Human Molecular Genetics

This Article Abstract FREE Full Text (PDF) A corrigendum has been published A corrigendum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (39) Request Permissions Google Scholar Articles by Schröder, R. Articles by Vicart, P. Search for Related Content PubMed PubMed Citation Articles by Schröder, R. Articles by Vicart, P. Social Bookmarking          What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 6 657-669
© 2003 Oxford University Press

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

Rolf Schröder^1,*,, Bertrand Goudeau^2,, Monique Casteras Simon², Dirk Fischer¹, Thomas Eggermann³, Christoph S. Clemen⁴, Zhenlin Li⁵, Jens Reimann¹, Zhigang Xue⁵, Sabine Rudnik-Schöneborn³, Klaus Zerres³, Peter F. M. van der Ven⁶, Dieter O. Fürst⁶, Wolfram S. Kunz⁷ and Patrick Vicart²

¹Department of Neurology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, ²Laboratoire Cytosquelette et Développement, Faculté de Médecine Pitié-Salpêtrière, Paris, France, ³Institute of Human Genetics, RWTH Aachen, 52057 Aachen, Germany, ⁴Center for Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany, ⁵Biologie Moléculaire de la Différenciation, Université Paris 7, France, ⁶Department of Cell Biology, University of Potsdam, 14471 Potsdam, Germany and ⁷Department of Epileptology, University Hospital Bonn, 53105 Bonn, Germany

Received November 15, 2002; Revised December 20, 2002; Accepted January 9, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities (OMIM no. 125660) (1–7). Desmin, the main intermediate filament (IF) protein in skeletal and cardiac muscle cells, is a structural component of the extrasarcomeric cytoskeleton which forms a three-dimensional scaffold around myofibrillar Z-discs, thereby interlinking neighbouring myofibrils and connecting the myofibrillar apparatus to nuclei, the subsarcolemmal cytoskeleton and cytoplasmic organelles such as mitochondria (8–14).

A milestone in our current understanding of desmin function was the generation of desmin (-/-) mice by two laboratories independently (15,16). Although desmin (-/-) mice are viable and fertile, they develop progressive muscle weakness and dystrophic alterations in both cardiac and skeletal muscle. Since severe structural changes with disorganization and deranged alignment of myofibrils, as well as sarcolemmal disruption, were most prominent in highly used striated muscles, it was concluded that the lack of desmin results in an increased susceptibility of muscle fibres to physical strain during muscle contraction (14–16). Even in the abcence of desmin, interconnecting filamentous structures were observed between neighbouring myofibrils and from the Z-discs of most peripheral myofibrils to the overlying sarcolemma (9). In normal striated muscle, the filamentous intermyofibrillar cytoskeleton is composed of a network of various components comprising the IF proteins desmin, synemin and paranemin, the molecular chaperone B-crystallin, and the multifunctional cytoskeletal linker plectin (8,14,17–23). The latter protein has been attributed to play an essential role in the proper spacing, stabilization and subcellular attachment of intermediate filaments (20,24).

Mutations in the human desmin, B-crystallin and plectin gene all give rise to a progressive skeletal myopathy (1,3,24–27). This indicates that these essential members of the extrasarcomeric cytoskeleton have complementary (but not interchangeable) roles in the structural and functional maintenance of striated muscle fibres in response to physical stress. All three disorders, which are morphologically characterized by myofibrillar abnormalities, and abnormal cytoplasmic accumulation of desmin-immunoreactive material, share their structural myofibrillar and intermyofibrillar abnormalities with the large group of the so-called myofibrillar myopathies. These disorders comprise sporadic and familial neuromuscular conditions of considerable clinical and genetic heterogeneity. In a recently reported series of 53 patients with myofibrillar myopathy only three cases could be attributed to either desmin or B crystallin mutations (27), indicating that the vast majority of myofibrillar myopathies are due to so far unidentified gene defects. Accordingly, familial myopathies mapping to chromosomes 12q, 10q23 and 2q24–31 are not yet genetically defined (28,29). Furthermore, mutation analysis of the human genes encoding the IF proteins synemin, paranemin and syncoilin (a novel desmin-binding protein) will be a central issue in deciphering the complex genetic and pathophysiological background of myofibrillar myopathies (30,31).

Although the primary event in the pathogenesis of myopathies caused by desmin B-crystallin and plectin mutations seems clearly related to structural and functional defects of the three-dimensional extrasarcomeric intermediate filament cytoskeleton, the exact molecular mechanisms leading to progressive muscle damage in these disorders are unclear. A possible explanation was indicated by recent studies in desmin (-/-) and plectin (-/-) mice, that suggested that perturbations of the desmin/plectin cytoskeleton are associated with an abnormal distribution of mitochondria and respiratory function abnormalities (13,14,32–34).

In the present study, we report the functional consequences of the first desmin insertion mutation in a patient and in cultured cells. We demonstrate that the K239fsX242 mutation leads to a dual phenotype in diseased skeletal muscle with focal derangements of the extrasarcomeric cytoskeleton charac-terized by prominent desmin, synemin, plectin and B-crystallin–immunoreactive protein aggregates, whereas others areas within the same muscle fibre display a normal cross-striated distribution of these proteins. Using transfection studies in SW13 and BHK21 cells, we show that the K239fsX242 desmin mutant is incapable of forming a desmin intermediate filament network, induces a collapse of a pre-existing desmin cytoskeleton with consecutive changes in the subcellular distribution of mitochondria, and leads to the abnormal aggregation of cytoplasmic proteins. Changes in the subcellular distribution of mitochondria in BHK21 cells that express the desmin mutant are mirrored in the diseased skeletal muscle fibres of our patient, that showed an irregular succinate dehydrogenase (SDH) staining pattern in a large number of fibres. Furthermore, our analysis of mitochondrial function in isolated saponin-permeablized skeletal muscle fibres from our patient provided evidence for an in vivo inhibition of complex I activity. Our results strongly indicate that primary intermediate filament pathology in human skeletal muscle induces mitochondrial abnormalities which may contribute to the vicious circle leading to progressive muscle dysfunction in human desminopathies and other forms of myofibrillar myopathies.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Clinical phenotype
A 40-year-old Caucasian male patient presented with a history of slowly progressive muscle weakness since the age of 18. Although muscle weakness was initially restricted to the distal anterior leg muscle compartment, weakness and atrophy gradually spread over the years to his arm and proximal leg muscles. In his mid-twenties, he first developed episodes of cardiac arrhythmia and at the age of 31 a defibrillator device was implanted because of episodes with malignant ventricular tachycardia. He has two healthy children but lost one child at the age of 9 years, who was in a persistent vegetative state for 8 years after a cardiac arrest of unknown cause. Otherwise the family history was negative for neuromuscular or cardiac disorders. In June 2001 neurological examination demonstrated generalized muscle weakness and wasting which was pronounced in distal muscle groups. Repeated laboratory testing revealed normal creatine kinase levels. Needle electromyography demonstrated positive sharp waves and fibrillation potentials as well as short-duration polyphasic motor unit potentials with decreased amplitudes in all muscles analysed.

