Nebulin alters cross-bridge cycling kinetics and increases thin filament activation. A novel mechanism for increasing tension and reducing tension cost

Nebulin is a giant filamentous F-actin-binding protein (∼800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (-log[Ca²⁺]) and force-ATPase relations, as well as the rate of tension re-development (k_tr) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons ^-1 mm^-1 s^-1) and reductions in k_tr (4.7 versus 7.3 s^-1), calcium sensitivity (pCa₅₀ 5.74 versus 5.90), and cooperativity of activation (n_H 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.