Desmin

Desmin is the muscle-specific member of the intermediate filament family of cytoskeletal proteins. In mature striated muscle, desmin filaments surround the Z-discs, interconnect them to each other, and link the entire contractile apparatus to the sarcolemma, cytoplasmic organelles and the nuclear membrane, probably through plectin (Refs Reference Lazarides34, Reference Price35, Reference Hermannn and Aebi36). In cardiac muscle, desmin is increased at intercalated discs and is the major component of Purkinje fibres. Desmin is encoded by a single-copy gene (DES) that encompasses nine exons and codes for 476 amino acids (Ref. Reference Li37). Desmin comprises three domains: the N- and C-terminal (head and tail) structures and the central α-helical core. The central core is composed in turn of four consecutive helical segments: 1A, 1B, 2A and 2B (Refs Reference Weber and Geisler38, Reference Brown, Cohen and Parry39, Reference Herrmann40, Reference Strelkov41). Mice lacking desmin have severe cardiovascular lesions and skeletal myopathy (Refs Reference Li42, Reference Milner43, Reference Tornell44).

αB-crystallin

αB-crystallin is a chaperone that binds to unfolded proteins under stress conditions to prevent aberrant secondary structures and aggregation. In skeletal muscle, αB-crystallin binds to desmin and actin. αB-crystallin is encoded by the gene CRYAB; it encompasses three exons and codes for 175 amino acids (Refs Reference Brakenhoff45, Reference Golenhofen46, Reference Horwitz47).

Myotilin

Myotilin is expressed exclusively in skeletal and cardiac muscle and is a component of sarcomeric Z-disc (Ref. Reference Salmikangas48); it interacts with several other Z-disc proteins including α-actinin, filamin C, F-actin and FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disc) (Refs Reference Salmikangas48, Reference van der Ven49, Reference Salmikangas50, Reference von Nandelstadh51, Reference Gontier52). The myotilin gene (MYOT) encompasses ten exons and codes for 498 amino acids (Ref. Reference Salmikangas48). Mice lacking myotilin have no apparent morphological skeletal or cardiac muscle disorders, and they develop normally. These observations suggest either that myotilin does not participate in muscle development and basal function maintenance or that other proteins serve as structural and functional compensatory molecules when myotilin is absent (Ref. Reference Moza53).

ZASP

ZASP (Z-band alternatively spliced PDZ-motif-containing protein) is a sarcomeric protein that plays a major role in maintaining Z-disc stability in striated and cardiac muscle. ZASP directly binds to the C-terminal region of α-actinin. The ZASP gene (LDB3) encompasses 18 exons, which are differentially spliced to form several isoforms: exon 4 is expressed without exon 6 in three cardiac isoforms, and exon 6 is expressed without exon 4 in skeletal muscle isoforms (Refs Reference Faulkner54, Reference Au55, Reference Huang56). Zebrafish lacking cypher/ZASP show severe developmental defects, including thinning of the cardiac ventricular wall (Ref. Reference Van der Meer57). Drosophila larvae lacking ZASP do not form Z-discs and do not recruit α-actinin to the Z-disc (Ref. Reference Jani and Schöck58). Targeted deletion of ZASP in mouse causes skeletal and cardiac myopathy and premature death from failure of multiple striated muscles (Ref. Reference Zhou59). The yeast two-hybrid assay and pull-down assay demonstrated that the Asp626Asn mutation of Cypher/ZASP, which causes dilated cardiomyopathy in Japanese patients, increased the affinity of the LIM domain for protein kinase C, thus suggesting a novel mechanism for impaired cardiac muscle function (Ref. Reference Arimura60).

