Expression of messenger RNA of the cardiac isoforms of troponin T and I in myopathic skeletal muscle

In the absence of clinical signs, elevated values of the cardiac isoforms of troponin T (cTnT) and I (cTnI) can be found in the serum samples of some patients with skeletal muscle myopathies; the cause is unclear. We studied the messenger RNA (mRNA) expression of cTnT and cTnI in the skeletal muscles of 24 patients with histologically proven myopathies and in 18 patients in whom a myopathy could be excluded. For cTnT- and cTnI-mRNA determination, we designed specific primer pairs for nested polymerase chain reaction. After amplification, the products were digested with 2 restriction enzymes and visualized. We found cTnT mRNA in 7 skeletal muscle biopsy specimens (6 patients with Duchenne muscular dystrophy, 1 patient with a primary sarcoglycanopathy) and cTnI mRNA in 6 (5 with Duchenne muscular dystrophy, 1 patient with a histologically negative biopsy). The mRNA of the cardiac isoforms, cTnT and cTnI, is expressed in the skeletal muscles of patients with Duchenne muscular dystrophy, but also in some other myopathies. Further studies are needed to show whether the mRNA is translated into the protein, but serum levels of cTnT and cTnI in patients with Duchenne muscular dystrophy would seem to indicate this.     

Myoferlin, a candidate gene and potential modifier of muscular dystrophy

Dysferlin, the gene product of the limb girdle muscular dystrophy (LGMD) 2B locus, encodes a membrane-associated protein with homology to Caenorhabditis elegans fer-1. Humans with mutations in dysferlin ( DYSF ) develop muscle weakness that affects both proximal and distal muscles. Strikingly, the phenotype in LGMD 2B patients is highly variable, but the type of mutation in DYSF cannot explain this phenotypic variability. Through electronic database searching, we identified a protein highly homologous to dysferlin that we have named myoferlin. Myoferlin mRNA was highly expressed in cardiac muscle and to a lesser degree in skeletal muscle. However, antibodies raised to myoferlin showed abundant expression of myoferlin in both cardiac and skeletal muscle. Within the cell, myoferlin was associated with the plasma membrane but, unlike dysferlin, myoferlin was also associated with the nuclear membrane. Ferlin family members contain C2 domains, and these domains play a role in calcium-mediated membrane fusion events. To investigate this, we studied the expression of myoferlin in the mdx mouse, which lacks dystrophin and whose muscles undergo repeated rounds of degeneration and regeneration. We found upregulation of myoferlin at the membrane in mdx skeletal muscle. Thus, myoferlin ( MYOF ) is a candidate gene for muscular dystrophy and cardiomyopathy, or possibly a modifier of the muscular dystrophy phenotype.     

Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.     

Distinction between Duchenne and other muscular dystrophies by ribosomal protein synthesis.

Ribosome concentration, ribosome distribution on sucrose density gradients, and in-vitro ribosomal amino-acid incorporation (noncollagen and collagen synthesis) were studied in muscle biopsy samples obtained from 30 patients with Duchenne muscular dystrophy, seven patients with Becker muscular dystrophy, and 10 with facioscapulohumeral muscular dystrophy. Ribosome concentration was normal in Duchenne and facioscapulohumeral and decreased in Becker muscular dystrophy. Distribution of ribosomes in sucrose density gradients showed abnormalities (sharp monosomal peak and fewer polyribosomes) only in Duchenne muscular dystrophy and was normal in the other two types. In-vitro amino-acid incorporation of ribosomes in Duchenne muscular dystrophy revealed high collagen and low noncollagen synthesis of the heavy polyribosomes. This abnormality is controlled by an undetermined enzymatic factor belonging to the soluble enzyme fraction. Supplementation of the dystrophic heavy polyribosomes with normal soluble enzymes restored the synthesis of collagen to that of the controls. Heavy polyribosomes extracted from normals or from carriers produce proportionately more collagen in the presence of soluble enzyme fraction from Duchenne muscular dystrophy than in the presence of their homologous enzymes. In Becker muscular dystrophy, both noncollagen and collagen synthesis of the heavy polyribosomes were increased, under the influence of ribosomal factors. The different protein synthesis in Duchenne and Becker muscular dystrophies suggests that these conditions are non-allelic. In facioscapulohumeral muscular dystrophy the changes in protein synthesis occurred only in the early stage of the disease and consisted of increased noncollagen synthesis of the light polyribosomes, while the heavy polyribosomes had normal activity including collagen synthesis. This reaction was controlled by ribosomal factors.     