Mutation analysis
Mutation analysis using direct nucleotide sequencing of PCR products amplified from all nine coding exons of the desmin gene revealed a novel heterozygous single adenine insertion mutation (c.5141_5143insA; GenBank ID 181539, accession M63391) in the very 3' end of exon 3 (Fig. 1A and B). This mutation resides in a part of the gene that encodes in the evolutionary highly conserved 1B domain of the alpha-helical coiled-coil rod domain of the desmin protein. It causes a frame shift and a premature termination signal located four codons downstream of the insertion side, thereby leading to a truncated desmin molecule with a predicted molecular weight of 27 kDa (K239fsX242; Swiss-Prot P17661). This mutation was not detected in 50 normal control chromosomes. We did not have the chance to perform a desmin mutation analysis in any other family members. However, the negative family history for cardiac or neuromuscular diseases might indicate that the detected monoallelic insertion is due to a de novo mutation in the reported patient.

View larger version (16K): [in this window] [in a new window]   Figure 1. Comparison of nucleotide sequences of PCR-amplified desmin genomic DNA from the patient with distal myopathy and cardiac arrhythmia (A) and a normal control subject (B) revealed a novel heterozygous single adenine insertion mutation (c.5141_5143insA) residing in exon 3 of the desmin gene. (C) Desmin protein domain structure: desmin has a tripartite structure comprising a central -helical coiled-coil rod domain flanked by non-helical head and tail domains. The central rod domain, formed by four (1A, 1B, 2A, 2B) -helical segments separated by three short polypeptide linkers, has been shown to play a pivotal role in the dimerization/polymerization of desmin polypeptides which is the basis for the formation of the three-dimensional desmin intermediate filament cytoskeleton. The detected mutation (arrowhead) resides in the C-terminal region of exon 3, which encodes the evolutionary highly conserved 1B domain of the alpha- helical coiled-coil desmin rod domain (K239fsX242).

 
Morphological analysis
Morphological analysis of a muscle biopsy taken from the left vastus lateralis muscle showed mild myopathic changes consisting of slightly increased endomysial connective tissue, increased fibre diameter variability and roundness of muscle fibres. Furthermore, haematoxylin and eosin and Gomori trichrome stains revealed multiple basophilic inclusions, which could be detected in the subsarcolemmal region and, to a lesser extent, in the cytoplasm of the vast majority of fibres (Fig. 2A and B). While multiple muscle fibres displayed areas of attenuated or even absent SDH-staining indicating a focal depletion of mitochondria, other fibers showed focal areas with increased SDH-staining indicating focal mitochondrial proliferation (Fig. 2C). Cytochrome c oxidase (COX)-negative fibres were present (<2% of all fibres; Fig. 2D), but no classical ragged-red fibres were observed.

View larger version (155K): [in this window] [in a new window]   Figure 2. Morphological analysis of diseased skeletal muscle. Haematoxylin and eosin (A) and Gomori trichrome (B) staining revealed multiple basophilic inclusions in the subsarcolemmal region (arrowheads) and, to a lesser extent, in the cytoplasm of muscle fibres (arrows; bar=50 µm). Succinate dehydrogenase staining (C) showed multiple fibres with areas of attenuated or even absent SDH-staining (arrows and arrowheads) indicating a focal depletion of mitochondria (*succinate dehydrogenase-negative fibre; bar=50 µm). COX staining (D) (*COX-negative fibre; bar=50 µm). Ultrastructural analysis of skeletal muscle showed intermyofibrillar (E) and subsarcolemmal granulofilamentous protein aggregates (bar=0.5 µm). (F) Immunogold electron microscopy with the monoclonal anti-desmin antibody (mab-D33) and a secondary antibody coupled to 10 nm gold particles, showed a dense labelling of pathological protein aggregates, indicating the presence of wild-type desmin in these inclusions (bar=0.2 µm).

 
Ultrastructural analysis demonstrated Z-disc streaming (data not shown), and the presence of intermyofibrillar and subsarcolemmal desmin-immunoreactive granulofilamentous protein aggregates (Fig. 2E and F). Furthermore, multiple fibres displayed areas with a focal clustering of mitochondria in areas with pathological protein aggregates (Fig. 2E), whereas other regions with granufilamentous inclusion showed a reduction or even abscence of mitochondria (data not shown). In areas with Z-band streaming, we noted a reduction of mitochondria in the vincinity of some but certainly not all of these myofibrillar lesions.

Indirect immunofluorescence analysis of isolated muscle fibres showed an intense labelling of these pathological aggregates with antibodies against desmin, synemin, B-crystallin (Fig. 3) and, to a somewhat lesser extent, with antibodies against plectin and ubiquitin (data not shown). However, it is noteworthy that other areas within the same muscle fibres showed a normal cross-striated distribution of desmin, synemin, B-crystallin and plectin. Furthermore, double immunolabelling with antibodies against desmin in conjunction with antibodies against fast-, slow- and fetal myosin demonstrated that the abnormal desmin inclusions were not specific for a certain fibre type, since inclusions were detected in type I, type II and regenerating fibres (data not shown).

View larger version (80K): [in this window] [in a new window]   Figure 3. Double indirect immunofluorescence staining of isolated muscle fibres using antibodies against desmin (A, D), synemin (B), and B-crystallin (E) and colocalization pictures (C, F). Note the presence of multiple desmin-, synemin- and B-crystallin-immunoreactive pathological protein aggregates in some areas, whereas other regions within the same muscle fibre display a normal transverse cross-striated staining pattern of these components of the intermyofibrillar cytoskeleton (bar=25 µm).

 
Desmin protein expression
To study the consequences of the K239fsX242 desmin mutation on desmin protein expression, we performed one- and two-dimensional gel electrophoresis of total protein extracts from normal and diseased skeletal muscle in conjunction with western blotting. Both the polyclonal desmin antiserum (Fig. 4A), and the monoclonal desmin antibody (data not shown) showed a strong labelling of a single 53 kDa band in both normal and diseased muscle with no apparent differences in the intensity of the signals. However, no mutated desmin protein with a predicted molecular weight of 27 kDa could be detected by using these two desmin antibodies. Two-dimensional gel electrophoresis and immunoblotting analysis of total protein extracts from normal human skeletal muscle detected desmin isoforms in a range of pH 5.4–5.9 (calculated pI is 5.2). In contrast, our analysis of diseased muscle showed a spectrum with a range of immunolabelled desmin isoforms between pH 5.4 and 5.8 (Fig. 4B), indicating the absence of the less acidic desmin variants. This shift was confirmed using IPG stripes with a linear pH range from 3 to 10 (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 4. Expression of desmin analyzed by one- and two-dimensional gel electrophoresis of total protein extracts from normal (C) and diseased skeletal muscle (DM). (A) Immunoblotting after one-dimensional 12% SDS–PAGE using the polyclonal desmin antiserum showed a strong labelling of a single 53 kDa band in both normal and diseased muscle with no apparent changes in their signal intensities. Note that the 27 kDa truncated desmin molecule is not detected by our antibody. (B) Two-dimensional gel electrophoresis analysis of total protein extracts showed a labelling of desmin isoforms in a range of pH 5.4–5.9 (calculated pI is 5.2) in normal human skeletal muscle (C). In contrast, in diseased muscle (DM) the antibody detects a more acidic spectrum of desmin variants between pH 5.4 and 5.8.