Filamin C

Filamin C is a muscle-specific member of the filamin family of proteins (Refs Reference Himmel61, Reference van der Flier and Sonnenberg62). Filamins have an actin-binding domain at their N-terminus, and a C-terminal Ig-like domain that enables the protein to dimerise. In addition to its role in organising actin filaments, filamin C interacts with several other proteins, including γ and δ sarcoglycans, myotilin, calsarcin and β1 integrin. These interactions suggest that filamin C plays an important role during myofibrillogenesis (Refs Reference Thompson63, Reference van der Ven64, Reference Takada65, Reference Stossel66). The filamin C gene (FLNC) encompasses 46 exons and codes for 2705 amino acids (Refs Reference Xie67, Reference van der Ven68). Filamin-C-deficient mice have defects in primary myogenesis resulting in reduced myotubes and reduced muscle fibres; as a result, animals die shortly after birth (Ref. Reference Dalkilic69).

Desminopathies

MFMs caused by desmin mutations are disabling conditions that most often present with distal muscle weakness, often associated with a cardiopathy, in young adults (Refs Reference Goldfarb7, Reference Goldfarb12, Reference Muñoz-Mármol13, Reference Dalakas14, Reference Dagvadorj26, Reference Olivé27, Reference Goldfarb and Laing28, Reference Sjoberg70, Reference Li71, Reference Sjoberg72, Reference Park73, Reference Dagvadorj74, Reference Fidzianska75, Reference Vrabie76, Reference Bar77, Reference Dalakas78). Age at onset usually varies between 15 and 40 years. Autosomal dominant inheritance occurs in the majority of cases, but de novo mutations are also frequently found. By contrast, autosomal recessive mutations have been reported in only a few cases (Refs Reference Goldfarb12, Reference Muñoz-Mármol13).

Muscular involvement may be manifested as a distal myopathy with later involvement of the proximal muscles, or as limb-girdle myopathy with distal progression. In some families the disease presents as a scapuloperoneal syndrome (Ref. Reference Walter79). Respiratory impairment is frequently observed during the course of the illness. Involvement of bulbar and facial muscles is a late complication in some patients, or may even be present at the disease onset (Refs Reference Olivé27, Reference Vrabie76). Cardiomyopathy may be manifested as hypertrophic, dilated or restrictive. In addition, atrioventricular conduction abnormalities with recurrent episodes of syncopes and arrhythmias are frequently seen in desminopathies.

The variable clinical spectrum of desminopathies depends on several factors, including the position of the mutation in the desmin gene. Patients with mutations in the 2B segment of DES tend to show primarily skeletal muscle pathology, whereas patients with mutations in the 1B segment and tail region show predominant cardiopathy (Ref. Reference Goldfarb and Laing28). The prognosis is usually poor; pacemaker requirement and/or cardiac transplantation are indicated in cases with severe cardiopathy.

αB-crystallinopathies

The first reported family with crystallinopathy presented with proximal and distal myopathy, involvement of the velopharyngeal muscles, respiratory failure, cardiopathy and cataracts, with age of onset in the 30s. The disease was transmitted with an autosomal dominant inheritance (Refs Reference Vicart15, Reference Fardeau80, Reference Fardeau81). Two further cases were later identified: one presented with cervical and limb-girdle weakness and respiratory failure; the other had proximal and distal limb weakness. Both patients had peripheral neuropathy; cardiac involvement and lens opacities were absent (Ref. Reference Selcen and Engel29). Another patient, bearing a different mutation in CRYAB, suffered from dilated cardiomyopathy without apparent skeletal muscle involvement (Ref. Reference Inagaki, Hayashi and Arimura82).

Myotilinopathies

The first MYOT-associated disease mutations were identified in two families suffering from limb-girdle muscular dystrophy type 1A (LGMD1A) (Refs Reference Hauser83, Reference Hauser84). Affected individuals presented between the ages of 18 and 58 years with proximal weakness in the lower extremities, later spreading to involve distal muscles. In some cases, nasal speech was a prominent symptom. The pattern of inheritance was autosomal dominant (Refs Reference Hauser83, Reference Hauser84).

