Abnormalities in the expression of nebulin in chromosome-2 linked nemaline myopathy

doi:10.1016/S0960-8966(00)00172-3

Neuromuscular Disorders

Volume 11, Issue 2, March 2001, Pages 146-153

https://doi.org/10.1016/S0960-8966(00)00172-3 Get rights and content

Abstract

Nemaline myopathy is clinically and genetically heterogeneous. The most common autosomal recessive form affecting infants (NEM2) links to chromosome 2q, and is caused by mutations in the gene for nebulin. We have examined the immunocytochemical expression of nebulin in skeletal muscle in 11 cases of nemaline myopathy, from ten families, with linkage compatible to chromosome 2q.22, the locus for nebulin. Mutations in the gene for nebulin have been found in eight of these cases. Immunolabelling with polyclonal antibodies to C-terminal regions of nebulin was compared with antibodies to fibre-type-specific myofibrillar proteins, including myosin heavy chain isoforms and α-actinin isoforms. No cases showed a complete absence of C-terminal nebulin, and no enhancement of labelling of the rods was seen with conventional fluorescence microscopy. In control muscle an antibody to the M176–181 repeat region of nebulin showed higher expression in fibres with slow myosin, while ones to the serine-rich domain and to the SH3 domain showed uniform expression. In some cases of nemaline myopathy differences in these patterns were observed. Two siblings with a homozygous mutation in exon 185, that produces a stop codon, showed an absence of labelling only with the SH3 antibody, and other cases showed uneven labelling with this antibody or some fibres devoid of label. Fibre type correlations also showed differences from controls, as some fibres had a fast isoform of one protein but a slow isoform of another. These results indicate that analysis of nebulin expression may detect abnormalities in some cases linked to the corresponding locus and may help to direct molecular analysis. In addition, they may also be relevant to studies of fibre type plasticity and diversity in nemaline myopathy.

Introduction

At least four genes are responsible for the various forms of nemaline myopathy [1]. Three of these genes have been identified: the genes for slow α-tropomyosin on chromosome 1q21, nebulin on chromosome 2q.22, and actin on chromosome 1q42. All familial cases with the ‘typical’ form, with onset in infancy and slow, or no, progression [1], have shown linkage compatible to the nebulin region on chromosome 2q; mutations in the nebulin gene have been identified in some of these cases [2].

Nebulin is a very large protein (600–900 kDa) that spans the thin filaments of the sarcomere and is thought to act as a molecular ruler for actin. Nebulin shows species and developmental diversity, and several isoforms are expressed [3]. We have studied the immunocytochemical expression of nebulin in cases of children and young adults with nemaline myopathy linked to 2q, using antibodies to C-terminal regions located in the Z-disc. As isoforms of nebulin relate to muscle fibre types [3], we also studied the expression of isoforms of other myofibrillar proteins, to examine possible abnormalities.

Section snippets

Methods

Nemaline myopathy was diagnosed in 11 cases aged from 5 days to 24 years at age of biopsy, by clinical assessment and by the presence in skeletal muscle biopsies of the typical rod-like structures that stain red with the modified Gomori trichrome technique [4]. Two cases were siblings. In some cases a diagnosis had been reached on an earlier biopsy and a second sample was obtained for nebulin studies. Nine of the patients met the clinical criteria for the ‘typical’ congenital form that presents

Nebulin in control muscle

A striation pattern was visible in longitudinally orientated fibres with all three antibodies to nebulin (Fig. 2). Previous studies have shown that the C-terminal domain of nebulin, that contains the epitopes for these antibodies, is associated with the Z-disc (Fig. 1) [3]. The antibodies to the serine-rich and SH3 domains labelled all fibres uniformly, but the antibody to M176–181 showed higher labelling of fibres expressing slow myosin (Fig. 2, Fig. 3).

Nebulin in 2q cases of nemaline myopathy

No enhancement of nebulin labelling of

Discussion

The cases of nemaline myopathy presented all show linkage results compatible with linkage to the region of the nebulin gene on chromosome 2q, and in eight patients one, or both, mutations have been identified; six of these mutations are expected to be truncating [2]. The two patients with missense mutations are expected to have another, as yet unidentified mutation; it remains to be seen whether these will also be truncating. Our results show that despite truncating mutations nebulin can be

Acknowledgements

We thank Mrs L. Feng for technical assistance and Mrs K Davidson for photographic assistance. We are grateful to Drs Fardeau, Urtizberea and Odent for referring the biopsy of patient 1, and Professor Hanefeld and Dr Bönnemann for referring the biopsy of the patient 4 to us. KP is supported by grants to CWP from the Association Francaise contre les Myopathies, the Swedish Cultural Foundation of Finland, the Finska Läkaresällskapet, and the Medicinska understödsföreningen Liv och Hälsa. For

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Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.
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Citation Excerpt :
Biopsies from patients with recessive NEB mutations usually do not show an absence of protein. Exceptions to this are seen in some severe neonatal cases where an absence is detectable with immunostaining for the nebulin SH3 domain-specific antibody.52 Rods restricted to slow/type I fibers are seen in TPM3-related cases, as α-tropomyosin 3 is the predominant isoform in slow/type I skeletal muscle fibers.53
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Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2)
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Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is clinically characterized by muscle weakness. However, the mechanisms underlying this weakness are poorly understood. Here, we studied the contractile phenotype of skeletal muscle from NM patients with nebulin mutations (NEM2). SDS–PAGE and Western blotting studies revealed markedly reduced nebulin protein levels in muscle from NM patients, whereas levels of other thin filament-based proteins were not significantly altered. Muscle mechanics studies indicated significantly reduced calcium sensitivity of force generation in NM muscle fibers compared to control fibers. In addition, we found slower rate constant of force redevelopment, as well as increased tension cost, in NM compared to control fibers, indicating that in NM muscle the rate of cross-bridge attachment is reduced, whereas the rate of cross-bridge detachment is increased. The resulting reduced fraction of force generating cross-bridges is expected to greatly impair the force generating capacity of muscle from NM patients. Thus, the present study provides important novel insights into the pathogenesis of muscle weakness in nebulin-based NM.

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Abnormalities in the expression of nebulin in chromosome-2 linked nemaline myopathy

Abstract

Introduction

Section snippets

Methods

Nebulin in control muscle

Nebulin in 2q cases of nemaline myopathy

Discussion

Acknowledgements

Neuromusc Disord

J Mol Med

Neuromusc Disord

Neuromusc Disord

Neuromusc Disord