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

Abstract

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.

Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function

“…This implies that the Z-disk fragment assists primarily in the stabilization of the Z-disks. Neb cKO mice have shortened thin filament lengths and decreased actomyosin interactions [32,33,36,37,82,83], which do not appear to be positively impacted by the presence of the Z-disk fragment. As nebulin is a massive, multi-functional protein, treatment of nemaline myopathy might not be attainable to a sufficient degree with only a Z-disk fragment and additional studies will be needed first.…”

Nebulin: big protein with big responsibilities

“…They found that the ratio of myofiber stiffness during maximum activation and rigor is similar in Neb cKO and wild-type animals (62% vs. 68%, respectively) suggesting that an equal number of cross-bridges are activated during contraction. Thus, Kawai et al provides evidence which opposes previous studies, suggesting a reduction in the force generated by each cross-bridge instead of a reduced number of cross-bridges as the underlying cause for contractile dysfunction in nebulin deficient muscle (Kawai et al 2018). However, the data presented by Kawai et al (2018) was obtained exclusively from type 1 fibers obtained from skinned soleus and the data was not normalised to the total number of myofilaments per muscle cross-section.…”

“…This is critical for maintaining Z-disk structure and myofibril alignment, in particular during muscle stretch and activation. Nebulin deficient muscle shows an increase in lateral displacement between myofibrils (which was more pronounced at larger sarcomere lengths) and in sarcomere disruption during forceful contractions (Kawai et al 2018;Tonino et al 2010). Myofibrillar misalignment and myofibril splitting as well as fragmented Z-disks were often observed in Neb KO mice and these abnormalities were only present once muscle had been active after birth (Bang et al 2006).…”

“…As discussed above, changes in thin filament length and structural disorganisation are an obvious cause of weakness as they will reduce the number of available ordered myosin/actin interactions. However, by limiting the influence of abnormal thin filament lengths and sarcomeric disorganisation on contractility measurements, a number of studies provided strong evidence that nebulin is also directly involved in regulating actin-myosin cross-bridge cycling (see Table 2; Bang et al 2006Bang et al , 2009Chandra et al 2009;Joureau et al 2017;Kawai et al 2018;Lawlor et al 2011;Lee et al 2013;Li et al 2015;Ochala et al 2011;Ottenheijm et al 2010Ottenheijm et al , 2013. For example, contractile dysfunction persisted at shorter sarcomere lengths at which the filament overlap allows for efficient force generation (Chandra et al 2009;Ottenheijm et al 2010), and in models with thin filament lengths and sarcomeric structure comparable to healthy muscle (Bang et al 2009;Ochala et al 2011).…”

“…For example, contractile dysfunction persisted at shorter sarcomere lengths at which the filament overlap allows for efficient force generation (Chandra et al 2009;Ottenheijm et al 2010), and in models with thin filament lengths and sarcomeric structure comparable to healthy muscle (Bang et al 2009;Ochala et al 2011). Kawai et al (2018) determined that a force deficit of 69% remains after correcting for the lack in overlap in nebulin deficient fibers (which caused a theoretical reduction in force generating cross-bridges of ~ 75.4% in Neb KO compared to wild-type). Contractile measurements on structurally intact individual myofibrils clearly showed that structural damage of nebulin deficient muscle was not the only factor in causing muscle weakness, but that contractile dysfunction is intrinsic to the contractile apparatus (Ottenheijm et al 2013).…”