More recently, MYOT-associated disease mutations were identified in a subset of patients with familial or sporadic MFM (Refs Reference Selcen and Engel16, Reference Olivé30). The pattern of inheritance was autosomal dominant in some cases but other patients did not have a family history of disease. The age of disease onset varied from 42 to 77 years (Ref. Reference Olivé30). Distal muscle weakness of the lower limbs was the commonest presentation but other cases presented with proximal weakness. In the majority of patients, foot drop due to anterior tibialis weakness was the first symptom of the disease. However, results of muscle computed tomography studies indicate that the disease actually started in the posterior leg muscles (Ref. Reference Olivé30). In the upper extremities, wrist and finger extensors and deltoids were the most affected muscles. Peripheral neuropathy and cardiomyopathy was frequently observed in some cases (Ref. Reference Selcen and Engel16), but not in others (Ref. Reference Olivé30). A MYOT mutation has been shown to be the cause of spheroid body myopathy in a large family presenting at a mean age of 40 with proximal muscle weakness (Ref. Reference Foroud85). A few other patients with myotilin mutations have been identified (Refs Reference Fischer86, Reference Berciano87).

Considering all these data, late-onset distal or mixed distal and proximal myopathy is the most frequent clinical phenotype associated with myotilin mutations, whereas peripheral neuropathy, cardiomyopathy and respiratory insufficiency are observed only occasionally.

ZASPopathies

Mutations in LDB3 were identified after screening a series of 54 cases with MFM (Ref. Reference Selcen and Engel17). Three heterozygous mutations were detected in 11 cases; two were found in exon 6, whereas another was found in exon 9. Dominant inheritance occurred in seven patients. The age at onset was between 44 and 73 years. Furthermore, the Ala165Val ZASP mutation has been recently identified as the cause of autosomal dominant distal myopathy described by Markesbery et al. Haplotype studies in this family and in five other unrelated families of European ancestry carrying the identical mutation revealed common markers at the locus, suggesting the existence of a founder mutation (Ref. Reference Griggs88). In addition, mutations in exon 4 and exon 6 of the ZASP gene have been identified in patients suffering from dilated cardiomyopathy (Ref. Reference Arimura60).

Patients with zaspopathy present after the age of 40 with distal leg weakness, with the disorder slowly progressing to involve intrinsic hand muscles and mild proximal weakness. As for myotilinopathies, muscle imaging studies indicate early involvement of the posterior calf muscles. Cardiopathy and peripheral neuropathy are additional features in some patients.

Filaminopathy

Members of a large German pedigree suffering from limb-girdle myopathy were shown to carry a heterozygous nonsense mutation in the filamin C gene (Ref. Reference Vorgerd18). Four additional families carrying the same mutation were later identified. Haplotype analyses strongly indicate a founder mutation. The mean age at onset of clinical symptoms was 44 years (with a range of 24 to 57 years). Muscular involvement was slowly progressive, following a pattern of LGMD. Patients frequently developed respiratory muscle weakness, and about a third suffered from cardiac abnormalities comprising conduction blocks, tachycardia, diastolic dysfunction and left ventricular hypertrophy (Ref. Reference Kley89).

Myotilinopathy

Muscle biopsies in myotilinopathies show variable myopathic changes including variation in fibre size, fibre splitting, and internal nuclei, as well as variable degrees of fatty and fibrous tissue replacement. Typically, muscle fibres contain nonhyaline and dense hyaline deposits of various shape and size. These structures are best visualised with modified trichrome stain as dark blue or red-blue inclusions and are eosinophilic on haematoxylin and eosin staining. Small cytoplasmic bodies and nemaline-like bodies often clustered in groups are frequently observed. The hyaline inclusions show strong congophilia (Refs Reference Selcen and Engel16, Reference Olivé30). Spheroid bodies may be present. Rimmed and nonrimmed vacuoles are found in the majority of cases. Oxidative enzyme and ATPase activities are absent in the fibre areas containing the inclusions (Refs Reference Selcen and Engel16, Reference Olivé30). Immunohistochemical studies reveal accumulation of myotilin, desmin, dystrophin and many other proteins in muscle fibres containing cytoplasmic inclusions (Refs Reference Selcen and Engel16, Reference Olivé30, Reference Fischer86). Under electron microscopy, streaming of the Z-line is a prominent feature of early lesions in myotilinopathy. More-damaged fibres show major myofibrillar abnormalities with disruption of the sarcomeres, which are replaced by remnants of myofibrils, and electron-dense material. Mitochondrial abnormalities are usually observed at the periphery of disrupted sarcomeres. Nuclear and sarcoplasmic filamentous structures, such as those described in inclusion body myositis, can be found in myotilinopathies (Ref. Reference Fischer86). Autophagic vacuoles filled with polymorphous and dense osmiophilic material, myelin-like inclusions and membranous structures are common in myotilinopathy (Refs Reference Selcen and Engel16, Reference Olivé30). Characteristic myopathology in myotilinopathies is shown in Figure 2.