 
Transfection studies
The functional consequences of the novel desmin insertion mutation were studied by means of transfection studies in SW13 cells that do not contain an endogenous intermediate filament cytoskeleton, and BHK21 cells that express desmin, vimentin and keratin intermediate filaments. Our western blot studies with two different desmin antibodies failed to detect the mutated 27 kDa desmin protein. To overcome these problems in the visualization of mutant desmin in our transfection studies, wild-type and K239fsX242 mutant human desmin cDNA were cloned into a myc-tag expression vector which allows the immunodetection of recombinant wild-type and mutant desmin protein by using an anti-myc tag antibody. As demonstrated in Figure 5A and B, transfection of SW13 cells with wild-type desmin cDNA carrying a N-terminal myc-tag resulted in the formation of a desmin filament network similar to that observed in cells transfected with wild-type desmin cDNA lacking the N-terminal myc-tag. These findings indicate that the N-terminal myc-tag does not interfere with the assembly and formation of desmin filaments. In contrast, immunodetection of myc in myc/K239fsX242 transfected SW13 cells showed that the mutant desmin is incapable of assembling a three-dimensional desmin intermediate filament cytoskeleton but leads to a speckled pattern of myc-immunoreactive cytoplasmic protein aggregates (Fig. 5C). In BHK21 cells, endogenous and myc-tagged labelled desmin networks give similar patterns (Fig. 5D–F), whereas transfection with myc/K239fsX242 disrupts the endogenous desmin cytoskeleton in BHK21 cells (Fig. 5G–I).

View larger version (83K): [in this window] [in a new window]   Figure 5. Transfection studies in SW13 (A–C) and BHK21 cells (D–I). (A) Immunodetection of desmin 48 h after transfection with wild-type human desmin cDNA in SW13 cells (mab-D33). Note the formation of a desmin cytoskeleton. (B) Immunodetection of myc 48 h after transfection with wild-type human desmin cDNA carrying an N-terminal myc-tag in SW13 cells (anti-myc antibody). Note the formation of a normal three-dimensional desmin cytoskeleton in SW13 cells. (C) Immunodetection of myc 48 h after transfection with K239fsX242 mutant desmin carrying an N-terminal myc-tag. Note that the desmin mutant is incapable of forming a desmin intermediate filament network but leads to abnormal myc-immunoreactive protein aggregates. (D) Immunodetection of the transfected and endogenous desmin network in BHK21 cells 72 h after the transfection with myc-tagged wild-type human desmin cDNA. (E) Immunodetection of myc in the same cells transfected with myc-tagged wild-type human desmin cDNA. (F) Visualization of endogenous and myc-labelled wild-type desmin BHK21 transfected cells. (G) Desmin immunolabelling reveals the disruption of the endogenous desmin cytoskeleton and the formation of pathological protein aggregates in BHK21 cells 72 h after transfection with the K239fsX242 desmin mutant. (H) Immunodetection of myc in pathological protein aggregates in the same cells. (I) Colocalization of endogenous desmin and myc-labelled mutant desmin in pathological protein aggregates in the same cells.

 
Upon transfection of these cells with myc/K239fsX242 aggregates of myc-immunoreactive material were observed 48 h after transfection but the endogenous desmin cytoskeleton still had a normal appearance (not shown). Seventy-two hours following transfection, when expression levels of the mutant protein are significantly higher, the endogenous desmin cytoskeleton was completely disrupted, and endogenous wild-type desmin was found together with mutant desmin in aggregates (Fig. 5G–I). This indicates that the ratio of wild-type desmin to mutant desmin determines the effects of the expression of the mutated desmin. Furthermore, these experiments show that in cells with pre-existing desmin intermediate filaments the mutated desmin can induce a collapse of the desmin cytoskeleton.

To address the issue whether the disruption of the endogenous desmin cytoskeleton exerts an effect on the subcellular distribution of Mitochondria, transfected BHK21 cells were incubated with the mitotracker^® fluorescent dye, which allows a specific labelling of mitochondria. In myc/wild-type desmin transfected BHK21 cells, mitochondria were localized throughout the cytoplasm with a somewhat denser concentration in the perinuclear area when compared to the cell periphery (Fig. 6A–C). In contrast, cells transfected with the myc/K239fsX242 desmin mutant showed a dramatic increased clustering of mitochondria in the perinuclear region where mitochondria seemed to be entrapped in the aggregates of the disrupted desmin filament cytoskeleton (Fig. 6D–F).

View larger version (81K): [in this window] [in a new window]   Figure 6. Transfection studies in BHK21 cells with respect to the subcellular distribution of mitochondrias. (A) Immunodetection of myc 72 h after transfection of wild-type human desmin cDNA carrying an N-terminal myc-tag. (B) Visualization of mitochondria using the Mitotracker® fluorescent dye in the same cells. Mitochondria are labelled throughout the cytoplasm with a denser aggregation in the perinuclear region when compared with the cell periphery. (C) Dual visualization of mitochondria and myc-tagged wild-type human desmin. (D) Immunodetection of myc in pathological protein aggregates after transfection with the K239fsX242 desmin mutant. (E) Note the markedly altered subcellular distribution of mitochondria with clustering of mitochondria in the perinuclear region in response to the collapse of the endogenous desmin network in BHK21 cells transfected with the K239fsX242 desmin mutant. (F) Dual visualization of mitochondria and myc-tagged mutant desmin. Note the clustering of mitochondria in the perinuclear region as well as the entrapment of mitochondria in pathological protein aggregates.

 
Mitochondrial function
Mitochondrial function was studied in skeletal muscle homogenates and isolated muscle fibers from the reported patient. As shown in Table 1, biochemical analysis of respiratory chain enzyme activities in skeletal muscle homogenates showed normal levels of complex I, complex IV and citrate synthase, as well as normal complex I/citrate synthase and COX/citrate synthase ratios. However, our analysis of mitochondrial function in isolated saponin-permeabilized skeletal muscle fibres, which more closely represent the in vivo situation, showed decreased maximal rates of respiration with the NAD-dependent substrate combination glutamate and malate, and a higher amytal sensitivity of respiration (Table 2). This indicates an in vivo inhibition of complex I activity in skeletal muscle harbouring the K239fsX242 desmin insertion mutation.

View this table: [in this window] [in a new window]   Table 1. Analysis of respiratory chain enzyme activities in skeletal muscle homogenates from diseased muscle (DM) showed normal values for CI (complex I), COX (cytochrome c oxidase) and CS (citrate synthase) as well as normal CI:CS and COX:CS ratios.

 

View this table: [in this window] [in a new window]   Table 2. Rates of respiration (four independent measurements) with the NAD-dependent substrate combination glutamate and malate, succinate, TMPD (N,N,N,N-tetra-methyl-paraphenylen-diamine) plus ascorbate. Note the 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 in skeletal muscle harbouring the K239fsX242 desmin insertion mutation. Using a two-sided t-test, we found a significant (P<0.05) difference between diseased and normal control muscle fibres. Ci (amytal)=flux control coefficient with amytal; Ci (KCN)=flux control coefficient with KCN

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Morphological and biochemical consequences of the novel K239fsX242 desmin mutation
Over the past 4 years, an increasing number of genetically proven cases with myopathy/cardiomyopathy caused by mutations in the desmin gene have been reported (1–7,35). Apart from a single case with a missense mutation residing in the desmin tail domain, all other pathogenic desmin gene alterations (missense mutations, n=8; deletion mutation, n=1; splice site mutations, n=2) were found in the evolutionary highly conserved -helical coiled-coil rod domain.

