Muscular dystrophies comprise at least 34 conditions, characterized by progressive skeletal muscle weakness and degeneration. The loci affected include mutations in both muscle-specific genes and genes that are more widely expressed such as LMNA and EMD, responsible for EDMD (Emery–Dreifuss muscular dystrophy). LMNA encodes A-type lamins, whereas EMD encodes emerin, both located in the nuclear envelope. Mutation or loss of A-type lamins or emerin in the terminally differentiated myonuclei of muscle fibres results in muscle damage. Importantly, since LMNA and EMD are also expressed by the resident skeletal muscle stem cells, the satellite cells, the mutations that cause muscle damage may also directly compromise the regenerative response. Thus EDMD is different from dystrophic conditions such as Duchenne muscular dystrophy, where the mutated gene is only expressed in the muscle fibres. In this brief review, we examine the evidence that myoblasts carrying EDMD-causing mutations are compromised, and discuss the possibility that such dysfunction results in reduced efficiency of muscle regeneration, so actively contributes to disease progression.
Overview of skeletal muscle
Voluntary movement in vertebrates is achieved by the co-ordinated control of skeletal muscles, the functional unit of which is the myofibre, a highly specialized syncytium sustained by hundreds of post-mitotic myonuclei. Skeletal muscle is an adult stem cell system, whereby these functionally specialized differentiated cells are maintained and replaced by a self-renewing stem cell population. The resident stem cell of adult skeletal muscle is the satellite cell, which lies quiescent on the surface of the myofibre, beneath the overlying basal lamina . Satellite cells are activated to generate myoblasts that proliferate and eventually undergo myogenic differentiation to provide new myonuclei. Thus, in normal skeletal muscle, satellite cells are responsible for both the routine needs of muscle homoeostasis and the more sporadic demands of myofibre hypertrophy, repair or regeneration . Satellite cells also self-renew, thus maintaining a population of quiescent undifferentiated precursors that are available to respond to repeated demand [3,4].
Among the conditions primarily affecting skeletal muscle, the muscular dystrophies are among the most debilitating. Muscular dystrophies are characterized by progressive skeletal muscle weakness and degeneration, although they vary in age of onset, muscles affected and severity . These genetic conditions affect 1 in 3500 live births and currently comprise at least 34 different clinical disorders . The genes responsible for muscular dystrophies are a disparate group. They range from mutations in predominantly muscle-specific genes such as DMD (encoding dystrophin), which causes DMD (Duchenne muscular dystrophy) and Becker muscular dystrophy , to genes also expressed in many other tissues, such as LMNA (encoding A-type lamins) mutations, responsible for AD-EDMD [autosomal dominant EDMD (Emery–Dreifuss muscular dystrophy)]  and limb-girdle muscular dystrophy 1B . Interestingly, other laminopathies can also be relatively tissue-restricted, including cardiomyopathies, neuropathies, lipodystrophies and dermopathies, or more systemic, such as severe premature aging syndromes [10,11]. Although more widely expressed than LMNA, mutations in EMD (encoding emerin) only cause the tissue-restricted X-EDMD (X-linked EDMD) .
In addition to tissue distribution, a further classification can be based upon the subcellular localization of the mutated protein. Dystrophin, for example, is cytoskeletal, being a key constituent of the DAPC (dystrophin-associated protein complex), while the sarcoglycan complex of the DAPC, is transmembrane . The DAPC translates force from the intracellular cytoskeleton and contractile apparatus, across the plasmalemma, to the extracellular matrix. It is therefore intuitive that dysfunction of the DAPC could lead to myofibre damage and hence a muscle disorder . On the other hand, LMNA encodes A-type lamins and EMD encodes emerin, proteins located in the nuclear envelope, and it is far less clear how these mutations cause EDMD. Thus different molecular mechanisms are responsible for generating dystrophic phenotypes .
The role of lamins and emerin
Lamins are type V intermediate filaments, grouped into A-type and B-type. The single LMNA gene encodes four A-type lamins, the major of which are lamins A and C, while LMNB1 and LMNB2 encode the three B-type lamins. Lamins are major constituents of the nuclear lamina, a proteinaceous network underlying the inner nuclear membrane, important for maintaining nuclear architecture and stability [16–18]. Furthermore, carefully orchestrated lamin depolymerization and repolymerization is required for nuclear disassembly and reassembly during mitosis. The nuclear envelope not only has this fundamental structural role, but also is an important signalling platform. Lamins bind the chromatin core histones, RNA polymerase II, RNA-splicing factors and many transcription factors, suggesting that perturbed organization of the nuclear lamina could also adversely affect many nuclear processes .
