Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers

Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb⁺) and conditional Neb knockout (Neb^-) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca²⁺ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb⁺ fibers and 68% in Neb^- fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb- fibers than in Neb⁺ fibers, the increase in force per CB in the presence of Neb is ~1.5 fold. The equilibrium constant of the CB detachment step (K₂), its rate (k₂), and the rate of the reverse force generation step (k_-4) are larger in Neb⁺ fibers than in Neb- fibers. The rates of the force generation step (k₄) and the reversal detachment step (k_-2) change in the opposite direction. These effects can be explained by Le Chatelier's principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.