Mutation analysis in our patient with the clinical phenotype of a distal myopathy and malignant cardiac arrhythmias revealed a heterozygous single adenine insertion mutation (c.5141_5143insA) in exon 3 which encodes the C-terminal part of the -helical 1B segment of the central rod domain (Fig. 1C). This, to our knowledge first, desmin insertion mutation leads to a frame shift and premature termination of translation only four codons downstream of the insertion site, thereby leading to a truncated desmin mutant lacking the last 227 amino acids (K239fsX242).

Standard histological and indirect immunofluorescence analysis of cross-sections from desmin myopathy muscle showed mild myopathic changes and multiple subsarcolemmal and cytoplasmic desmin-immunoreactive inclusions, which are the light microscopic equivalent of the desmin-immunoreactive granulofilamentous protein aggregates seen at the ultrastructural level. These pathological protein aggregates were also stained by antibodies against the intermediate filament synemin, the intermediate filament-associated protein plectin and the small heat shock protein B-crystallin, all components of the extrasarcomeric intermediate filament system. Although pathological protein aggregates were a prominent feature in the majority of muscle fibres, analysis of isolated skeletal muscle fibres also showed widespread areas with a normal appearing desmin, synemin, plectin and B-crystallin staining pattern, indicating that the structural organization of the extrasarcomeric cytoskeleton is disturbed in some but not all areas of diseased muscle fibres. This concept is further corroborated by a recently published paper on the cytoskeletal derangements in a patient harboring a L345P desmin missense mutation (22).

Western blot analysis with two different desmin antibodies showed labelling of a single 53 kDa band with equal signal intensities in both normal and diseased skeletal muscle, indicating that the normal desmin allele is sufficient to produce largely normal amounts of the protein. In contrast, two-dimensional gel electrophoresis showed slight differences between normal and diseased muscle. While normal skeletal muscle showed labelling of wild-type desmin spots between pH 5.4 and 5.9, analysis of diseased muscle revealed a shift of immunolabelled wild-type desmin to more acidic variants. Taking into account that the pI of desmin is essentially determined by phosphorylation, these findings indicate a higher degree of phosphorylation of 53 kDa wild-type desmin in desmin myopathy muscle when compared with normal controls. Although the functional significance of this finding in desmin myopathy muscle awaits further experimental elucidation, it should be noted that in vitro assembly studies demonstrated an inhibitory effect of phosphorylation on the polymerization of desmin polypeptides (36,37).

Since similar changes in the desmin phosphorylation pattern were also observed in muscle specimens from patients with pathogenic mutations in the alphaB-crystallin gene (25,38), the alterations seem to be a secondary effect of the disorganization of the desmin cytoskeleton.

The mutated desmin (predicted molecular mass 27 kDa) could be detected neither with monoclonal antibody D33 nor with the polyclonal desmin antiserum. This confirms the observation that both desmin antibodies are directed against epitopes downstream of the K239fsX242 mutation (39,40). Unfortunately, desmin-specific antibodies that recognize the aminoterminal 27 kDa portion of the protein do not seem to be available. As a consequence, the immunological detection of desmin on the light microscopic as well as at the ultrastructural level is restricted to wild-type 53 kDa desmin derived from the normal allele. Thus, the expression level and subcellular distribution of mutant desmin in diseased muscle remains elusive.

The K239fsX242 mutant desmin is incapable of forming a desmin intermediate filament network, induces a collapse of a pre-existing desmin cytoskeleton and leads to abnormal cytoplasmic protein aggregation
Analysis of myc/Des K239fsX242 transfected SW13 showed that the mutant desmin is incapable of assembling a (de novo) filament network and leads to abnormal cytoplasmic myc-immunoreactive protein aggregates. In BHK21 cells, which contain an endogenous desmin intermediate filament network, transfection with myc/K239fsX242 leads to a disruption of this pre-existing desmin cytoskeleton, and abnormal protein aggregation reminiscent of the desmin-containing aggregates in the patient's muscle. The fact that this effect in BHK21 cells was only observed in cells with a high level of mutated desmin, after 72 h of transfection, points to a dosage effect. These findings, which are in line with previous transfection studies using mutated desmin variants (2–6), indicate that the K239fsX242 desmin mutant exerts a dominant negative effect on the processes that are required for maintaining a normal intermediate filament network formation, at least in cultured cells. The observed dosage effect has already been found in in vitro experiments that demonstrated that the phenotype of the desmin filaments that are assembled from mixtures of wild-type and mutant desmin depends on the stoichiometry of both proteins (2). These data might explain the observed variations in ultrastructural findings in the muscle fibres of patients suffering from a desminopathy.

From intermediate filament pathology to mitochondrial dysfunction: a relevant aspect in human desminopathies?
Varying degrees of mitochondrial dysfunction have been reported in a wide range of inflammatory as well as hereditary neuromuscular disorders (24,41–45). To date, the exact pathologic mechanisms leading to secondary mitochondrial dysfunction, as well as the functional relevance of a secondary ‘metabolic crisis’, in various muscle diseases is an unresolved issue (42). However, with respect to desminopathies there are good reasons to contemplate a more specific relationship between intermediate filament pathology and mitochondrial dysfunction. Several ultrastructural studies have documented a structural relationship between the intermediate filament cytoskeleton and mitochondrial dysfunction (13,46,47). Furthermore, recent studies in desmin (-/-) mice showed that the targeted ablation of desmin leads to alterations in the spatial distribution of mitochondria, and to structural and biochemical mitochondrial abnormalities in striated muscle (14,32–34).

To analyse putative effects of the K239fsX242 desmin mutant on mitochondrial localization, we studied their distribution in transfected BHK21 cells using the Mitotracker^® fluorescent dye. In myc/K239fsX242 transfected cells, we observed marked changes in the subcellular distribution with massive clustering of mitochondria in the perinuclear region that coincided with the disruption of the pre-existing desmin cytoskeleton and formation of pathological protein aggregates. These changes in the subcellular distribution of mitochondria are mirrored, at least in part, in the skeletal muscles from our patient. SDH staining, a histochemical marker for mitochondria, showed patchy enzyme activity in multiple muscle fibres with areas of attenuated or even absent SDH activity, indicating focal depletion of mitochondria. Interestingly, we also observed focal areas with increased activity, indicating focal mitochondrial proliferation or aggregation. In this context, it is important to note that the focal derangements of the desmin cytoskeleton due to the K239fsX242 desmin mutation are present in some but not all areas of individual muscle fibres, which in turn might explain focal differences in the subcellular distribution of mitochondria within a single muscle fibre.

Our biochemical analysis of respiratory chain enzyme activities in desmin myopathy skeletal muscle homogenates showed normal results, which rules out any gross abnormalities of cell respiration. However, analysis of mitochondrial function in isolated saponin-permeabilized skeletal muscle fibres, an optimal tool for the detection of subtle changes of oxidative phosphorylation that more closely represents the in vivo situation, showed decreased maximal rates of respiration with the NAD-dependent substrate combination glutamate and malate as well as a higher amytal sensitivity of respiration. Considering the observed normal enzyme activities in skeletal muscle homogenates, these findings suggest an in vivo inhibition of complex I activity in intact skeletal muscle fibres. In analogy to the findings in desmin (-/-) mice, these mild changes of oxidative phosphorylation in diseased muscle may be attributed to a reduced focal diffusion of mitochondrial substrates caused by the myofibrillar disorganization and/or pathological protein aggregates. We speculate that the focal depletion of mitochondria and/or focal disturbance in ATP production might further weaken the structural integrity and functional interplay of neighbouring myofibrils, and thus aggravate the focal myofibrillar changes caused by the lack of an intact intermyofibrillar cytoskeleton. Furthermore, mitochondrial abnormalities might result in the non-lysosomal degradation of pathological protein aggregates via the ATP-dependent 26S-proteasome pathway. These aspects will have to be addressed in future experimental studies.