Emerin is a member of the LEM [LAP2 (lamina-associated polypeptide 2), emerin and MAN1]-domain family of lamin-binding proteins residing in the inner nuclear membrane. The emerin and lamin A/C complex cross-links chromatin, through the nuclear lamina, to the inner nuclear membrane. Emerin also binds a conserved chromatin protein barrier-to-autointegration factor, transcriptional repressors and F-actin (filamentous actin), and is thought to play roles in gene expression, nuclear stability and the cell cycle .
Nuclear envelope proteins also connect nuclear components to the cytoskeleton. In particular, SUN [Sad1 and UNC (uncoordinated)]-domain proteins are anchored to the inner nuclear membrane and bind nesprins, anchored to the outer nuclear membrane and in contact with the cytoskeleton. Together, SUN-domain proteins and nesprins are referred to as the LINC (linker of nucleoskeleton and cytoskeleton) complex . Since emerin is a known binding partner of lamins, and has now also been shown to bind both nesprin-1α and nesprin-2β, it too can be considered part of the LINC complex [21,22]. Thus a nuclear envelope rendered defective by the presence of mutated proteins, could also impact on the cytoskeleton/cytoplasm.
The satellite cell in dystrophic muscle
The mutations that cause muscular dystrophies result in chronic muscle damage and degeneration, which repeatedly induce a regenerative response mediated by satellite cells. Their role in repairing/regenerating dystrophic muscle in conditions such as DMD and sarcoglycanopathies is initially efficient (Figure 1). Indeed, regeneration maintains near normal muscle function in the mdx mouse model of DMD throughout life, but, if the satellite cell compartment is compromised, there is a more severe dystrophic phenotype . Since the repaired/regenerated myofibres express the same mutated genes, however, they too are subject to the same stresses and again become damaged. These chronic rounds of repair/regeneration are thought to eventually lead to replicative senescence in many satellite cells . Moreover, the evolving dystrophic muscle micro-environment, particularly increasing fibrosis, presents physical barriers to efficient myofibre repair/regeneration. Thus in DMD, for example, although the regenerative capacity of the muscle is gradually compromised and becomes ineffective in many muscles, this is a secondary effect of the disease-causing mutation .
Model of how satellite cell dysfunction could contribute to EDMD
In EDMD, it is thought that a combination of mechanisms result in nuclear envelope defects causing muscle damage. The mutated proteins cause nuclear fragility, which renders the myonuclei more susceptible to mechanical stress and damage caused by myofibre contraction, with a possible contribution from the disrupted LINC complex. Mutations also disrupt the role of the nuclear envelope as a signalling platform, affecting the dynamics of muscle gene expression [10,19,25]. Crucially though, emerin is present in the nuclear envelope of satellite-cell-derived myoblasts of adult muscle (Figure 2), whereas A-type lamins are located in both the nuclear lamina and nucleoplasm of the adult-derived C2 myoblastic cell line . Thus the primary mutation not only causes myofibre damage and degeneration of post-mitotic myofibres, but also could directly compromise satellite cell performance, and so efficiency of myofibre repair and regeneration (Figure 1). Therefore satellite cell dysfunction may contribute directly to disease progression in EDMD.
Expression of emerin in proliferating satellite cells
Control of myogenic differentiation is affected in EDMD
Analysis of the transcriptomes from AD-EDMD and X-EDMD muscle biopsies show that the pRb1 (retinoblastoma protein 1)/MyoD pathway is perturbed . pRb is central to the co-ordination of cell-cycle exit and initiation of myogenic differentiation, and is in part regulated by phosphorylation by cyclin/cyclin-dependent kinase complexes . In brief, pRb is inactivated by hyperphosphorylation in proliferating myoblasts, but, upon differentiation cues, becomes dephosphorylated and able to interact with proteins, especially members of the E2F transcription factor family, to prevent cell-cycle progression. In addition, pRb also acts in multiple ways to facilitate activation of muscle-specific genes . Importantly, hypophosphorylated pRb1 removes HDAC1 (histone deacetylase 1) from the muscle-specific transcription factor MyoD, allowing its acetylation by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. MyoD, together with MEF2 (myocyte-enhancing factor 2), then activates genes leading to irreversible cell-cycle exit and completion of the differentiation programme. Indeed, Cre-mediated targeted deletion of pRb in mouse myoblasts prevents their fusion into myotubes, with high degrees of apoptosis resulting . It is proposed that normal emerin and A-type lamin interactions are required to facilitate interactions between MyoD, pRb1 and CBP/p300. Disturbance of these interactions in EDMD by mutations in A-type lamins, or lack of emerin, results in disruption of downstream transcriptional regulation necessary for myoblasts to exit the cell cycle and differentiate. The increased levels of proteins controlling acetylation [CBP, EP300 (E1A-binding protein 300), CRI-1 (CBP/EP300 inhibitory protein 1) and NAP1L1 (nucleosome assembly protein 1-like 1)] in EDMD suggest compensatory up-regulation to overcome this block .
