A Novel Mouse Model of Nebulin-Based Nemaline Myopathy

Abstract

Nebulin - a giant sarcomeric protein - plays a pivotal role in skeletal muscle contractility by regulating thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which NEB exon 55 is deleted (NEBΔex55), a mutation known to frequently occur in NM patients.NEBΔex55 mice are born close to Mendelian ratio's, but show growth retardation after birth. Electronmicroscopy shows nemaline rods - a hallmark feature of NM - in muscle fibers from NEBΔex55 mice. Western blotting studies with nebulin-specific antibodies reveal much reduced nebulin levels in muscle from NEBΔex55 mice. Immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is reduced in muscle fibers from NEBΔex55 mice. In line with reduced thin filament length, the maximal force generating capacity of skinned muscle fibers is reduced in NEBΔex55 mice with a more pronounced reduction at longer sarcomere lengths. Finally, in NEBΔex55 mice the regulation of contraction is impaired, as evidenced by marked changes in cross bridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. This reduced calcium sensitivity was observed only at short sarcomere lengths, suggesting that nebulin might play a role in the length dependence of activation.In conclusion, we have generated the first nebulin-based NM model. Our data indicate that the phenotype of NEBΔex55 mice closely recapitulates that observed previously by us in patients harboring this particular mutation.