The genetic and molecular basis of muscular dystrophy: roles of cell–matrix linkage in the pathogenesis

Muscular dystrophies are a heterogeneous group of genetic disorders. In addition to genetic information, a combination of various approaches such as the use of genetic animal models, muscle cell biology, and biochemistry has contributed to improving the understanding of the molecular basis of muscular dystrophy's etiology. Several lines of evidence confirm that the structural linkage between the muscle extracellular matrix and the cytoskeleton is crucial to prevent the progression of muscular dystrophy. The dystrophin–glycoprotein complex links the extracellular matrix to the cytoskeleton, and mutations in the component of this complex cause Duchenne-type or limb-girdle-type muscular dystrophy. Mutations in laminin or collagen VI, muscle matrix proteins, are known to cause a congenital type of muscular dystrophy. Moreover, it is not only the primary genetic defects in the structural or matrix proteins, but also the primary mutations of enzymes involved in the protein glycosylation pathway that are now recognized to disrupt the matrix–cell interaction in a certain group of muscular dystrophies. This group of diseases is caused by the secondary functional defects of dystroglycan, a transmembrane matrix receptor. This review considers recent advances in understanding the molecular pathogenesis of muscular dystrophies that can be caused by the disruption of the cell–matrix linkage.     

Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta/alternative macrophage activation pathway

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.     

Skeletal myopathy in transgenic mice carrying human prototype c-Ha-ras gene

Skeletal myopathy was found in almost all-transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mouse). Microscopically, variation of the muscle fiber size, centrally placed nuclei, regenerating fibers, and interstitial fibrosis were evident; hyalinization and necrosis were sometimes observed in the skeletal muscle (femoralis and pectoralis) of the rasH2 mice. Inflammatory changes in the skeletal muscle or abnormality of adjacent peripheral nerve were not observed. The features were essentially similar to those of muscular dystrophy. Although the severity was relatively mild compared to 34-week-old rasH2 mice, the skeletal myopathy was also observed in younger male (10 weeks of age) rasH2 mice. In nontransgenic littermates, skeletal myopathy was not observed. The mRNA of human c-Ha-ras product was detected in femoral muscle from the rasH2 mice by RT-PCR. In conclusion, these data suggest that skeletal myopathy is occurring in almost all rasH2 mice. Integration of c-Ha-ras gene is thought to be crucial to pathogenesis of skeletal myopathy in the rasH2 mice. Further characterization of the muscular lesion and its pathogenesis are needed to explore the possibility of rasH2 mouse becoming a new model for muscular dystrophy.     

Myofiber degeneration in autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) (LGMD1B)

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene that code for the nuclear membrane protein lamin A/C. We investigated skeletal muscle fibers from several muscles for cytoplasmic degenerative changes in three patients with genetically confirmed Emery-Dreifuss muscular dystrophy. Methods included quantitative light and electron microscopy and PCR-based mutational analysis. Results: The degenerative pathway was characterized by the gradual replacement of individual myofibers by connective tissue. Early stages of degeneration typically involved only a segment of the cross-sectional area of a myofiber. Intermediate stages consisted of myofiber shrinkage due to "shedding" of peripheral cytoplasmic portions into the endomysial space, and fragmentation of the myofibers by interposed collagen fibrils. Empty basement membrane sheaths surrounded by abundant deposits of extracellular matrix marked the end stage of the degenerative process. The nuclear number-to-cytoplasmic area in myofibers of one patient increased with increasing cross-sectional area, suggesting that satellite cell fusion with myofibers may have compensated for myofiber shrinkage. The pattern of degeneration described herein differs from muscular dystrophies with plasma membrane defects (dystrophinopathy, dysferlinopathy) and explains the frequently found absence of highly elevated serum creatine kinase levels in autosomal dominant Emery-Dreifuss muscular dystrophy.     

