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At least 19 recordsLinked to original sources
PubMed2003

On noxious desmin: functional effects of a novel heterozygous desmin insertion mutation on the extrasarcomeric desmin cytoskeleton and mitochondria.

Recent studies in desmin (-/-) mice have shown that the targeted ablation of desmin leads to pathological changes of the extrasarcomeric intermediate filament cytoskeleton, as well as structural and functional abnormalities of mitochondria in striated muscle. Here, we report on a novel heterozygous single adenine insertion mutation (c.5141_5143insA) in a 40-year-old patient with a distal myopathy. The insertion mutation leads to a frameshift and a truncated desmin (K239fs242). Using transfection studies in SW13 and BHK21 cells, we show that the K239fsX242 desmin mutant is incapable of forming a desmin intermediate filament network. Furthermore, it induces the collapse of a pre-existing desmin cytoskeleton, alters the subcellular distribution of mitochondria and leads to abnormal cytoplasmic protein aggregates reminiscent of desmin-immunoreactive granulofilamentous material seen in the ultrastructural analysis of the patient's muscle. Analysis of mitochondrial function in isolated saponin-permeablized skeletal muscle fibres from our patient showed decreased maximal rates of respiration with the NAD-dependent substrate combination glutamate and malate, as well as a higher amytal sensitivity of respiration, indicating an in vivo inhibition of complex I activity. Our findings suggest that the heterozygous K239fsX242 desmin insertion mutation has a dominant negative effect on the polymerization process of desmin intermediate filaments and affects not only the subcellular distribution, but also biochemical properties of mitochondria in diseased human skeletal muscle. As a consequence, the intermediate filament pathology-induced mitochondrial dysfunction may contribute to the degeneration/regeneration process leading to progressive muscle dysfunction in human desminopathies.

Adenine↗
PubMed2005

Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro.

Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Here, we report the pathogenic effects of a novel heterozygous R350P desmin missense mutation, which resides in the evolutionary highly conserved coil 2B domain of the alpha-helical coiled-coil desmin rod domain, on the assembly of desmin intermediate filaments (IF) in cultured cells and in vitro. By transfection experiments, we show that R350P desmin is incapable of de novo formation of a desmin IF network in vimentin-free BMGE+H, MCF7 and SW13 cells and that it disrupts the endogenous vimentin cytoskeleton in 3T3 fibroblast cells. Hence, transfected cells displayed abnormal cytoplasmic protein aggregates reminiscent of desmin-positive protein deposits seen in the immunohistochemical and ultrastructural analysis of skeletal muscle derived from the index patient of the affected family. To study the functional effects of the R350P desmin mutation at the protein level, we performed in vitro assembly studies with wild-type (WT) and mutant desmin protein. Our analysis revealed that the in vitro assembly process of R350P desmin is already disturbed at the unit length filament level and that further association reactions generate huge, tightly packed protein aggregates. On assessing the pathogenic effects of R350P desmin in various mixtures with WT desmin, we show that a ratio of 1 : 3 (R350P desmin/WT desmin) is sufficient to effectively block the normal polymerization process of desmin IFs. Our findings indicate that the heterozygous R350P desmin mutation exerts a dominant negative effect on the ordered lateral arrangement of desmin subunits. This disturbance of the lateral packing taking place in the first phase of assembly is ultimately leading to abnormal protein aggregation.

3T3 Cells↗
PubMed2000

Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene.

