Thin-filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

Thin-filament regulation of isometric force redevelopment (ktr) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca2+-activated force (rFmax) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal ktr was unaffected until rFmax was <0.2 of pre-extracted Fmax. In contrast, reconstitution with cTnC or xsTnC reduced maximal ktr to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rFmax of 0.68 ± 0.03 and 0.25 ± 0.02 Fmax. The ktr–pCa relation of fibres containing sTnC: xxsTnC mixtures (rFmax > 0.2 Fmax) was little effected, though ktr was slightly elevated at low Ca2+ activation. The magnitude of the Ca2+-dependent increase in ktr was greatly reduced following cTnC or xsTnC reconstitution because ktr at low levels of Ca2+ was elevated and maximal ktr was reduced. Solution Ca2+ dissociation rates (koff) from whole Tn complexes containing sTnC (26 ± 0.1 s−1), cTnC (38 ± 0.9 s−1) and xsTnC (50 ± 1.2 s−1) correlated with ktr at low Ca2+ levels and were inversely related to rFmax. At low Ca2+ activation, ktr was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating ktr at any [Ca2+] and suggest Ca2+ activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment

Single-myosin crossbridge interactions with actin filaments regulated by troponin-tropomyosin

Striated muscle contraction is governed by the thin filament regulatory proteins troponin and tropomyosin. Here, we investigate the molecular mechanisms by which troponin–tropomyosin inhibits myosin's interactions with the thin filament in the absence of calcium by using a laser trap. The displacement events for a single-myosin molecule interacting with a reconstituted thin filament were shorter (step size = 5 nm) and prolonged (69 ms) compared with actin alone (11 nm and 26 ms, respectively). However, these changes alone do not account for the degree of inhibition of thin filament movement observed in an ensemble assay. Our investigations of single- and multiple-myosin molecules with regulated thin filaments suggest the primary basis for this inhibition derives from a ≈100-fold decrease in the probability of myosin attaching to actin. At higher myosin concentrations, short bursts of motility are observed in a laser trap consistent with the strong binding of a single-myosin crossbridge, resulting in cooperative binding of other cycling crossbridges. We confirmed this cooperativity in the in vitro motility assay by observing thin filament translocation in the absence of calcium but at low [ATP], consistent with rigor activation. We have developed a simple mechanistic model that reproduces and provides insight into both the observed single-myosin molecule and ensemble data in the absence of Ca(2+). These data support the hypothesis that thin filament inhibition in the absence of Ca(2+) is largely achieved by modulating the rate of attachment and/or transition from the weakly to strongly bound state