Since mitochondrial abnormalities usually increase with age, mitochondrial dysfunction might progress undetected until an as yet undefined threshold is reached. At this point a ‘metabolic crisis’ is induced which contributes to progressive muscle dysfunction in primary desmin myopathies and other forms of myofibrillar myopathies.

In conclusion, we report for the first time a desmin insertion mutation. This mutation leads to a distal myopathy associated with malignant cardiac arrhythmias. The K239fsX242 desmin mutation has a dominant negative effect on the polymerization of desmin intermediate filaments, which leads to focal disruptions of the extrasarcomeric intermediate filament cytoskeleton and pathological protein aggregates in diseased human muscle fibres.

The occurrence only focal pathological changes might be explained by focal differences in the wild-type:mutant desmin ratio. The focal changes of the desmin cytoskeleton seem to induce focal myofibrillar disarray and abnormalities in the subcellular distribution and biochemical properties of mitochondria. Our transfection experiments provided evidence that at least the mitochondrial abnormalities are a direct cause of the expression of truncated desmin.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mutation analysis
Genomic DNA of the patient was isolated from peripheral lymphocytes by standard procedures. The complete coding region and intron–exon boundaries of the desmin gene were screened for variations by direct sequencing of PCR products. Information on primers and PCR conditions can be obtained from the authors upon request. PCR was carried out in a 25 µl volume, containing 80 ng genomic DNA, 50 pmol of each primer, 50 mM KCl, 10 mM Tris–HCl pH 8.3, 1.5 mM MgCl₂, 0.01% gelatin, 200 µM of each dNTP, and 1 U Taq polymerase (Gibco BRL). Sequencing of PCR products was carried out using the BigDyeTermination-Cycle Sequencing System (Applied Biosystem), the sequencing products were analyzed on an ABI377 DNA Sequencer (Applied Biosystems).

Muscle biopsy
A diagnostic biopsy was taken from the left vastus lateralis muscle of the patient. Normal control muscle was obtained from a patient who underwent muscle biopsy for diagnosis of neuromuscular symptoms but was ultimately deemed to be normal by means of combined clinical, electrophysiological and histological criteria.

Morphological analysis
Single fibre preparation from normal human skeletal muscle was performed as described previously (48). Cryostat sections (9 µm) of snap-frozen, unfixed muscle were stained for haematoxylin and eosin, oil red O, periodic acid-Schiff, Gomori trichrome, nicotin-adenine dinucleotide tetrazolium-H reductase, myofibrillar adenosine triphosphatase at pH 4.2, 4.6 and 9.4, cytochrome c oxidase, and SDH. These experiments were performed by standard procedures.

Antibodies
The following primary antibodies were used in this study: (1) mouse monoclonal anti-desmin antibody D33 (Dako, Germany); (2) rabbit polyclonal anti-desmin antiserum PDE 2203 (Euro-Diagnostica, The Netherlands); (3) rabbit polyclonal anti-B-crystallin antiserum (Chemicon, USA); (4) P2 guinea pig serum, an antiserum directed against the carboxy-terminal repeat domain 6 of human plectin (12); (5) rabbit polyclonal anti-synemin antiserum (23). (6) mouse monoclonal anti-fast myosin heavy chain antibody (Sigma, Germany); (7) mouse monoclonal anti-slow myosin heavy chain antibody (Sigma, Germany); (8) mouse monoclonal anti-fetal myosin heavy chain antibody (Sigma, Germany); (9) mouse monoclonal anti-ubiquitin antibody (Sigma, Germany); and (10) rabbit polyclonal anti-myc antibody (Sigma, Germany). Isotype-specific secondary antibodies conjugated with fluorescein isothiocyanate, Cy2, or Texas Red were applied according to the recommendations of the manufacturers (Southern Biotechnology Associates, USA; Jackson Immunoresearch Laboratories, USA; Molecular Probes, USA).

Indirect immunofluorescence
Indirect immunofluorescence analysis of human skeletal muscle was performed as described previously (20,49) and pictures were acquired digitally with a Nikon E800 microscope (Nikon, Düsseldorf, Germany), and a Zeiss Axiophot microscope (Zeiss, Germany).

Concerning functional studies, transfected cells were washed in PBS and fixed for 5 min in methanol–acetone (7:3 vol:vol) at 4°C, 48 and 72 h after transfection. Following washing steps in PBS, cells were processed for indirect immunofluorescence. Coverslips were mounted in Mowiol and pictures of transfected cells were acquired digitally with an Olympus BX60 microscope (Olympus, Germany).

Immunoelectron microscopy
Desmin immunogold electron microscopy of skeletal muscle was performed as described previously (24). The primary antibody against desmin was diluted 1:20. A secondary anti-rabbit antibody coupled to 10 nm colloidal gold particles was purchased from Amersham Pharmacia (Freiburg, Germany).

Gel electrophoresis and western blotting
For one-dimensional gel electrophoresis, preparation of total protein extracts, SDS–PAGE using 12% polyacrylamide gels, protein transfer and visualization of proteins on membranes were carried out as described previously (12).

Two-dimensional gel electrophoresis was performed using an adaptation of the protocols described by Goerg et al. (50) and Rabilloud et al. (51) and the Amersham Pharmacia Biotech protocol. For protein extraction, 4 mg of each muscle specimen were cut at -80°C, lysed in 200 µl lysis buffer [7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 2% IPG-buffer, 2% DTT, 1 mM PMSF, complete mini protease inhibitor (Roche), bromophenol blue], homogenized and centrifuged at 16 000 g. For the first dimension 18 cm IPG-stripes (pH 4–7 linear, Amersham) were rehydrated (7 M urea, 2 M thiourea, 2% CHAPS, 2% IPG-buffer, 13 mM DTT, bromophenol blue) and samples of the supernatants were loaded using cups placed at the anodic side. Isoelectric focusing was performed at 18°C (Multiphor II horizontal, Amersham) using the following voltage gradient (power supply EPS 3501XL, Amersham): linear on 400 V in 1 min, 400 V for 4 h, linear on 3500 V in 5 h, 3500 V for 13 h. After IEF, stripes were equilibrated for 15 min each in buffer 1 (6 M urea, 50 mM Tris–HCl, 2% SDS, 30% glycerin, 1% DTT, bromophenol blue, pH 8.8) and buffer 2 (6 M urea, 50 mM Tris–HCl, 2% SDS, 30% glycerin, 4% iodacetamide, bromophenol blue, pH 6.8), SDS–PAGE was carried out using 12% polyacrylamide gels according to Laemmli (52).

Immunodetection of desmin using the polyclonal antiserum was performed with a peroxidase-coupled secondary antibody (1:30 000) and the ‘Supersignal’ enhanced chemoluminescence system (Pierce, Rockford, IL, USA).