BACKGROUND: Myofibrillar myopathies, often referred to as desmin-related myopathies, are a heterogeneous group of inherited or sporadic distal-onset skeletal myopathies associated with cardiomyopathy. Among the myofibrillar proteins that characteristically accumulate within the muscle fibers of affected patients, the one found most consistently is desmin, a muscle-specific intermediate-filament protein responsible for the structural integrity of the myofibrils. Skeletal and cardiac myopathy develops in mice that lack desmin, suggesting that mutations in the desmin gene may be pathogenic. METHODS: We examined 22 patients from 8 families with dominantly inherited myofibrillar or desmin-related myopathy and 2 patients with sporadic disease and analyzed the desmin gene for mutations, using complementary DNA (cDNA) amplified from muscle-biopsy specimens and genomic DNA extracted from blood lymphocytes. Restriction-enzyme analysis was used to confirm the mutations. Expression vectors containing normal or mutant desmin cDNA were introduced into cultured cells to determine whether the mutant desmin formed intermediate filaments. RESULTS: Six missense mutations in the coding region of the desmin gene that cause the substitution of an amino acid were identified in 11 patients (10 members of 4 families and 1 patient with sporadic disease); a splicing defect that resulted in the deletion of exon 3 was identified in the other patient with sporadic disease. Mutations were clustered in the carboxy-terminal part of the rod domain, which is critical for filament assembly. In transfected cells, the mutant desmin was unable to form a filamentous network. Seven of the 12 patients with mutations in the desmin gene had cardiomyopathy. CONCLUSIONS: Mutations in the desmin gene affecting intermediate filaments cause a distinct myopathy that is often associated with cardiomyopathy and is termed "desmin myopathy." The mutant desmin interferes with the normal assembly of intermediate filaments, resulting in fragility of the myofibrils and severe dysfunction of skeletal and cardiac muscles.

Adult↗
PubMed1992

Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm.

The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells.

Animals↗
PubMed1996

Human desmin gene: cDNA sequence, regional localization and exclusion of the locus in a familial desmin-related myopathy.

Desmin is a muscle-specific intermediate filament that is encoded by a gene assigned to human chromosome 2q35. Desmin-related myopathies are inherited disorders characterized by an intrasarcoplasmic accumulation of desmin. Recently, the knockout of the desmin gene was shown to generate a myopathic syndrome in transgenic mice, suggesting that functional abnormality of desmin may generate similar clinical symptoms in mouse and human. To determine the potential role of the desmin gene in a well-defined desmin-related myopathy (autosomal dominant form of Fardeau), human desmin cDNAs obtained from affected and unaffected individuals were cloned, sequenced and compared. No obvious mutation was detected. A BssHII restriction fragment length polymorphism (RFLP) was identified in exon 6 of the desmin gene. This RFLP was associated with a previously identified EcoRV RFLP in exon 4 to generate a tetra-allelic system, which was tested for linkage to the desmin-related myopathy in three families. The human desmin gene was localized within an 11-cM interval on chromosome 2q using a panel of radiation hybrids. This 11-cM region was clearly excluded by linkage analysis in the three desmin-related myopathy families using a set of highly polymorphic microsatellite markers. These results suggest that the desmin gene is not primarily involved in this disease.

Alternative Splicing↗
PubMed1996

Characterization of ADP-ribosylation sites on desmin and restoration of desmin intermediate filament assembly by de-ADP-ribosylation .

Desmin is an intermediate filament protein that can be ADP-ribosylated by arginine-specific mono(ADP-ribosyl) transferase. Stoichiometric modification of desmin by the transferase causes inhibition of assembly of desmin into 10-nm intermediate filaments (Huang et al., 1993, Biochem. Biophys. Res. Commun. 197, 570-577). In this work, the sites of modification that can affect disassembly have been identified. ADP-ribosylated desmin (1.2 mol ADP-ribose/mol desmin) was digested with lysyl endopeptidase followed by trypsin. Two ADP-ribosylated peptides were obtained, sequenced by Edman degradation, and analyzed by the use of matrix-assisted laser desorption/ionization mass spectrometry. Arginines 48 and 68 of desmin's head domain were shown to be sites of modification, with arginine 48 the major ADP-ribosylation site. ADP-ribosylated desmin (4 mol ADP-ribose/mol desmin) was treated with ADP-ribosylarginine hydrolase. Removal of more than three ADP-ribose groups results in partial restoration of desmin's ability to form intermediate filaments. It is necessary to remove all ADP-ribose groups from desmin to restore its complete ability to form intermediate filaments. The fact that the effect of ADP-ribosylation on the filament-forming properties of desmin is fully reversible suggests that ADP-ribosylation alone is responsible for the changes noted in desmin.

ADP Ribose Transferases↗
PubMed2000

Bundle formation of smooth muscle desmin intermediate filaments by calponin and its binding site on the desmin molecule.

