Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy

Abstract

Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, 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 (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.

Current and future therapeutic approaches to the congenital myopathies

“…These studies have revealed that the absence of certain proteins (e.g. nebulin or skeletal alpha-actin), or the presence of mutated amino acids in skeletal alpha-actin and tropomyosin, alter Ca 2+ binding, thin filament conformation and/or the interaction between myosin and actin molecules [86,95,[97][98][99][100][101][102][103][104][105][106][107][108], resulting in reduced force generation at the sarcomere and, ultimately, skeletal muscle weakness.…”

KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

“…The Previous work has shown definitively that mutations in NEB in patients and loss of Neb in mice results in NM, suggesting that decreased NEB in KO mice could contribute to the NM phenotype (1,4,(36)(37)(38). A pervading belief is that NEB acts as a molecular ruler to regulate thin filament lengths based on shortened thin filament lengths seen in Neb-deficient mice (36,37,47). However, more recent studies argue that NEB deficiency contributes directly to a loss in muscle contractility due to dysregulation of actin-myosin crossbridge formation (45,47,48).…”

“…A pervading belief is that NEB acts as a molecular ruler to regulate thin filament lengths based on shortened thin filament lengths seen in Neb-deficient mice (36,37,47). However, more recent studies argue that NEB deficiency contributes directly to a loss in muscle contractility due to dysregulation of actin-myosin crossbridge formation (45,47,48). Accordingly, we see over a 50% loss in hind limb strength of P1 KO mice, which have approximately 50% reduction in NEB, without any evidence of sarcomere defects.…”

The Small-Molecule Fast Skeletal Troponin Activator, CK-2127107, Improves Exercise Tolerance in a Rat Model of Heart Failure

“…This mechanism of action leads to a significantly higher force output at submaximal levels of motor nerve stimulation in animals and humans (Russell et al, 2012;Hansen et al, 2014). Tirasemtiv and its structural analogs have demonstrated efficacy in improving muscle strength in preclinical disease models of neuromuscular origin, including a mouse model of nemaline myopathy (Lee et al, 2013;Ottenheijm et al, 2013), rat model of myasthenia gravis (Russell et al, 2012), and mouse model of amytrophic lateral sclerosis (Hwee et al, 2014). Furthermore, in clinical trials, tirasemtiv improved respiratory function and other measures of skeletal muscle strength and endurance in patients with amytrophic lateral sclerosis (Shefner et al, 2012), myasthenia gravis, and exercise-limiting calf claudication (Bauer et al, 2014).…”