Site-directed mutagenesis
The mutated human desmin cDNA which includes the A insertion was obtained using a mutated oligonucleotide for site-directed mutagenesis into a pcDNA3 vector (Invitrogen, Germany) containing the human desmin cDNA (pcDNA3/Des) (53). The following synthetic oligonucleotides were used: DESForward, 5'-CCACGCGCACCAACGAGAAGGTGG-3'; DESReverse, 5'-CGGATATCCCTGAGGGCGGCAGTG-3'; INSAF, 5'-GATCGCGTTCCTTAAAGAAAGTGCATGA-3'; INSAR, 5'-TCATGCACTTTCTTTAAGGAACGCGATC-3'.The A insertion is underlined. The wild-type and mutant cDNA in plasmid pcDNA3 were excised and cloned into a Myc-tag expression vector (54) to generate the myc/Des and myc/K239fsX242 constructs. Sequencing of cloned DNAs was performed using the DyePrimer Sequencing apparatus according to the manufacturer's instructions (Perkin-Elmer Cetus, USA) on an automated DNA sequencer.

Cell culture and DNA transfection
The expression vectors were transfected into SW13 and BHK21 cell lines. The SW13 (vim-) cells do not express desmin, vimentin and keratin, which makes them an optimal model system for intermediate filament expression studies in cultured cells. Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Gibco). One day before transfection, cultured cells were trypsinized and plated on glass coverslips placed in a culture dish. Transfection was carried out using Fugene 6 (Roche, Germany) according to the instructions of the manufacturer. Expression vectors pcDNA3/Des, myc/Des or myc/K239fsX242 (1.5 µg) were added to each dish and the cells were incubated overnight.

Mitochondrial function
Measurements of respiratory chain enzyme activities of NADH [CoQ₁ oxidoreductase (complex I), cytochrome c oxidase, citrate synthase] in skeletal muscle homogenates as well as analysis of mitochondrial function in isolated saponin-permeablized skeletal muscle fibres were performed as described previously (20,48,55).

	ACKNOWLEDGEMENTS

 
The excellent technical assistance of Mrs K. Kappes-Horn, Mrs M. Altenschmidt Mering and Mrs K. Tolksdorf is gratefully acknowledged. Furthermore, we thank Doctor Fernando Rodrigues-Lima for the generous gift of the myc-tag plasmid and helpfull discussions. This project is part of the German network on muscular dystrophies (MD-NET, research project R12, 01GM0302) funded by the German ministry of education and research (BMBF, Bonn, Germany). Part of this work was supported by the Association Française contre les Myopathies (AFM). This article is dedicated to Professor H.H. Goebel on the occasion of his 65th birthday.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +49 2282875964; Fax: +49 2282875964; Email: rolf.schroeder{at}ukb.uni-bonn.de

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Goldfarb, L.G., Park, K.Y., Cervenakova, L., Gorokhova, S., Lee, H.S., Vasconcelos, O., Nagle, J.W., Semino-Mora, C., Sivakumar, K. and Dalakas, M.C. (1998) Missense mutations in desmin associated with familial cardiac and skeletal myopathy. Nat. Genet., 19, 402–403.[CrossRef][Web of Science][Medline]

2. Munoz-Marmol, A.M., Strasser, G., Isamat, M., Coulombe, P.A.,Yang, Y., Roca, X., Vela, E., Mate, J.L., Coll, J., Fernandez-Figueras, M.T. et al. (1998) A dysfunctional desmin mutation in a patient with severe generalized myopathy. Proc. Natl Acad. Sci. USA, 95, 11312–11317.[Abstract/Free Full Text]

3. Dalakas, M.C., Park, K.Y., Semino-Mora, C., Lee, H.S., Sivakumar, K. and Goldfarb, L.G. (2000) Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene. New Engl. J. Med., 342, 770–780.[Abstract/Free Full Text]

4. Park, K.Y., Dalakas, M.C., Goebel, H.H., Ferrans,V.J., Semino-Mora, C., Litvak, S., Takeda, K. and Goldfarb, L.G. (2000) Desmin splice variants causing cardiac and skeletal myopathy. J. Med. Genet., 37, 851–857.[Abstract/Free Full Text]

5. Sjoberg, G., Saavedra-Matiz, C.A., Rosen, D.R., Wijsman, E.M., Borg, K., Horowitz, S.H. and Sejersen,T. (1999) A missense mutation in the desmin rod domain is associated with autosomal dominant distal myopathy, and exerts a dominant negative effect on filament formation. Hum. Mol. Genet., 8, 2191–2198.[Abstract/Free Full Text]

6. Sugawara, M., Kato, K., Komatsu, M., Wada, C., Kawamura, K., Shindo, P.S., Yoshioka, P.N., Tanaka, K., Watanabe, S. and Toyoshima, I. (2000) A novel de novo mutation in the desmin gene causes desmin myopathy with toxic aggregates. Neurology, 55, 986–990.[Abstract/Free Full Text]

7. Goudeau, B., Dagvadorj, A., Rodrigues-Lima, F., Nedellec, P., Casteras-Simon, M., Perret, E., Langlois, S., Goldfarb, L. and Vicart, P. (2001) Structural and functional analysis of a new desmin variant causing desmin-related myopathy. Hum. Mutat., 18, 388–396.[CrossRef][Web of Science][Medline]

8. Price, M.G. and Lazarides, E. (1983) Expression of intermediate filament-associated proteins paranemin and synemin in chicken development. J. Cell Biol., 97, 1860–1874.[Abstract/Free Full Text]

9. Carlsson, L., Li, Z.L., Paulin, D., Price, M.G., Breckler, J., Robson, R.M., Wiche, G. and Thornell, L.E. (2000) Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice. Histochem. Cell Biol., 114, 39–47.[Web of Science][Medline]

10. Granger, B.L. and Lazarides, E. (1980) Synemin: a new high molecular weight protein associated with desmin and vimentin filaments in muscle. Cell, 22, 727–738.[CrossRef][Web of Science][Medline]

11. Hemken, P.M., Bellin, R.M., Sernett, S.W., Becker, B., Huiatt, T.W. and Robson, R.M. (1997) Molecular characteristics of the novel intermediate filament protein paranemin. Sequence reveals EAP-300 and IFAPa-400 are highly homologous to paranemin. J. Biol. Chem., 272, 32489–32499.[Abstract/Free Full Text]

12. Schröder, R., Warlo, I., Herrmann, H., Van Der Ven, P.F., Klasen, C., Blumcke, I., Mundegar, R.R., Furst, D.O., Goebel, H.H. and Magin, T.M. (1999) Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle. Eur. J. Cell Biol., 78, 288–295.[Web of Science][Medline]

13. Reipert, S., Steinbock, F., Fischer, I., Bittner, R.E., Zeold, A. and Wiche, G. (1999) Association of mitochondria with plectin and desmin intermediate filaments in striated muscle. Exp. Cell Res., 252, 479–491.[CrossRef][Web of Science][Medline]

14. Carlsson, L. and Thornell, L.E. (2001) Desmin-related myopathies in mice and man. Acta Physiol Scand., 171, 341–348.[CrossRef][Web of Science][Medline]

15. Li, Z., Colucci-Guyon, E., Pincon-Raymond, M., Mericskay, M., Pournin, S., Paulin, D. and Babinet, C. (1996) Cardiovascular lesions and skeletal myopathy in mice lacking desmin. Dev. Biol., 175, 362–366.[CrossRef][Web of Science][Medline]