Collagen VI related muscle disorders

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two conditions which were previously believed to be completely separate entities. BM is a relatively mild dominantly inherited disorder characterised by proximal weakness and distal joint contractures. UCMD was originally described as an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. Here we review the clinical phenotypes of BM and UCMD and their diagnosis and management, and provide an overview of the current knowledge of the pathogenesis of collagen VI related disorders.     

Laminin-111 protein therapy reduces muscle pathology and improves viability of a mouse model of merosin-deficient congenital muscular dystrophy

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A.     

免疫荧光检测抗肌萎缩蛋白诊断肌营养不良症的临床应用

目的 采用免疫荧光技术对Duchenne型肌营养不良症（Duchenne muscular dystrophy,DMD),Becker型肌营养不良症（Becker muscular dystrophy,BMD)，面肩肱型肌营养不良症（facioscapulohumeral muscular dystrophy,FSHD)以及神经性肌萎缩患者骨骼肌细胞膜的dystrophin蛋白进行检测，为临床诊断、分类肌营养不良症提供简便的实验方法。方法 对47例患者选择3种dystrophin 的鼠抗单克隆抗体、羊抗和兔抗多克隆抗体，分别进行免疫荧光技术检测。结果 16例DMD患者均为阴性染色；11例BMD患者为弱阳性染色；10例FSHD和10例神经性肌萎缩患者均为阳性染色。结论 检测肌营养不良症患者骨骼肌膜dystrophin蛋白，有助于肌营养不良症的临床诊断和分型。     

Proteomics reveals drastic increase of extracellular matrix proteins collagen and dermatopontin in the aged mdx diaphragm model of Duchenne muscular dystrophy

Duchenne muscular dystrophy is a lethal genetic disease of childhood caused by primary abnormalities in the gene coding for the membrane cytoskeletal protein dystrophin. The mdx mouse is an established animal model of various aspects of X-linked muscular dystrophy and is widely used for studying fundamental mechanisms of dystrophinopathy and testing novel therapeutic approaches to treat one of the most frequent gender-specific diseases in humans. In order to determine global changes in the muscle proteome with the progressive deterioration of mdx tissue with age, we have characterized diaphragm muscle from mdx mice at three ages (8-weeks, 12-months and 22-months) using mass spectrometry-based proteomics. Altered expression levels in diaphragm of 8-week vs. 22-month mice were shown to occur in 11 muscle-associated proteins. Aging in the mdx diaphragm seems to be associated with a drastic increase in the extracellular matrix proteins, collagen and dermatopontin, the molecular chaperone αB-crystallin, and the intermediate filament protein vimentin, suggesting increased accumulation of connective tissue, an enhanced cellular stress response and compensatory stabilization of the weakened membrane cytoskeleton. These proteomic findings establish the aged mdx diaphragm as an excellent model system for studying secondary effects of dystrophin deficiency in skeletal muscle tissue.     

Muscleblind-like 2 (Mbnl2) -deficient mice as a model for myotonic dystrophy.

Myotonic dystrophy (DM), the most common adult-onset muscular dystrophy, is caused by CTG or CCTG microsatellite repeat expansions. Expanded DM mRNA microsatellite repeats are thought to accumulate in the nucleus, sequester Muscleblind proteins, and interfere with alternative mRNA splicing. Muscleblind2 (Mbnl2) is a member of the family of Muscleblind RNA binding proteins (that also include Mbnl1 and Mbnl3) that are known to bind CTG/CCTG RNA repeats. Recently, it was demonstrated that Mbnl1-deficient mice have characteristic features of human DM, including myotonia and defective chloride channel expression. Here, we demonstrate that Mbnl2-deficient mice also develop myotonia and have skeletal muscle pathology consistent with human DM. We also find defective expression and mRNA splicing of the chloride channel (Clcn1) in skeletal muscle that likely contributes to the myotonia phenotype. Our results support the hypothesis that Muscleblind proteins and specifically MBNL2 contribute to the pathogenesis of human DM.     