Figure 2.

Muscle biopsy findings in a patient with a myotilin (Ser55Phe) mutation. Serial cryostat consecutive sections stained with haematoxylin and eosin (a) and modified trichrome stain (b) showing polymorphous hyaline inclusions and nonrimmed vacuoles. The abnormal fibres display strong desmin (c), αB-crystallin (d), myotilin (e) and ubiquitin immunoreactivity (f). Under electron microscopy, areas of sarcomeric disorganisation are replaced by remnants of electron-dense granular and filamentous material (g–i). These findings have been previously reported (see Ref. 30). Magnifications: a–h, ×400. Bars: g, 2 µm; h, 0.5 µm; i, 0.2 µm.

Desminopathy

Muscle biopsies of affected muscles in desminopathy show nonspecific variation in the fibre size, small atrophic fibres, increased numbers of internal nuclei, and variable numbers of rimmed vacuoles. The most characteristic feature is the presence of amorphous eosinophilic regions that stain dark green or bluish with modified trichrome and show strong immunoreactivity for desmin, αB-crystallin and many other proteins. These areas are typically devoid of oxidative enzymes and ATPase activities. Necrosis and inflammatory infiltrates are rare. Electron microscopy reveals accumulation of granulofilamentous material under the sarcolemma or among the myofibrils, and small aggregates of electron-dense material between the myofibrils. Areas in the vicinity of these aggregates show variable streaming of the Z-line. In addition, disorganisation of the myofibrillar network is evident in some cases. As in myotilinopathies, autophagic vacuoles with polymorphous and myelin-like debris, and accumulation of mitochondria, may be present (Refs Reference Goebel4, Reference Olivé8, Reference Dalakas14, Reference Park73, Reference Fidzianska75, Reference Vrabie76, Reference Bar77, Reference Schröder90). Characteristic myopathology in desminopathies is shown in Figure 3.

Figure 3.

Muscle biopsy findings in a patient with a desmin (Ile367Phe) mutation. Cryostat consecutive sections stained with modified trichrome stain (a), haematoxylin and eosin (b) and NADH (c), showing several fibres containing bluish, amorphous cytoplasmic inclusions devoid of oxidative enzyme activity. Abnormal fibre regions display strong desmin (d), αB-crystallin (e), and faint myotilin (f) and ubiquitin (g) immunoreactivity. Under electron microscopy, a matrix of granulofilamentous material is observed among the myofibrils (h and i). These findings have been previously reported (see Ref. 27). Magnifications: a–g, ×400. Bars h and I, 1 µm.

αB-crystallinopathy

Optical and electron microscopy findings in muscle biopsies in αB-crystallinopathy were indistinguishable from desminopathy; for example, desmin and αB-crystallin-immunoreactive inclusions and granulofilamentous material are present in both disorders (Refs Reference Vicart15, Reference Selcen and Engel29, Reference Fardeau81). However, immunohistochemical studies in two cases have shown an apparent lack of ubiquitin, gelsolin and α1-antichymotrypsin in αB-crystallinopathy (Ref. Reference Selcen and Engel29).

ZASPopathies

Muscle biopsies of affected muscles in cases associated with mutations in the ZASP gene show similar abnormalities to those seen in myotilinopathies (Ref. Reference Selcen and Engel17). Interestingly, only mild ZASP accumulation is observed (Ref. Reference Griggs88).

