Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress
Introduction
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common form of inherited muscle disease following Duchenne and myotonic dystrophy [1]. FSHD is characterized by an asymmetric, progressive weakness of the facial and pectoral girdle, often with subsequent involvement of the foot dorsiflexors and pelvic girdle muscles [2]. The disorder is inherited in an autosomal dominant fashion with nearly complete penetrance [1]. FSHD results from a deletion of integral copies of a 3.3 kb tandem repeat unit on the long arm of chromosome 4 (D4Z4) [3]. A ‘short’ EcoRI fragment (<38 kb) containing D4Z4 and the probe locus p13E11 segregates with the disease, while the size of this polymorphic locus in the normal population ranges between 38 and 300 kb [4]. All known genes in this region map proximal to the 3.3 kb repeats. The severity of the disease is highly variable but a general inverse correlation exists between the size of the EcoRI fragment and the presentation of severe symptoms [5], [6]. Pleiotropic manifestations of FSHD include progressive high tone hearing loss, retinal vasculopathy and, in several patients with only a single 3.3 kb repeat, severe neurological deficits including mental retardation and epilepsy [1], [7], [8].
Although the specific gene(s) responsible for FSH muscular dystrophy have not been identified, the disease is postulated to result from a perturbation in telomeric chromatin structure. D4Z4 exhibits many characteristics of heterochromatin such as hypermethylation, regions of sequence identity to known classes of heterochromatic repeats, cross-hybridization to regions known to be heterochromatic, histone H4 subtype association similar to telomeric regions, and a genomic location immediately adjacent to the telomere [9], [10], [11], [12], [13]. Integral deletions of D4Z4 likely disrupt the heterochromatic nature of the 4q subtelomere, with consequent dysregulation of genes residing proximal to the 3.3 kb tandem repeat. This position effect hypothesis in FSHD is further supported by data demonstrating that translocation of a ‘small’ EcoRI fragment containing highly homologous 3.3 kb-like repeats from chromosome 10q26 onto 4qter results in FSHD as well [14].
Attempts to identify the gene(s) involved in FSHD have been thwarted by the repetitive nature of the genomic region and by the unusual molecular genetics underlying this muscular dystrophy. However, the recent development of oligonucleotide microarray (GeneChip) technology presents a powerful tool to examine cellular pathophysiology [15]. Expression monitoring of thousands of genes simultaneously provides a biochemical ‘fingerprint’ of disease. This approach is especially useful for diseases, such as FSHD, in which dysregulated gene expression is thought to be causal. Concomitantly, methods for the propagation and differentiation of muscle satellite cells as myoblast cell lines have allowed investigation of the early stages of muscle development in culture. The current study takes advantage of these developments to reveal biochemical pathways altered in FSHD.
Interestingly, our preliminary studies of myoblasts from FSHD patients identified a phenotype that is morphologically distinct from normal myoblasts. The FSHD myoblasts, as described in this study, exhibit a ‘vacuolar/necrotic’ phenotype. ‘Necrosis’ is a term that is frequently used to describe the morphologic changes in cells which are not undergoing programmed cell death/apoptosis. TUNEL+ staining in FSHD myoblasts reveals no evidence that apoptosis is occurring (Figlewicz, unpublished data). Therefore the degradative process is more accurately categorized as necrosis, and the morphologic appearance of some of the myoblasts reflects the changes typically seen in necrosis: swelling of the nucleus and the cytoplasm, and vacuolation. The necrotic/vacuolar phenotype of FSHD myoblasts indicates that aberrant gene expression may occur early on in FSHD skeletal muscle development, and warrants global gene expression profiling of myoblasts prior to differentiation into myotubes.
Preliminary studies in our laboratory also indicated that a necrotic phenotype similar to that of FSHD myoblasts could be elicited in normal myoblasts by a 24-h exposure to the superoxide anion generator paraquat but not the protein kinase C inhibitor, staurosporine. As previous studies by others have already demonstrated that dystrophin-deficient (mdx) myoblasts do not exhibit increased susceptibility to oxidative stress [16], we chose to look at the response to oxidative stress in other muscle diseases for which mutations in the dystrophin-dystroglycan complex do not underlie the pathology. Thus, this study was undertaken to examine not only the susceptibility to oxidative stress in FSHD myoblasts, but to determine whether this is a defining characteristic of FSHD proliferative-stage myoblasts or whether we could associate the defect with another functional class of muscle disease. Disease controls were therefore chosen specifically to examine (1) muscle diseases with an inflammatory component (dermatomyositis), (2) ion-channel disorders (myotonia congenita), (3) adult-onset dominant myopathies with inclusions (desmin storage myopathy) and (4) trinucleotide repeat disorders (myotonic dystrophy 2/PROMM). While an inflammatory process has been observed in some cases of FSHD [1], the presence and degree of inflammation is not a consistent feature in this disease. An inflammatory myopathy is therefore included amongst other muscle disease controls with distinct pathologic mechanisms.
