Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2)
Introduction
Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy, with an estimated incidence of ∼1 per 50,000 live births (Wallgren-Pettersson, 1990). NM is characterized at the muscle’s ultrastructural level by the presence of rod-shaped structures (nemaline rods) in affected muscle fibers (Morris et al., 1990). Clinically, the most prominent feature of NM is generalized muscle weakness that greatly affects the daily-life activities, and the quality of life of these patients (North et al., 1997).
NM is a genetically heterogeneous disorder of the skeletal muscle thin filament caused by mutations in any one of at least six different genes, all encoding thin filament proteins of the sarcomere: ACTA1 (actin), TPM3 and TPM2 (α- and β-tropomyosin), TNNT1 (troponinT), CFL2 (cofilin-2), and NEB (nebulin), for a review see Sanoudou and Beggs (2001). Despite detailed knowledge of the underlying genetic basis for NM in many patients, the mechanisms underlying muscle weakness in NM patients are poorly understood.
Mutations in the nebulin gene are the most common cause of NM (patients referred to as NEM2), accounting for ∼50% of all NM cases (Pelin et al., 1999). Nebulin is a giant protein (MW ∼800 kDa) expressed at high levels in skeletal muscle. A single nebulin molecule spans the thin filament with its C-terminus anchored at the Z-disk and its N-terminal region directed towards the thin filament pointed end (Wang and Wright, 1988). Previous studies (Witt et al., 2006, Bang et al., 2006) revealed that nebulin-deficient murine muscle fibers have thin filaments that vary in length (Witt et al., 2006) and that are on average shorter than in wildtype muscle (Witt et al., 2006, Bang et al., 2006), supporting the notion that nebulin is important in establishing thin filament length. Thin filament length is an important aspect of muscle function as the extent of overlap between thick and thin filaments determines the sarcomere’s force generating capacity: short thin filaments reduce overlap and impair force generation. In accordance with a role for nebulin in establishing thin filament length, we have shown that similar to the nebulin knockout (KO) mouse model, human NM patients with nebulin-deficiency also have shorter and non-uniform thin filament lengths, which can partly account for the observed muscle weakness in nebulin-based NM (Ottenheijm et al., 2009).
Recent studies on nebulin knockout mouse models suggest that nebulin’s role in muscle function extends beyond a purely structural one, and involves a role in the regulation of cross-bridge cycling kinetics and thin filament activation. It was found that nebulin increases the fraction of force generating cross-bridges that is available in the overlap zone (Chandra et al., 2009, Bang et al., 2009) and enhances the force response to submaximal calcium concentrations (Chandra et al., 2009). Thus, in the nebulin KO mouse model reduced active tension and calcium sensitivity of force generation has been reported. Here we tested whether similar characteristics are present in muscle from NM patients with nebulin gene mutations. We found a reduced rate of force development as well as increased tension cost in nebulin-deficient muscle fibers from NM patients. Moreover, force generation in response to submaximal calcium concentrations was significantly decreased. These findings suggest altered cross-bridge cycling kinetics and thin filament activation in nebulin-deficient fibers from NM patients, and provide a novel mechanism for muscle weakness in nebulin-based NM.
Section snippets
Muscle biopsies from nemaline myopathy patients
Skeletal muscle specimens, remaining from diagnostic procedures or obtained during clinically indicated surgical procedures, were collected from four nemaline myopathy patients following informed consent supervised by the Children’s Hospital Boston institutional review board, and from four unaffected control subjects, and stored frozen and unfixed at −80 °C until use (Table 1). All four NM patients had mutations in the nebulin gene, including three patients who were homozygous for the previously
Nebulin protein levels in NEM2 patients and controls
We studied skeletal muscle of four NM patients with nebulin mutations and four control subjects with no history of skeletal muscle disease (for control subject and patient characteristics see Table 1). Three of the patients had homozygous deletions of exon 55 of the nebulin gene and the fourth patient had a heterozygous single base-pair deletion resulting in a premature stop codon (p.Ser1908AlafxX8) and a second unidentified mutation.
To study protein levels of nebulin, as well as the levels of
Discussion
Although mutations in six different genes have been implicated in NM, mutations in the nebulin gene are the most common cause of NM (Pelin et al., 1999). Recent work indicated that in the absence of nebulin in murine muscle, cross-bridge cycling kinetics are altered to reduce force production (Chandra et al., 2009, Bang et al., 2009), and myofilament calcium sensitivity is reduced (Chandra et al., 2009). Here, we have studied NEM2 patients who have NM caused by nebulin gene mutations, resulting
Acknowledgments
Thanks to Danielle Pier and Hal Schneider for nebulin gene mutation detection and for Eric Rogers for gel and Western blot analysis. Special thanks to all the patients, their family members, and referring physicians and health care providers, particularly genetic counselor Joanne Taylor, MS CGC, without whom this study would not have been possible. This work was supported by a VENI grant from the Dutch Organization for Scientific Research to C.A.C.O, NIH RO1 AR053897 to H.G., NIH R01 AR044345,
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