy GGP07133, STREP Project ‘‘BIGHEART’’ 241577 EC and NIH HL22461.