16. Milner, D.J., Weitzer, G., Tran, D., Bradley, A. and Capetanaki, Y. (1996) Disruption of muscle architecture and myocardial degeneration in mice lacking desmin. J. Cell Biol., 134, 1255–1270.[Abstract/Free Full Text]

17. Bellin, R.M., Sernett, S.W., Becker, B., Ip,W., Huiatt, T.W. and Robson, R.M. (1999) Molecular characteristics and interactions of the intermediate filament protein synemin. Interactions with alpha-actinin may anchor synemin-containing heterofilaments. J. Biol. Chem., 274, 29493–29499.[Abstract/Free Full Text]

18. Bellin, R.M., Huiatt, T.W., Critchley, D.R. and Robson, R.M. (2001) Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres. J. Biol. Chem., 276, 32330–32337.[Abstract/Free Full Text]

19. Schweitzer, S.C., Klymkowsky, M.W., Bellin, R.M., Robson, R.M., Capetanaki,Y. and Evans, R.M. (2001) Paranemin and the organization of desmin filament networks. J. Cell Sci., 114, 1079–1089.[Abstract]

20. Schröder, R., Furst, D.O., Klasen, C., Reimann, J., Herrmann, H. and van der Ven, P.F. (2000) Association of plectin with Z-discs is a prerequisite for the formation of the intermyofibrillar desmin cytoskeleton. Lab. Invest., 80, 455–464.[Web of Science][Medline]

21. Schröder, R., Pacholsky, D., Reimann, J., Matten, J., Wiche, G., Furst, D.O. and van der Ven, P.F. (2002) Primary longitudinal adhesion structures: plectin-containing precursors of costameres in differentiating human skeletal muscle cells. Histochem. Cell Biol., 118, 301–310.[Web of Science][Medline]

22. Carlsson, L., Fischer, C., Sjoberg, G., Robson, R.M., Sejersen, T. and Thornell, L.E. (2002) Cytoskeletal derangements in hereditary myopathy with a desmin L345P mutation. Acta Neuropathol. (Berl.), 104, 493–504.[Medline]

23. Titeux, M., Brocheriou, V., Xue, Z., Gao, J., Pellissier, J.F., Guicheney, P., Paulin, D. and Li, Z. (2001) Human synemin gene generates splice variants encoding two distinct intermediate filament proteins. Eur. J. Biochem., 268, 6435–6449.[Web of Science][Medline]

24. Schröder, R., Kunz, W.S., Rouan, F., Pfendner, E., Tolksdorf, K., Kappes-Horn, K., Altenschmidt-Mehring, M., Knoblich, R., van der Ven, P.F., Reimann, J. et al. (2002) Disorganization of the desmin cytoskeleton and mitochondrial dysfunction in plectin-related epidermolysis bullosa simplex with muscular dystrophy. J. Neuropathol. Exp. Neurol., 61, 520–530.[Web of Science][Medline]

25. Vicart, P., Caron, A., Guicheney, P., Li, Z., Prevost, M.C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.M. et al. (1998) A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat. Genet., 20, 92–95.[CrossRef][Web of Science][Medline]

26. Nakano, S., Engel, A.G., Waclawik, A.J., Emslie-Smith, A.M. and Busis, N.A. (1996) Myofibrillar myopathy with abnormal foci of desmin positivity. I. Light and electron microscopy analysis of 10 cases. J. Neuropathol. Exp. Neurol., 55, 549–562.[Web of Science][Medline]

27. Selcen, D., Ohno, K. and Engel, A.G. (2002) Analysis of 53 patients with myofibrillar myopathy. J. Neurol. Sci., 199, 102.

28. Engel, A.G. (1999) Myofibrillar myopathy. Ann. Neurol., 46, 681–683.[CrossRef][Web of Science][Medline]

29. Goebel, H.H. and Fardeau, M. (2002) Desmin-Protein Surplus Myopathies, 96th European Neuromuscular Centre (ENMC)-sponsored International Workshop held 14–16 September 2001, Naarden, The Netherlands. Neuromusc. Disord., 12, 687–692.[CrossRef][Medline]

30. Newey, S.E., Howman, E.V., Ponting, C.P., Benson, M.A., Nawrotzki, R., Loh, N.Y., Davies, K.E. and Blake, D.J. (2001) Syncoilin, a novel member of the intermediate filament superfamily that interacts with alpha-dystrobrevin in skeletal muscle. J. Biol. Chem., 276, 6645–6655.[Abstract/Free Full Text]

31. Poon, E., Howman, E.V., Newey, S.E. and Davies, K.E. (2002) Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex. J. Biol. Chem., 277, 3433–3439.[Abstract/Free Full Text]

32. Milner, D.J., Mavroidis, M., Weisleder, N. and Capetanaki,Y. (2000) Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function. J. Cell Biol., 150, 1283–1298.[Abstract/Free Full Text]

33. Kay, L., Li, Z., Mericskay, M., Olivares, J., Tranqui, L., Fontaine, E., Tiivel, T., Sikk, P., Kaambre, T., Samuel, J.L. et al. (1997) Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton. Biochim. Biophys. Acta, 1322, 41–59.[Medline]

34. Linden, M., Li, Z., Paulin, D., Gotow, T. and Leterrier, J.F. (2001) Effects of desmin gene knockout on mice heart mitochondria. J. Bioenerg. Biomembr., 33, 333–341.[CrossRef][Web of Science][Medline]

35. Park, K.Y., Dalakas, M.C., Semino-Mora, C., Lee, H.S., Litvak, S., Takeda, K., Ferrans, V.J. and Goldfarb, L.G. (2000) Sporadic cardiac and skeletal myopathy caused by a de novo desmin mutation. Clin. Genet., 57, 423–429.[CrossRef][Web of Science][Medline]

36. Geisler, N. and Weber, K. (1988) Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain. EMBO J., 7, 15–20.[Web of Science][Medline]

37. Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y. and Sato, C. (1988) Intermediate filament reconstitution in vitro. The role of phosphorylation on the assembly-disassembly of desmin. J. Biol. Chem., 263, 5970–5978.[Abstract/Free Full Text]

38. Rappaport, L., Cintard, F., Samuel, J.L., Delcayre, C., Marotte, F., Tome, F. and Fardeau, M. (1988) Storage of phosphorylated desmin in a familial myopathy. FEBS Lett., 231, 421–425.[CrossRef][Web of Science][Medline]

39. Debus, E., Weber, K. and Osborne, M. (1983) Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein. EMBO J., 2, 2305–2312.[Web of Science][Medline]

40. Raats, J.M., Hendderik, J.B., Verdijk, M., van Oort, F.L., Gerards, W.L., Ramaekers, F.C. and Bloemendal, H. (1991) Assembly of carboxy-terminally deleted desmin in vimentin-free cells. Eur. J. Cell Biol., 56, 84–103.[Web of Science][Medline]

41. Horvath, R., Fu, K., Johns, T., Genge, A., Karpati, G. and Shoubridge, E.A. (1998) Characterization of the mitochondrial DNA abnormalities in the skeletal muscle of patients with inclusion body myositis. J. Neuropathol. Exp. Neurol., 57, 396–403.[Web of Science][Medline]

42. Chen, Y.W., Zhao, P., Borup, R. and Hoffman, E.P. (2000) Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology. J. Cell Biol., 151, 1321–1336.[Abstract/Free Full Text]

43. Wong, K.T., Dick, D. and Anderson, J.R. (1996) Mitochondrial abnormalities in oculopharyngeal muscular dystrophy. Neuromusc. Disord., 6, 163–166.[CrossRef][Web of Science][Medline]

44. Scholte, H.R., Luyt-Houwen, I.E., Busch, H.F. and Jennekens, F.G. (1985) Muscle mitochondria from patients with Duchenne muscular dystrophy have a normal beta oxidation, but an impaired oxidative phosphorylation. Neurology, 35, 1396–1397.[Free Full Text]

45. Reimann, J., Kunz, W.S., Vielhaber, S., Kappes-Horn, K. and Schröder, R. (2002) Mitochondrial dysfunction in myofibrillar myopathy. Neuropathol. Appl. Neurobiol.(in press).

