The differential gene expression profiles of proximal and distal muscle groups are altered in pre-pathological dysferlin-deficient mice

doi:10.1016/j.nmd.2005.09.002

Neuromuscular Disorders

Volume 15, Issue 12, December 2005, Pages 863-877

https://doi.org/10.1016/j.nmd.2005.09.002 Get rights and content

Abstract

The selective pattern of muscle involvement is a key feature of muscular dystrophies. Dysferlinopathy is a good model for studying this process since it shows variable muscle involvement that can be highly selective even in individual patients. The transcriptomes of proximal and distal muscles from wildtype C57BL/10 and dysferlin deficient C57BL/10.SJL-Dysf mice at a prepathological stage were assessed using the Affymetrix oligonucleotide-microarray system. We detected significant variation in gene expression between proximal and distal muscle in wildtype mice. Dysferlin defiency, even in the absence of pathological changes, altered this proximal distal difference but with little specific overlap with previous microarray analyses of dysferlinopathy. In conclusion, proximal and distal muscle groups show distinct patterns of gene expression and respond differently to dysferlin deficiency. This has implications for the selection of muscles for future microarray analyses, and also offers new routes for investigating the selectivity of muscle involvement in muscular dystrophies.

Introduction

Microarray analysis as a tool to study muscular dystrophy has been used in an increasing number of studies in both patients and animal models with mutations in a variety of different muscular dystrophy associated genes. These studies have identified some secondary changes, which appear to be common to muscular dystrophy in general. The compilation of particular expression profiles from patients and animal models of specific types of muscular dystrophy may eventually delineate reproducible ‘molecular signatures’ of disease.

While it is well recognised that a characteristic and subtly different pattern of muscle involvement can be observed in various forms of muscular dystrophy, the mechanism by which some muscles are apparently more or less susceptible to the effects of the underlying mutation remains entirely unknown. One possibility is that a broad range of normal expression profiles in different skeletal muscle groups might condition their response to disease. If the patterns of gene expression in these muscle groups responded differently to the presence of a muscular dystrophy associated mutation, this would provide support for this hypothesis.

Dysferlinopathy (MIM*603009) is a good model of variability in muscular dystrophy. Mutations in dysferlin give rise to a number of clinically distinct presentations including a proximal, limb-girdle form of muscular dystrophy and two forms of distal myopathy, Miyoshi myopathy (MM) and distal myopathy with anterior tibial involvement (DMAT) [1], [2]. A very highly selective pattern of muscle involvement is seen in individual affected patients and identical dysferlin mutations have been shown to cause a variable phenotype within the same family or population group [2], [3]. However no genetic variants, which modify this phenotype are currently known. This variable but selective muscle involvement in dysferlinopathy suggests that it can act as a paradigm for understanding selectivity of muscle involvement in muscular dystrophies in general. Due to the obvious barriers to studying different human muscle groups we chose to study gene expression patterns in two muscle groups in a new mouse model of dysferlinopathy and its matched wildtype strain.

The SJL mouse strain, a well characterised model of dysferlinopathy, has a number of phenotypes unrelated to the dysferlin mutation. Since SJL descends from a wild-derived strain of Swiss mice which are genetically distinct from the common laboratory mouse [4], [5], [6] there is not an appropriate control strain available. In order to overcome these disadvantages we transferred the dysferlin mutation from SJL onto C57BL/10 by repeated backcrossing. Affected mice develop a muscular dystrophy which appears comparable to human dysferlinopathy and shows more severe pathological changes in the affected limb muscles than the SJL strain.

We have used oligonucleotide microarray analysis to demonstrate that gene expression indeed varies in the proximal and distal musculature of wildtype mice and that dysferlin deficiency even at a prepathological stage alters this pattern. Changes in gene expression in the mutant animals show some overlap with previously reported studies of muscular dystrophy, indicating that changes at the transcript level are an early marker of the dystrophic process.

Section snippets

Animals

The dysferlin mutation from SJL was detected by PCR using standard techniques as previously described [5]. C57BL/10.SJL-Dysf mice were generated by repeatedly backcrossing the dysferlin mutation from the SJL strain onto C57BL10ScSnHim. Offspring of N2- and all further generations were genotyped for the dysferlin mutation and only heterozygous animals were used for breeding. To obtain homozygous animals to be used for experiments, offspring from generations N8 and N9 were mated with littermates.