EDMD-causing mutations affect cell-cycle exit and differentiation in mouse models
X-EDMD-causing mutations predominately result in a loss of function (∼85% null) . Skeletal muscle of Emd-null mice has no apparent pathology, but there is altered motor co-ordination [30,31]. Examination of muscle regeneration in Emd-null mice again reveals that the pRb1/MyoD pathway is affected. A transient up-regulation of CBP, Cri-1, Nap1l1, MyoD, Rb1, Hdac1 and Lmna, and a reduction in the transcription of some MyoD target genes, is observed. Emd-null myoblast fusion in vitro and muscle regeneration in vivo are both delayed, but not prevented, with uncoordinated dephosphorylation of pRb1 . Similarly, siRNA (small interfering RNA)-mediated knockdown of emerin in myoblasts reduces myogenic differentiation . Interestingly, knockdown of the emerin-binding protein Lmo7 in HeLa cells also alters CBP, NAP1L1 and p130 levels . Where X-EDMD is caused by non-null mutations, emerin localization to the inner nuclear membrane and its cell-cycle-dependent phosphorylation pattern can be altered [34,35]. For example, Del236–241 emerin increases cell-cycle length in both COS7 and HeLa cells . Effects of these mutations on myoblast function have yet to be determined.
Lmna-null mice develop an acute muscular dystrophy, dying within weeks of birth . Again the pRb1/MyoD pathway is affected, as permanent myoblast cell lines from Lmna-null mice have reduced levels of MyoD and both hyper- and hypho-phosphorylated pRb1, together with reduced downstream targets desmin and M-cadherin. Exit from the cell cycle is perturbed and differentiation potential is impaired .
A useful model of adult myogenesis is the immortalized C2 myogenic cell line, obtained from injured adult mouse muscle . Myogenic differentiation in C2C12 (a C2 subclone) involves remodelling of the lamin-A/C–LAP2α nucleoskeleton, such that A-type lamins relocate from the nucleoplasm to the nuclear lamina following differentiation . In proliferating myoblasts, lamin C and LAP2α are associated with hypophosphorylated pRb, and so this remodelling of the lamina removes lamin C from the LAP2α–pRb complex to promote pRb function. Disease-causing LMNA mutations tend to be autosomal-dominant, associated with either a gain- or loss-of-function . Transfection of C2 cells with the EDMD-causing mutation lamin A W520S prevents up-regulation of myogenin and subsequent differentiation . It is proposed that the mutated A-type lamin interferes with this reorganization of the nuclear lamina, and so relocation of lamin C, necessary for correct pRb function during cell-cycle exit and differentiation. Similarly, lamin A R453W-expressing permanent C2C12 cell lines do not up-regulate myogenin upon induction of differentiation and fail to dephosphorylate pRb efficiently and down-regulate PCNA (proliferating-cell nuclear antigen). Cell-cycle exit is not well co-ordinated, with inefficient fusion into myotubes and increased apoptosis . Interestingly, myogenic differentiation is not affected by a lamin A mutation causing Dunnigan-type familial partial lipodystrophy, indicating a specific effect of EDMD-causing mutations on myoblast function .
Human disease-causing LMNA missense mutations have also been targeted to the Lmna locus in mouse . Of these, LmnaH222P/H222P mice show a clear dystrophic muscle phenotype in both skeletal and cardiac muscles, with increased connective tissue and atrophic, hypertrophic and regenerating myofibres . Some satellite cells exhibit pathological nuclear morphological changes, but a thorough examination of satellite cell function is currently lacking.