46. Stromer, M.H. and Bendayan, M. (1990) Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria. Cell Motil. Cytoskeleton, 17, 11–18.[CrossRef][Web of Science][Medline]

47. Georgatos, S.D. and Maison, C. (1996) Integration of intermediate filaments into cellular organelles. Int. Rev. Cytol., 164, 91–138.[Medline]

48. Vielhaber, S., Kunz, D., Winkler, K., Wiedemann, F.R., Kirches, E., Feistner, H., Heinze, H.J., Elger, C.E., Schubert, W. and Kunz, W.S. (2000) Mitochondrial DNA abnormalities in skeletal muscle of patients with sporadic amyotrophic lateral sclerosis. Brain, 123, 1339–1348.[Abstract/Free Full Text]

49. Schröder, R., Mundegar, R.R., Treusch, M., Schlegel, U., Blumcke, I., Owaribe, K. and Magin, T.M. (1997) Altered distribution of plectin/HD1 in dystrophinopathies. Eur. J. Cell Biol., 74, 165–171.[Web of Science][Medline]

50. Goerg, A., Posrel, W. and Guenther, S. (1988) The current state of two-dimensional electrophoresis with immobilised pH gradients. Electrophoresis, 9, 531–546.[CrossRef][Web of Science][Medline]

51. Rabilloud, T., Adessi, C., Giraudel, A. and Lunardi, J. (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 18, 307–316.[CrossRef][Web of Science][Medline]

52. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[CrossRef][Medline]

53. Vicart, P., Dupret, J.M., Hazan, J., Li, Z., Gyapay, G., Krishnamoorthy, R., Weissenbach, J., Fardeau, M. and Paulin, D. (1996) Human desmin gene: cDNA sequence, regional localization and exclusion of the locus in a familial desmin-related myopathy. Hum. Genet., 98, 422–429.[CrossRef][Web of Science][Medline]

54. Fensome, A.C., Rodrigues-Lima, F., Josephs, M., Paterson, H.F. and Katan, M. (2000) A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species. J. Biol. Chem., 275, 1128–1136.[Abstract/Free Full Text]

55. Saks, V.A., Veksler, V.I., Kuznetsov, A.V., Kay, L., Sikk, P., Tiivel, T., Tranqui,L., Olivares, J., Winkler, K., Wiedemann, F. et al. (1998) Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol. Cell Biochem., 184, 81–100.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				P. Panagopoulou, C. H. Davos, D. J. Milner, E. Varela, J. Cameron, D. L. Mann, and Y. Capetanaki Desmin mediates TNF-{alpha}-induced aggregate formation and intercalated disk reorganization in heart failure J. Cell Biol., October 20, 2008; 181(5): 761 - 775. [Abstract] [Full Text] [PDF]

				D. Fischer, R. A. Kley, K. Strach, C. Meyer, T. Sommer, K. Eger, A. Rolfs, W. Meyer, A. Pou, J. Pradas, et al. Distinct muscle imaging patterns in myofibrillar myopathies Neurology, September 2, 2008; 71(10): 758 - 765. [Abstract] [Full Text] [PDF]

				M. R. Stone, A. O'Neill, R. M. Lovering, J. Strong, W. G. Resneck, P. W. Reed, D. M. Toivola, J. A. Ursitti, M. B. Omary, and R. J. Bloch Absence of keratin 19 in mice causes skeletal myopathy with mitochondrial and sarcolemmal reorganization J. Cell Sci., November 15, 2007; 120(22): 3999 - 4008. [Abstract] [Full Text] [PDF]

				T. Lowe, R. A. Kley, P. F.M. van der Ven, M. Himmel, A. Huebner, M. Vorgerd, and D. O. Furst The pathomechanism of filaminopathy: altered biochemical properties explain the cellular phenotype of a protein aggregation myopathy Hum. Mol. Genet., June 1, 2007; 16(11): 1351 - 1358. [Abstract] [Full Text] [PDF]

				M. C. Walter, P. Reilich, A. Huebner, D. Fischer, R. Schroder, M. Vorgerd, W. Kress, C. Born, B. G. Schoser, K. H. Krause, et al. Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P Brain, June 1, 2007; 130(6): 1485 - 1496. [Abstract] [Full Text] [PDF]

				E. Moric-Janiszewska and G. Markiewicz-Loskot Review on the genetics of arrhythmogenic right ventricular dysplasia Europace, May 1, 2007; 9(5): 259 - 266. [Abstract] [Full Text] [PDF]

				M. R.G. Taylor, D. Slavov, L. Ku, A. Di Lenarda, G. Sinagra, E. Carniel, K. Haubold, M. M. Boucek, D. Ferguson, S. L. Graw, et al. Prevalence of Desmin Mutations in Dilated Cardiomyopathy Circulation, March 13, 2007; 115(10): 1244 - 1251. [Abstract] [Full Text] [PDF]

				C. U. Hubbers, C. S. Clemen, K. Kesper, A. Boddrich, A. Hofmann, O. Kamarainen, K. Tolksdorf, M. Stumpf, J. Reichelt, U. Roth, et al. Pathological consequences of VCP mutations on human striated muscle Brain, February 1, 2007; 130(2): 381 - 393. [Abstract] [Full Text] [PDF]

				E. Arbustini, M. Pasotti, A. Pilotto, C. Pellegrini, M. Grasso, S. Previtali, A. Repetto, O. Bellini, G. Azan, M. Scaffino, et al. Desmin accumulation restrictive cardiomyopathy and atrioventricular block associated with desmin gene defects Eur J Heart Fail, August 1, 2006; 8(5): 477 - 483. [Abstract] [Full Text] [PDF]

				H. Bar, D. Fischer, B. Goudeau, R. A. Kley, C. S. Clemen, P. Vicart, H. Herrmann, M. Vorgerd, and R. Schroder Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro Hum. Mol. Genet., May 15, 2005; 14(10): 1251 - 1260. [Abstract] [Full Text] [PDF]

				D. Selcen and A. G. Engel Mutations in myotilin cause myofibrillar myopathy Neurology, April 27, 2004; 62(8): 1363 - 1371. [Abstract] [Full Text] [PDF]

				L. G. Goldfarb, P. Vicart, H. H. Goebel, and M. C. Dalakas Desmin myopathy Brain, April 1, 2004; 127(4): 723 - 734. [Abstract] [Full Text] [PDF]

				D. Selcen, K. Ohno, and A. G. Engel Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients Brain, February 1, 2004; 127(2): 439 - 451. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) A corrigendum has been published A corrigendum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (39) Request Permissions Google Scholar Articles by Schröder, R. Articles by Vicart, P. Search for Related Content PubMed PubMed Citation Articles by Schröder, R. Articles by Vicart, P. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics