Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle

Abstract

Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, thin filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die within 8–11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle thin filament lengths were up to 25% shorter compared with wild type, and thin filaments were uniform in length both within and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.

Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

“…Therefore, our results suggest that nebulin blocks phalloidin binding sites along the length of the actin thin filament. Knockdown of nebulin also resulted in an increase in the number of cells with nonstriated actin, an ϳ10% decrease in sarcomere lengths (Z-to-Z line distance) and an ϳ20% decrease in thin filament lengths (data not shown); the latter result is similar to what was reported previously in skeletal muscle isolated from nebulin knockout mice (Bang et al, 2006;Witt et al, 2006).…”

“…Therefore, our results suggest that nebulin blocks phalloidin binding sites along the length of the actin thin filament. Knockdown of nebulin also resulted in an increase in the number of cells with nonstriated actin, an ϳ10% decrease in sarcomere lengths (Z-to-Z line distance) and an ϳ20% decrease in thin filament lengths (data not shown); the latter result is similar to what was reported previously in skeletal muscle isolated from nebulin knockout mice (Bang et al, 2006;Witt et al, 2006).Remarkably, nebulin knockdown also resulted in a decrease of anti-CapZ staining intensity at the Z-disc, indicating that nebulin is required for Z-disc localization of CapZ ( Figure 8A). Two other integral Z-disc proteins, ␣-actinin ( Figure 8A) and N-terminal titin (data not shown), were only slightly affected by the knockdown of nebulin based on immunofluorescence staining, revealing that the Z-discs were intact.…”

“…Myofibrillogenesis was otherwise remarkably normal, although the increased presence of nonstriated thin filaments (indicative of elongated and/or disorganized filaments), decreased sarcomere lengths and myofibril misalignment suggests that myofibrillogenesis may have been slightly delayed. Nebulin knockdown also caused a decrease in thin filament lengths comparable with that observed in the skeletal muscle of nebulin knockout mice (Bang et al, 2006;Witt et al, 2006), which is consistent with nebulin functioning to determine the final lengths of the thin filaments.Using nebulin gene knockout mice, Witt et al (2006) found a much more subtle CapZ phenotype than the phenotype that we see here with siRNA-mediated knockdown. In their study, immunofluorescence microscopy revealed that CapZ was less intense and more broadly distributed at the Z-disc.…”

“…Although the size and susceptibility of nebulin to proteolysis have made some functional approaches very difficult, the evidence for its role in determining the final lengths of thin filaments is strong (for reviews, see Trinick, 1994;McElhinny et al, 2003;Horowits, 2006). Two independent studies of nebulin knockout mice confirm the importance of nebulin in thin filament length regulation (Bang et al, 2006;Witt et al, 2006). In both studies, skeltal muscle thin filaments were shorter.…”

“…Nebulin protein has been found in skeletal and cardiac muscle (although at lower levels in the latter), and transcripts have also been found in other tissues (Fock and Hinssen, 2002;Kazmierski et al, 2003;Bang et al, 2006). A single molecule of nebulin spans the entire length of the thin filament.…”