Characterisation of C57BL/10.SJL-Dysf mice

Dysferlin deficient SJL/HanHim mice were crossed to C57BL/10JHim and repeatedly backcrossed to C57BL/10. Following more than ten generations of backcrossing this strain should be more than 99.5% homozygous for C57BL/10 alleles and was therefore designated C57BL/10.SJL-Dysf. Very few dystrophic changes and no obvious inflammatory infiltrates could be detected in either proximal or distal muscles from the C57BL/10.SJL-Dysf animals at 21 days of age (Fig. 1A and B), but significant muscle

Discussion

It has been suggested that baseline transcriptional differences between wild type muscle groups may be a determinant of muscular dystrophy phenotypes [20]. There is increasing evidence that there are muscle group-specific transcriptomes and that anatomically distinct skeletal muscles may be much more different than has been previously appreciated [20], [21], [22], [23], [24]. Our hypothesis was that muscle specific transcriptomes might react differently in the presence of a disease associated

Summary and conclusions

This study has shown that considerable differences exist in the baseline expression profile between proximal and distal muscle groups, despite similar fibre type composition and histological appearance. In fact, greater disparity in expression was detected between wild-type proximal and distal than between the mutant and wild-type proximal muscles. It is likely that the differences we detected in the baseline transcription probably represent only a subset of the actual transcriptome

Acknowledgements

This work was carried out with financial support of the Deutsche Forschungsgemeinschaft (PO 699/2-1 to M.v.d.H.). We gratefully acknowledge the support of the Muscular Dystrophy Campaign (UK), Newcastle-upon-Tyne Hospitals Special Trustees, Association Francais contre les Myopathies and the Austrian Muscle Research Found (F.O., R.E.B). Many thanks to Alison Trainer for critical reading of the manuscript and to Richard Charlton for technical assistance.

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SJL/J mice were backcrossed onto the C57BL/10 background [14]. Despite initial claims of a more severe phenotype [62], the dystrophy in these B10.SJL-Dysfim/AwaJ mice (Table 1) appears to be very similar to the observed pathology in SJL/J. Dysferlin protein levels were also similar, 20–30% of control levels in neonatal muscle and 10% in adults (unpublished), compared with 15% in adult SJL/J muscle [14]. CK levels are also elevated [16], contrary to previous reports documenting normal serum CK but elevated pyruvate kinase levels [62].
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The differential gene expression profiles of proximal and distal muscle groups are altered in pre-pathological dysferlin-deficient mice

Abstract

Introduction

Section snippets

Animals

Characterisation of C57BL/10.SJL-Dysf mice

Discussion

Summary and conclusions

Acknowledgements

J Mol Diagn

Neuromuscul Disord

J Neuroimmunol

J Biol Chem

Neuromuscul Disord

Neuromuscul Disord

Cell

Trends Cell Biol

Biochim Biophys Acta

Neuromuscul Disord

Int J Dev Neurosci

Am J Pathol

Dysferlin, a novel skeletal muscle gene, is mutated in miyoshi myopathy and limb girdle muscular dystrophy

Nat Genet

Identical mutation in patients with limb girdle muscular dystrophy type 2B or miyoshi myopathy suggests a role for modifier gene(s)

Hum Mol Genet

Muscular dystrophy due to dysferlin deficiency in libyan jews, clinical and genetic features

Brain

Immunological disorders in SJL mice

Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B

Nat Genet

Cloning of the mouse dysferlin gene and genomic characterisation of the SJL-dysf mutation

NeuroReport

The lightcycler: a microvolume multisample fluorimeter with rapid temperature control

Biotechniques

Continuous fluorescence monitoring of rapid cycle DNA amplification

Biotechniques

Quantification of low-copy transcripts by continuous SYBR green I monitoring during amplification

Biotechniques

Clinical course in 30 dysferlinopathy patients: relevance of fiber type regenerative process and inflammatory response

Neurology

A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice

Hum Mol Genet

Defective membrane repair in dysferlin-deficient muscular dystrophy

Nature