The current study examines specific characteristics of FSHD myoblasts, including gene expression patterns, morphological phenotype and vulnerability to oxidative stress, and enables the characterization of FSHD as a distinct class of myopathy with aberrations in early stages of myogenesis.
Section snippets
Myoblast cell lines
Myoblast cell lines were derived from skeletal muscle biopsies taken from four FSHD patients, four myopathies other than FSHD (disease controls) and three individuals with no muscle disease (normal controls). Table 1 lists the sex, age, EcoRI fragment size (FSHD cell lines) and type of myopathy (disease controls) for each of these cell lines. All biopsies were obtained with informed consent, in accordance with protocols approved by the University of Rochester Research Subjects Review Board.
Expression of oxidative stress and extracellular matrix transcripts are altered in FSHD
Several categories of genes were consistently altered in FSHD myoblasts. These transcripts include enzymes involved in oxidative stress resistance, as well as extracellular matrix components (ECM) and tissue remodeling factors. The full set of expression data from each of the individual myoblast cell lines can be accessed electronically at http://www.ucihs.uci.edu/biochem/Winokur Specific transcripts involved in oxidative stress which are down-regulated in the FSHD myoblasts are listed in Table
Discussion
The pathogenetic mechanism in facioscapulohumeral muscular dystrophy remains complex and elusive 10 years after the mutation was initially described. This is in large part due to the unusual nature of the mutation itself, a tandem deletion of subtelomeric repeats which likely does not directly involve the responsible gene or genes. Recent attempts to understand this disorder have focused on identifying cellular pathways involved in FSHD in addition to unraveling the genetic mechanism. This
Acknowledgments
This study was supported by grants from the Muscular Dystrophy Association-USA (S.T.W.; R.T.; D.A.F.); the FSH Society, Inc. (S.T.W.; D.A.F.); a directed research Gift from the Shaw/Fischer families (S.T.W.), Collaborative UC/los Alamos Research (CULAR) (S.T.W.); and NIAMS Shannon Award RO1 AR45649 (D.A.F.). We thank Dr. James Olsen and Andy Strand for their contributions and advice on data analysis, and Drs. P. Sherrat and J. Hayes for the generous gift of GST-T2 antibodies.
References (33)
- et al.
Hypermethylation of the FSHD syndrome-associated D4Z4 repeat in normal and FSHD somatic cell populations but not in ICF syndrome cells
Mol Genet Metab
(2001) - et al.
Muscle cells from mdx mice have an increased susceptibility to oxidative stress
Neuromuscul Disord
(1998) - et al.
p21ras as a common signaling target of reactive free radicals and cellular redox stress
J Biol Chem
(1995) - et al.
Active genes in junk DNA? Characterization of DUX genes embedded within 3.3 kb repeated elements
Gene
(2001) - et al.
Facioscapulohumeral muscular dystrophy
- et al.
Genetic counselling in facioscapulohumeral muscular dystrophy
J Med Genet
(1991) - et al.
FSHD associated DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit
Hum Mol Genet
(1993) - et al.
Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy
Nat Genet
(1992) - et al.
Correlation between fragment size at D4F104S1 and age at onset or at wheelchair use, with a possible generational effect, accounts for much phenotypic variation in 4q35-facioscapulohumeral muscular dystrophy
Hum Mol Genet
(1995) - et al.
Evidence for anticipation and association of deletion size with severity of facioscapulohumeral muscular dystrophy
Ann Neurol
(1996)
Two cases of chromosome 4q35-linked early onset facioscapulohumeral muscular dystrophy with mental retardation and epilepsy
Neuropediatrics
Epilepsy and mental retardation in a subset of early onset 4q35-facioscapulohumeral muscular dystrophy
Neurology
Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystrophy
Hum Mol Genet
The DNA rearrangement associated with facioscapulohumeral muscular dystrophy involves a heterochromatin-associated repetitive element: implications for a role of chromatin structure in the pathogenesis of the disease
Chromosome Res
The distribution of somatic H1 subtypes is non-random on active vs. inactive chromatin: distribution in human fetal fibroblasts
Chromosome Res
High resolution fluorescence in situ hybridization to linearly extended DNA visually maps a tandem repeat associated with facioscapulohumeral muscular dystrophy immediately adjacent to the telomere of 4q
Hum Mol Genet
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