Other signalling pathways affecting satellite cell function in EDMD
Gene expression profiling of LmnaH222P/H222P and Emd−/y hearts revealed a common, abnormal, activation of the MAPK (mitogen-activated protein kinase) pathway, implicated in the development of cardiomyopathy [40,41]. Briefly, activation of MAPK signalling initiates a kinase cascade that results in the phosphorylation and activation of kinases including p38, JNKs (c-Jun N-terminal kinases) and ERKs (extracellular-signal-regulated kinases), which then phosphorylate target genes, including transcription factors. In satellite cells, inhibition of p38 leads to a failure to activate from quiescence and reversible cell-cycle exit, whereas p38 activation is also necessary to exit the cell cycle for differentiation [42,43]. Indeed, the pRb1/MyoD and the MAPK pathways are linked, since p38 kinase activity is required for expression of some target genes of MyoD .
Finally, alterations in the Notch and Wnt pathways are implicated in premature aging syndromes caused by mutations in the LMNA gene. Importantly, misregulation of the adult stem cell compartment occurs in premature aging syndromes [45,46]. Notch and Wnt/β-catenin signalling also regulate satellite cell function [47,48], and so disruption of these pathways by EDMD-causing mutations could also impinge upon efficient myofibre repair.
Could satellite cell dysfunction contribute to other muscular dystrophies?
The recent description of AD-EDMD patients with wild-type LMNA, but mutations in nesprin, indicate that altered nesprin function may also affect satellite cell function . Myoblasts from patients with FSHD (facioscapulohumeral dystrophy) have aberrant gene transcription and have perturbed differentiation [50,51]. Furthermore, FSHD shows a similar transcriptional profile to that of EDMD, suggesting that FSHD could also be classified as a nuclear envelope disease . Speculatively, could this association also imply that perturbed satellite cell function contributes to disease progression in FSHD?
The underlying cause of disease progression in EDMD may differ from many other dystrophy conditions, since the primary mutation can also directly affect satellite cell function. Thus loss of muscle strength and wasting due to myofibre damage and degeneration may be exacerbated by a compromised regenerative response (Figure 1). Consistent with this hypothesis, the pRb1/MyoD molecular pathway is perturbed in EDMD muscle, and cell-cycle exit and differentiation are disrupted in mouse models of EDMD.
Current theories propose polymorphisms and the intervention of modifying genes to explain the broad range of laminopathies. However, with regard to EDMD, it could be that the range of severity might, in part, also be related to the degree of myofibre degeneration, combined with how severely satellite cell function is affected by a particular mutation. Furthermore, the muscles affected are highly conserved within a specific disorder, yet the mutated proteins are expressed throughout the skeletal musculature. Could the documented heterogeneity in muscle regenerative potential , and satellite cell properties, also contribute to this phenomenon?
Research into how different disease-causing mutations in lamin A/C and emerin have an impact on myoblast function are already indicating possible novel therapeutic routes. For example, pharmacological inhibition of the MAPK pathway improved differentiation of C2C12 cells expressing R453W mutant lamin A , whereas viral-mediated expression of MyoD and desmin in Lmna-null myoblasts partially restored their differentiation potential . Thus understanding how compromised satellite cell function contributes to muscular dystrophies will not only help develop a better understanding of disease progression, but should also aid development of regenerative medicine therapies.
Nuclear Envelope Diseases and Chromatin Organization: Independent meeting held at New Hunt's House, King's College, Guy's Campus, London, U.K., 23–24 April 2008. Organized and Edited by Juliet Ellis (King's College London, U.K.).
CREB (cAMP-response-element-binding protein)-binding protein
CBP/EP300 inhibitory protein 1
dystrophin-associated protein complex
Duchenne muscular dystrophy
Emery–Dreifuss muscular dystrophy
autosomal dominant EDMD
E1A-binding protein 300
histone deacetylase 1
lamina-associated polypeptide 2
linker of nucleoskeleton and cytoskeleton
mitogen-activated protein kinase
nucleosome assembly protein 1-like 1
Sad1 and UNC (uncoordinated)
We thank Ana Perez-Ruiz for Figure 2. V.F.G. is supported by the Medical Research Council (grant number G0700307). J.E. would like to acknowledge the Muscular Dystrophy Campaign and the British Heart Foundation. The laboratory of P.S.Z. is supported by the Medical Research Council, the Muscular Dystrophy Campaign, the Association of International Cancer Research, Association Française contre les Myopathies and the MYORES Network of Excellence, contract 511978, from the European Commission 6th Framework Programme.