Filaminopathy

The histological findings in patients with MFM-associated mutation in the filamin C gene studied at different stages of the disease vary from mild myopathic changes to distinct muscular-dystrophy-like features. Rimmed and nonrimmed vacuoles are observed in a few cases. Some fibres contain pleomorphic hyaline, granular and amorphous deposits, ranging from red-blue to dark blue in trichrome stain. Most of them display congophilia and show strong filamin C, desmin, myotilin, αB-crystallin, dystrophin, Xin and sarcoglycan immunoreactivity. Core-like features are additional frequent findings (Refs Reference Vorgerd18, Reference Kley89).

At the ultrastructural level, major myofibrillar abnormalities including abnormal myofibrillar alignment, rod-body formation, and accumulation of granulofilamentous material similar to that seen in desminopathies are observed. In addition, sarcoplasmic paired helical filaments have been found (Refs Reference Vorgerd18, Reference Kley89).

Protein aggregates

A major morphological hallmark of MFMs is the presence of cytoplasmic accumulation of proteins forming insoluble aggregates in muscle cells. In addition to disease-specific mutant proteins (i.e. desmin in desminopathies, αB-crystallin in αB-crystallinopathies, myotilin in myotilinopathies), immunohistochemical studies have shown that protein aggregates in MFMs contain a wide variety of proteins (Box 1) (Refs Reference Nakano1, Reference De Bleecker, Engel and Ertl2, Reference Nakano3, Reference Goebel4, Reference Goebel, Goldfarb and Karpati6, Reference Goldfarb7, Reference Olivé8, Reference Ferrer10, Reference Dalakas14, Reference Selcen and Engel16, Reference Goebel and Warlo20, Reference Goebel and Muller21, Reference Olivé27, Reference Goldfarb and Laing28, Reference Olivé30, Reference Sjoberg70, Reference Vrabie76, Reference Schröder90, Reference Bar91, Reference Howman92, Reference Olivé93; see also Refs Reference Ferrer112, Reference Janué, Olivé and Ferrer136). The mechanisms leading to protein aggregation are not fully understood, but most mutant proteins are prone to aggregation, as revealed by studies in transgenic mice and in transfected cells (Ref. Reference Sherman and Goldberg23).

Box 1.

Proteins that accumulate in myofibrillar myopathies^a

^aFor more information see Refs Reference Nakano1, Reference De Bleecker, Engel and Ertl2, Reference Nakano3, Reference Goebel4, Reference Goebel, Goldfarb and Karpati6, Reference Goldfarb7, Reference Olivé8, Reference Ferrer10, Reference Dalakas14, Reference Selcen and Engel16, Reference Goebel and Warlo20, Reference Goebel and Muller21, Reference Olivé27, Reference Goldfarb and Laing28, Reference Olivé30, Reference Sjoberg70, Reference Vrabie76, Reference Schröder90, Reference Bar91, Reference Howman92, Reference Olivé93, Reference Ferrer112, Reference Janué, Olivé and Ferrer136.

Abbreviations: β-APP: β-amyloid precursor protein; CDK1–5, cyclin-dependent kinases 1–5; eNOS, endothelial nitric oxide synthase; LMP2, LMP7, MECL1, subunits associated with the immunoproteasome; iNOS, inducible nitric oxide synthase; N-CAM, neural cell adhesion molecule; nNOS, neuronal nitric oxide synthase; RAGE, receptor of advanced glycation end products; SNAP-25, synaptosomal-associated protein 25; SOD2, superoxide dismutase 2; UCHL1, ubiquitin C-terminal hydrolase L1.

Desmin transgenic mice expressing a deletion of seven amino acids (Arg173 to Glu179) show disruption of the cytoskeleton and accumulation of desmin and other proteins in the myocardium (Ref. Reference Wang94). Truncated desmin induces desmin–vimentin–cytokeratin coprecipitation, involution of intermediate filament networks, and nuclear fragmentation (Ref. Reference Yu95), and aberrant desmin attracts other proteins and produces high molecular weight insoluble aggregates (Ref. Reference Yu95). Cells transfected with mutant desmin may show variable disorganisation of filament assembly and abnormal protein deposits (Refs Reference Park73, Reference Schröder90, Reference Bar96, Reference Kaminska97, Reference Bar98).