Smooth muscle basic calponin, a major actin-, tropomyosin-, and calmodulin-binding protein, has been examined for its ability to interact with desmin intermediate filaments from smooth muscle cells using sedimentation analysis, turbidity changes, chemical cross-linking, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS), and electron microscopic observations. Calponin interacted with desmin intermediate filaments in a concentration-dependent manner in vitro. The binding of calponin to desmin produced dense aggregates at 30 degrees C. The dense aggregates were observed by electron microscopy to be composed of large anisotropic bundles of desmin filaments, indicating that calponin forms bundles of desmin filaments. The addition of calmodulin or S100 to the mixture of calponin and desmin caused the removal of calponin from the desmin filaments and inhibited bundle formation in the presence of Ca(2+), but not in the presence of EGTA. Calponin-related proteins including G-actin, tropomyosin, and SM22, had little effect on the binding of calponin to desmin filaments, whereas tubulin weakly inhibited the binding. Desmin had little influence on the calponin-actin and calponin-tubulin interactions using the zero-length cross-linker, EDC. Domain mapping with chymotryptic digestion showed that the binding site of calponin resides within the central a-helical rod domain of the desmin molecule. The chemical cross-linked products of calponin and synthetic peptides (TQ27, TNEKVELQELNDRFANYIEKVRFLEQQ; EE24, EEELRELRRQVDALTGQRARVEVE) derived from the rod domain were detected by MALDI TOF/MS. Furthermore, the calponin-desmin interaction was significantly inhibited by the addition of EE24, but only slightly by TQ27. These results suggest that calponin may act as a cross-linking protein between desmin filaments as well as among intermediate filaments, microfilaments and microtubules in smooth muscle cells.

Actins↗
PubMed2004

Cardiomyocyte-specific desmin rescue of desmin null cardiomyopathy excludes vascular involvement.

Mice deficient in desmin, the muscle-specific member of the intermediate filament gene family, display defects in all muscle types and particularly in the myocardium. Desmin null hearts develop cardiomyocyte hypertrophy and dilated cardiomyopathy (DCM) characterized by extensive myocyte cell death, calcific fibrosis and multiple ultrastructural defects. Several lines of evidence suggest impaired vascular function in desmin null animals. To determine whether altered capillary function or an intrinsic cardiomyocyte defect is responsible for desmin null DCM, transgenic mice were generated to rescue desmin expression specifically to cardiomyocytes. Desmin rescue mice display a wild-type cardiac phenotype with no fibrosis or calcification in the myocardium and normalization of coronary flow. Cardiomyocyte ultrastructure is also restored to normal. Markers of hypertrophy upregulated in desmin null hearts return to wild-type levels in desmin rescue mice. Working hearts were perfused to assess coronary flow and cardiac power. Restoration of a wild-type cardiac phenotype in a desmin null background by expression of desmin specifically within cardiomyocyte indicates that defects in the desmin null heart are due to an intrinsic cardiomyocytes defect rather than compromised coronary circulation.

Animals↗
PubMed2005

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+.

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.

Animals↗
PubMed1995

Desmin expression in rhabdomyosarcoma: influence of the desmin clone and immunohistochemical method.

AIM: To determine which, if any, of five commercially available desmin clones is most reliable at labelling desmin filaments and whether the enhanced polymer one step (EPOS) method of labelling is of any advantage in the routine diagnostic laboratory. METHODS: Thirty four rhabdomyosarcomas from the files at The Hospital for Sick Children, Great Ormond Street, London, were studied. Four different desmin clones, DE-R-11, D33, DE-U-10, and PDE, were applied to each using the conventional extravidin biotin peroxidase method. The D33 clone was also applied using the EPOS method. RESULTS: The EPOS method incorporating D33 persistently scored more cells as desmin positive and was positive in four cases which were negative on staining with the other clones. CONCLUSIONS: The D33 desmin clone used with the EPOS method is more reliable for identifying desmin filaments in tumours than other desmin antibodies tested. Different desmin clones using a routine technique label different rhabdomyosarcoma cells and therefore it is justifiable to use more than one clone.

Desmin↗
PubMed2000

A novel de novo mutation in the desmin gene causes desmin myopathy with toxic aggregates.

