Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy

Abstract

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods and undergo fiber-type switching toward oxidative types. Nebulin deficiency causes a large deficit in specific force, and mechanistic studies provide evidence that a reduced fraction of force-generating cross-bridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas (CSAs), worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even have hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin

“…To address the role of nebulin in determining cardiac muscle TFL, a mouse model was used in which exon 3 of the nebulin gene (containing the start codon) was floxed [15]. In pilot studies the floxed mouse was crossed with a mouse that expresses Cre recombinase under control of the muscle creatine kinase promoter (MCK Neb cKO), which causes deletion of nebulin in both skeletal and cardiac muscles (Figure S1 A and B).…”

“…S1E, left). However, because the MCK Neb cKO model shows severe skeletal muscle myopathy [15] it was important to exclude systemic changes caused by the skeletal muscle myopathy. We therefore also crossed the floxed mouse with the cardiac-specific αMHC Cre mouse and studied offspring heterozygous for the αMHC Cre transgene and homozygous for the nebulin floxed allele (αMHC Neb cKO).…”

“…A cardiac specific nebulin knockout mouse was produced by crossing the mouse in which loxP sites flank exon 3 of the nebulin gene (containing the start codon) [15] with a model that expresses Cre-recombinase under control of the cardiac specific αMHC promotor (tg(Myh6-cre)2182Mds/J from Jackson Laboratory). Details of the knockout strategy are provided in Figure S1A and B.…”

Exome sequencing reveals a nebulin nonsense mutation in a dog model of nemaline myopathy

“…Mouse models harboring mutations in ACTA1 (Nowak et al 2009; Nguyen et al 2011; Ravenscroft et al 2011), LMOD3 (Tian et al 2015), KLHL40 (Garg et al 2014), NEB (Bang et al 2006; Witt et al 2006; Li et al 2015), and TPM3 (Corbett et al 2001; de Haan et al 2002) are available for study of NM subtypes, but no large animal models have been described. Dog models have been instrumental in advancing therapeutic strategies for hereditary muscle disorders such as Duchenne muscular dystrophy and myotubular myopathy (Kornegay et al 2012; Childers et al 2014).…”

Sarcomere Dysfunction in Nemaline Myopathy

“…As a consequence, nebulin-deficient muscle has a narrower functional length range and operates at a shorter optimal length for force generation compared to wildtype muscle [36]. This observation was confirmed in various nebulin-deficient mouse models, including a model with the Neb Δ exon55 mutation [30], a frequently found mutation in NM [37], and in a conditional nebulin-deficient mouse model [31]. In line with this observation in nebulin-deficient mouse muscle, muscle fibers from NM patients with mutations in NEB , leading to nebulin deficiency, have shorter thin filament lengths and shorter optimal sarcomere length for force generation (Fig.…”