Dystrobrevin deficiency at the sarcolemma of patients with muscular dystrophy

Mutations in the genes encoding dystrophin or dystrophin-associated proteins are responsible for Duchenne muscular dystrophy or various forms of limb-girdle muscular dystrophies respectively. We have recently cloned the gene for the murine 87 kDa postsynaptic protein dystrobrevin, a dystrophin-associated protein. Anti-dystrobrevin antibodies stain the sarcolemma in normal skeletal muscle indicating that dystrobrevin co-localises with dystrophin and the dystrophin- associated protein complex. By contrast, dystrobrevin membrane staining is severely reduced in muscles of Duchenne muscular dystrophy patients, consistent with dystrobrevin being a dystrophin-associated protein. Interestingly, dystrobrevin staining at the sarcolemma is dramatically reduced in patients with limb-girdle muscular dystrophy arising from the loss of one or all of the sarcoglycan components. Normal dystrobrevin staining is observed in patients with other forms of limb- girdle muscular dystrophy where dystrophin and the rest of the dystrophin-associated protein complex are normally expressed and in other neuromuscular disorders. Our results show that dystrobrevin- deficiency is a generic feature of dystrophies linked to dystrophin and the dystrophin-associated proteins. This is the first indication that a cytoplasmic component of the dystrophin-associated protein complex may be involved in the pathogenesis of limb-girdle muscular dystrophy.      

Ullrich congenital muscular dystrophy: connective tissue abnormalities in the skin support overlap with Ehlers-Danlos syndromes

Ullrich congenital muscular dystrophy (UCMD) is caused by mutations in the three genes coding for the alpha chains of collagen VI and characterized by generalized muscle weakness, striking hypermobility of distal joints in conjunction with variable contractures of more proximal joints, and normal intellectual development. The diagnosis is supported by abnormal immunoreactivity for collagen VI on muscle biopsies. As patients with UCMD show clinical characteristics typical of classical disorders of connective tissue such as Ehlers-Danlos syndromes (EDS), we investigated the ultrastructure of skin biopsy samples from patients with UCMD (n=5). Electron microscopy of skin biopsies revealed ultrastructural abnormalities in all cases, including alterations of collagen fibril morphology (variation in size and composite fibers) and increase in ground substance, which resemble those seen in patients with EDS. Our findings suggest that there is a true connective tissue component as part of the phenotypic spectrum of UCMD and that there is considerable clinical as well as morphological overlap between UCMD and classic connective tissue disorders.     

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI.     

HISTOLOGICAL STUDY OF MUSCULAR CHANGES IN PROGRESSIVE MUSCULAR DYSTROPHY

Changes of skeletal muscle of 28 patients with progressive muscular dystrophy were studied light microscopically. Slowly progressive muscular atrophy with various forms of degeneration and more acute necrosis with incomplete regeneration were the principal changes. Fatty tissue infiltration and fibrosis of the interstitial tissue seemed to occur relatively late in the course of the disease. Incidence of necrosis and regeneration of muscle fibers are significantly higher in the Duchenne-type dystrophy and in an early stage, thus giving some quantitative difference concerning the genetic clinical types and duration of the disease, though no definite specific change is found for each type of muscular dystrophy.
Significance of these changes are discussed from a morphological standpoint and under consideration of biological speciality of muscle fiber. Regenerative substitution of necrotic muscle fiber performed by survived nuclei of the necrotic fiber itself in close association of myophago-cytosis appeared to be a peculiar process exhibited by skeletal muscle as a syncytial cell.