Transgenic mice expressing mutant myotilin (Thr57Ile) show myofibrillar aggregation, streaming of the Z-line, and vacuolisation. Mutant myotilin protein localises in the Z-disc and in the aggregates, together with other proteins associated with the Z-disc (Ref. Reference Garvey99). Overexpression of wild-type myotilin in LGMD1A mutant transgenic mice results in more-severe muscle degeneration, enhanced myofibrillar aggregation, and earlier onset of aggregation when compared with single-transgenic mutant mice (Ref. Reference Garvey100). However, expression of myotilin missense mutations Ser55Phe, Ser60Phe and Ser60Cys in transfected cells does not lead to gross changes in the total amount of myotilin or to aberrant post-translational modifications. These forms of mutated myotilin do not disrupt the endogenous desmin cytoskeleton or lead to pathological protein aggregation in transfected cells (Ref. Reference Fischer86).

Transgenic mice expressing mutant αB-crystallin show abnormal αB-crystallin and desmin aggregates in cardiomyocytes (Ref. Reference Wang101). Muscle cells transfected with mutant αB-crystallin also exhibit abnormal aggregates containing both proteins (Ref. Reference Vicart15).

The MFM-associated mutation Trp2710X in the filamin C gene produces a mutated filamin C polypeptide in which the mutant dimerisation domain is less stable and more susceptible to proteolysis. As a result, it does not dimerise properly and forms aggregates in vitro (Ref. Reference Löwe102).

Oxidative and nitrosative stress in MFMs

Free radical production is a widespread phenomenon occurring under physiological aerobic metabolism in eukaryotic cells. Skeletal muscles are particularly prone to accumulating oxidative damage to DNA, lipids and proteins over time (Refs Reference Stagsted, Bendixen and Andersen130, Reference Yan131). Increased protein carbonyl levels in muscles and increased protein oxidative damage have been described in various muscle diseases including Duchenne and other muscular dystrophies (Ref. Reference Rando132), mitochondrial myopathies (Ref. Reference Esposito133), and inflammatory myopathies (Ref. Reference Haslbeck134). Free radical damage to DNA has also been observed in Duchenne and Becker muscular dystrophies (Ref. Reference Rodriguez and Tarnapolsky135).

Increased levels of the advanced glycation end products (AGEs) N-carboxymethyl-lysine and N-carboxyethyl-lysine (glycoxidation markers), and increased levels of malondialdehyde-lysine and 4-hydroxynonenal adducts (lipoxidation markers), occur in muscle samples in myotilinopathies and desminopathies, as revealed by western blotting, immunohistochemistry, and double-labelling immunofluorescence and confocal microscopy (Ref. Reference Janué, Olivé and Ferrer136). In addition, RAGE (AGE receptor) immunoreactivity is found in the sarcolemma, as well as within the cytoplasm of abnormal muscle fibres in myotilinopathies and desminopathies. Superoxide dismutase (SOD2) expression is also activated in both groups of MFMs (Ref. Reference Janué, Olivé and Ferrer136).

Redistribution of nitric oxide synthases (NOS) has been demonstrated in dystrophinopathies, and it is likely related to the production of nitric oxide (NO) as a mediator to reduce cellular susceptibility to metabolic stress (Ref. Reference Buchwalow137). Recent studies point to the synergistic interaction between mechanical and oxidative/nitrosative stresses of sarcolemmal damage in dystrophinopathies (Ref. Reference Dudley138). Western blots of nitrotyrosine (N-Tyr) have shown increased nitration in myotilinopathies and desminopathies when compared with controls (Ref. Reference Janué, Olivé and Ferrer136). In addition, strong endothelial NOS (eNOS) and neuronal NOS (nNOS) immunoreactivities are observed at the sarcolemma as well as within the cytoplasm of muscle fibres containing protein deposits in myotilinopathies and desminopathies (Ref. Reference Janué, Olivé and Ferrer136).