Tropomyosin flexibility modulates Ca2+ sensitivity of thin filament and affects tension relaxation 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 Ca2+-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. We studied the mechanical consequences of the presence of chicken recombinant alpha tropomyosin, D137L, in rabbit skeletal muscle myofibrils, previously characterized in vitro. Endogenous Tm and Tn were replaced into rabbit skeletal muscle myofibrils with recombinant rabbit Tn (WT) and chicken alpha Tm (WT and L137D). Tm-Tn replacement was about 90 % (SDS-PAGE). Force recordings from small bundles of myofibrils show that at saturating Ca2+ (pCa 4.5), maximal tension was not affected by Tm flexibility nor were the rates of force activation (kACT) and force redevelopment (kTR). Interestingly, D137L Tm decelerates the rate of the fast phase of myofibril force relaxation. This is likely related to the higher ‘‘Ca2+-independent’’ tension observed in the D137L replaced myofibrils and then to the presence of some myosin heads able to cycle in the absence of Ca2+, resulting into slower relaxation and higher ‘‘resting’’ tension. Moreover, at submaximal Ca2+ (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 D137L Tm showed a 0.15 increase in pCa compared to WT and an increased apparent cooperativity. These results suggest that the presence in the sarcomere of D137L Tm could increase fractional occupancy of the thin filament Open State without Ca2+ (stronger affinity for Ca2+ than to the Blocked state) together with an increase in the cooperative unit size expected from a decrease in Tm flexibility. This result supports the hypothesis 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 STREP Project ‘‘BIG-HEART’’, 241577 EEC and NIH HL22461.