OBJECTIVE: To determine the cause and pathogenic mechanisms of a 21-year-old patient's cardioskeletal myopathy. The patient's muscle atrophy and weakness began in distal parts of limbs; cardiac and facial muscles were later involved. BACKGROUND: Desmin myopathy is a skeletal myopathy often associated with cardiomyopathy, caused by mutations in the desmin gene and characterized by desmin accumulation in affected muscle fibers, a leading marker of myofibrillar myopathies. Two kinds of deletions and seven missense mutations in the desmin gene have been identified. METHODS: Clinical examination, electron microscopy of muscle tissue, two-dimensional gel electrophoresis, DNA sequencing, restriction enzyme analysis, and gene transfection were performed. RESULTS: Electron microscopy showed disruption of sarcomeres at Z discs and electron-dense aggregates in biopsied skeletal and heart muscle. Two-dimensional gel electrophoresis of the patient's skeletal muscle proteins showed massive accumulation of desmin. The authors identified a novel desmin mutation, L385P in one allele in the carboxyl end of the rod domain 2B in the patient's leukocytes and skeletal muscle; neither parent had the mutation. Serologic study and DNA markers confirmed the de novo mutation. A peptide harboring desmin rod domains 2A and 2B with L385P tagged with green fluorescent protein induced cytoplasmic aggregates, nuclear DNA condensation, and cell death. CONCLUSIONS: A novel de novo mutation, L385P, causes desmin myopathy. An expression study indicated the toxic effect of the L385P mutation.

Adult↗
PubMed1990

Expression of desmin gene in skeletal and smooth muscle by in situ hybridization using a human desmin gene probe.

We have used a probe encoding for the human desmin gene to study the expression of the desmin gene in skeletal and smooth muscle by in situ hybridization. In human skeletal muscle, the results showed a strong and homogeneous level of desmin mRNA contrasting with the faintly immunostaining of the desmin protein. In smooth muscle cells of colon and uterus, in situ hybridization and immunofluorescence staining suggests that there are some cells which do not contain desmin. The optimal condition of desmin mRNA detection was in cryostat sections fixed with paraformaldehyde and in paraffin embedded tissue with the same fixative. The human desmin probe can be used as a marker of cell differentiation and a way to study the regulation of the expression of the desmin gene in pathological events.

Animals↗
PubMed2001

Structural and functional analysis of a new desmin variant causing desmin-related myopathy.

Desmin-related myopathy is a familial or sporadic disease characterized by skeletal muscle weakness and cardiomyopathy as well as the presence of intracytoplasmic aggregates of desmin-reactive material in the muscle cells. Previously, two kinds of deletions and eight missense mutations have been identified in the desmin gene and proven to be responsible for the disorder. The present study was conducted to determine structural and functional defects in a pathogenic desmin variant that caused a disabling disorder in an isolated case presenting with distal and proximal limb muscle weakness and cardiomyopathy. We identified a novel heterozygous Q389P desmin mutation located at the C-terminal part of the rod domain as the causative mutation in this case. Transfection of desmin cDNA containing the patient's mutation into C2.7, MCF7, and SW13 cells demonstrated that the Q389P mutant is incapable of constructing a functional intermediate filament network and has a dominant negative effect on filament formation. We conclude that Q389P mutation is the molecular event leading to the development of desmin-related myopathy.

Adult↗
PubMed1985

Characterization of the hamster desmin gene: expression and formation of desmin filaments in nonmuscle cells after gene transfer.

The structural organization of the hamster gene encoding the intermediate filament (IF) protein desmin has been determined. The gene, 6.5 kb in length, contains nine exons with a total length of 2169 nucleotides. Remarkably, the intervening sequences map at positions that fully correspond to those of the vimentin gene. The derived complete primary structure for hamster desmin (468 amino acids; 53,250 daltons) reveals striking species variations in the NH2-terminal domain of desmin. A plasmid containing the complete transcription unit of the desmin gene was transfected into hamster lens cells and into human epithelial (HeLa) cells. In both nonmuscle cell lines the desmin gene was biologically active. The synthesized desmin assembled into authentic IFs, as monitored by immunofluorescence. Double immunofluorescence staining showed that the newly formed desmin filaments colocalize with preexisting vimentin filaments, but not with preexisting keratin filaments.

Amino Acid Sequence↗
PubMed1990

Desmin-binding specificities of two desmin CNBr fragments that correspond to the headpiece domain and Helix 1B.

D88 and D109, two cyanogen bromide fragments of desmin which essentially correspond to the amino terminal headpiece domain and Helix 1B, respectively, bind to intact desmin with different topological specificities. D88, the headpiece domain fragment, binds only to the headpiece of intact desmin. In contrast, D109, which encompasses Helix 1B and most of the linker L10 binds to desmin even when its headpiece is removed. Additionally, these fragments only bind desmin if they are present during filament assembly; they do not bind pre-assembled desmin IF or tetramers. These observations suggest that, while alpha-helical coiled-coil interaction between rod domains provides the major driving force behind IF protein dimer formation, homophilic binding of head domains of these proteins may provide an additional stabilizing force and/or specify axial registration in certain IF proteins.

Animals↗
PubMed2003

Progressive skeletal myopathy, a phenotypic variant of desmin myopathy associated with desmin mutations.

Desmin myopathy is a familial or sporadic disorder characterized by the presence of desmin mutations that cause skeletal muscle weakness associated with cardiac conduction block, arrhythmia and heart failure. Distinctive histopathologic features include intracytoplasmic accumulation of desmin-reactive deposits and electron-dense granular aggregates in skeletal and cardiac muscle cells. We describe two families with features of adult-onset slowly progressive skeletal myopathy without cardiomyopathy. N342D point mutation was present in the desmin helical rod domain in patients of family 1, and I451M mutation was found in the non-helical tail domain in patients of family 2. Of interest, the same I451M mutation has previously been reported in patients with cardiomyopathy and no signs of skeletal myopathy. Some carriers of the I451M mutation did not develop any disease, suggesting incomplete penetrance. Expression studies demonstrated inability of the N342D mutant desmin to form cellular filamentous network, confirming the pathogenic role of this mutation, but the network was not affected by the tail-domain I451M mutation. Progressive skeletal myopathy is a rare phenotypic variant of desmin myopathy allelic to the more frequent cardio-skeletal form.

Alanine↗
PubMed1993

ADP-ribosylation of the intermediate filament protein desmin and inhibition of desmin assembly in vitro by muscle ADP-ribosyltransferase.

Arginine-specific mono(ADP-ribosyl)transferase purified from rabbit skeletal muscle catalyzes stoichiometric ADP-ribosylation of the intermediate filament protein, desmin. In contrast, cholera toxin catalyzes a much lower level of ADP-ribosylation of desmin. Modification results in potent inhibition of desmin's ability to assemble into filaments. Phosphorylation of desmin by the catalytic subunit of cAMP dependent protein kinase is also inhibited by ADP-ribosylation. ADP-ribosylation site(s) are located within the N-terminal head domain of desmin.

ADP Ribose Transferases↗
PubMed2005

Desmin accumulation restrictive cardiomyopathy and atrioventricular block associated with desmin gene defects.

BACKGROUND: Primary desminopathies are caused by desmin gene [DES (MIM*125660)] mutations. The clinical spectrum includes pure myopathies, cardiomuscular diseases and cardiomyopathies. Patients with restrictive cardiomyopathy (RCM) plus atrioventricular block (AVB) due to DES defects are frequently unrecognized unless desmin accumulation is specifically investigated in endomyocardial biopsy (EMB) by ultrastructural study. AIMS: To describe a cardiological phenotype characterized by RCM plus AVB due to desmin accumulation caused by DES defects. METHODS AND RESULTS: Desmin accumulation was diagnosed by means of ultrastructural and immunocytochemical studies of EMB in four unrelated probands with RCM and AVB. Candidate genes [DES and alphaB-crystallin (CRYAB)] were screened using sequence analysis. Four DES gene mutations were identified: three new (R16C, T453I and a 10 bp deletion at the exon-intron boundary of exon 3 disrupting the donor splice site) and one known (R406W). The disease was autosomal dominant in two families, recessive in one and associated with a de novo mutation in one. The mutations cosegregated with phenotype in all patients. CRYAB gene screening was negative. CONCLUSIONS: A cardiac phenotype characterized by RCM and AVB caused by desmin accumulation is associated with DES mutations. Although the mutations affected different domains, the cardiac phenotype was identical.

Adolescent↗