Desmin-related myopathies in mice and man.

Desmin, the main intermediate filament (IF) protein in skeletal and heart muscle cells, is of great importance as a part of the cytoskeleton. The IFs surround and interlink myofibrils, and connect the peripheral myofibrils with the sarcolemma. In myotendinous junctions and neuromuscular junctions of skeletal muscle fibres, desmin is enriched. In the heart, desmin is increased at intercalated discs, the attachment between cardiomyocytes, and it is the main component in Purkinje fibres of the conduction system. Desmin is the first muscle-specific protein to appear during myogenesis. Nevertheless, lack of desmin, as shown from experiments with desmin knockout (K/O) mice, does not influence myogenesis or myofibrillogenesis. However, the desmin knock-out mice postnatally develop a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. In other skeletal muscles the organization of myofibrils is remarkably unaffected. Thus, the main consequence of the lack of desmin is that the muscle fibres become more susceptible to damage. The loss of membrane integrity leads to a dystrophic process, with degeneration and fibrosis. In the heart cardiac failure develops, whereas in affected skeletal muscles regenerative attempts are seen. In humans, accumulations of desmin have been a hallmark for presumptive desmin myopathies. Recent investigations have shown that some families with such a myopathy have a defect in the gene coding for alphaB-crystallin, whereas others have mutations in the desmin gene. Typical features of these patients are cardiac affections and muscle weakness. Thus, mutations in the desmin gene is pathogenic for a distinct type of muscle disorder.     

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.     

Mice expressing L345P mutant desmin exhibit morphological and functional changes of skeletal and cardiac mitochondria

Desmin mutations underlie inherited myopathies/cardiomyopathies with varying severity and involvement of the skeletal and cardiac muscles. We developed a transgenic mouse model expressing low level of the L345P desmin mutation (DESMUT mice) in order to uncover changes in skeletal and cardiac muscles caused by this mutation. The most striking ultrastructural changes in muscle from DESMUT mice were mitochondrial swelling and vacuolization. The mitochondrial Ca(2+) level was significantly increased in skeletal and cardiac myocytes from DESMUT mice compared to wild type cells during and after contractions. In isolated DESMUT soleus muscles, contractile function and recovery from fatigue were impaired. A SHIRPA screening test for neuromuscular performance demonstrated decreased motor function in DESMUT compared to WT mice. Echocardiographic changes in DESMUT mice included left ventricular wall hypertrophy and a decreased left ventricular chamber dimension. The results imply that low levels of L345P desmin acts, at least partially, by a dominant negative effect on mitochondria.     

Desmin-lacZ transgene expression and regeneration within skeletal muscle transplants

The purpose of this study was to investigate the initiation and time course of the regeneration process in fragments of skeletal muscle transplants as a function of muscle tissue age at implantation. The appearance of desmin occurs at the very beginning of myogenesis. The transgenic desminnls lacZ mice used in the study bear a transgene in which the 1 kb DNA 5′ regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coliβ-galactosidase. The desmin lacZ transgene labels muscle cells in which the desmin synthesis programme has commenced. We implanted pectoralis muscle fragments from fetal transgenic embryos and mature and old transgenic mice into mature non-transgenic mice. Early events of myogenesis occurring during regeneration started sooner in transplants from 4-month-old (day 3 post-implantation) muscle than in those from 24-month-old (day 5-6 post-implantation) muscle, and they lasted longer in those from young (day 17 post-implantation) than in those from old (day 14 post-implantation) muscle fragments. In adult muscle, transgene activation proceeded from the periphery toward the centre of the transplant. In transplants from fetal 18-day-old pectoralis, myotubes with transgene activity were observed from day 1 to day 19. Desmin immunoreactivity, which appeared about one day after transgene activation, was followed by myosin expression. In adult transplants, the continuity of laminin labelling was disrupted around degenerative fibres, illustrating alteration of the extracellular matrix. Our data suggest that satellite cells from old muscle tissue have lower proliferative capacity and/or less access to trophic substances released by the host (damaged fibres, vascularization) than those from fetal or young adult muscle This revised version was published online in July 2006 with corrections to the Cover Date.     

Sporadic cardiac and skeletal myopathy caused by a de novo desmin mutation

Desmin myopathy is a familial or sporadic disorder characterized by intracytoplasmic accumulation of desmin in the muscle cells. We and others have previously identified desmin gene mutations in patients with familial myopathy, but close to 45% of the patients do not report previous family history of the disease. The present study was conducted to determine the cause of desmin myopathy in a sporadic patient presenting with symmetrical muscle weakness and atrophy combined with atrioventricular conduction block requiring a permanent pacemaker. A novel heterozygous R406W mutation in the desmin gene was identified by sequencing cDNA and genomic DNA. Expression of a construct containing the patient's mutant desmin cDNA in SW13 (vim-) cells demonstrated a high pathogenic potential of the R406W mutation. This mutation was not found in the patient's father, mother or sister by sequencing and restriction analysis. Testing with five microsatellite markers and four intragenic single nucleotide polymorphisms excluded alternative paternity. Haplotype analysis indicates that the patient's father was germ-line mosaic for the desmin mutation. We conclude that de novo mutations in the desmin gene may be the cause of sporadic forms of desmin-related cardiac and skeletal myopathy.     

Immunofluorescent localization of desmin and vimentin in developing cardiac muscle of Syrian hamster

The distributions of desmin and vimentin were examined in frozen sections of cardiac muscle from embryonic, newborn, and adult Syrian hamster by using immunofluorescent methods. Frozen sections of newborn and adult skeletal muscle were used for comparison. Cardiac myocytes from day 9 in utero embryos already show a clear association of desmin with the sarcomeric myofibrils. In newborn hearts, desmin is localized in the myofibrillar Z-line areas as well as in the peripheral cytoplasm of the cell. Three days after birth, desmin is associated with the intercalated discs. Thus, in adult cardiac muscle, desmin is present in both Z-bands and intercalated discs. Skeletal muscle of newborn and adult hamster also contains desmin associated with the Z-lines of myofibrils. Vimentin is associated with the myofibrils of day 9 in utero cardiac muscle cells. The protein remains associated with the myofibrillar Z-lines in the newborns and adults. No detectable staining for vimentin was observed in newborn or adult hamster skeletal muscle. The existence of vimentin as well as desmin in differentiated cardiac muscle may be a consequence of the somewhat more epithelial-like nature of cardiac cells as compared to skeletal muscle syncitia.     

Distribution and organization of desmin in cultured adult cardiac muscle cells: reflection on function

The cell-culture model for the study of desmin in adult cardiac muscle cells has provided insight into the function of desmin based on its distribution and structural organization. Initially, desmin emerged as a filamentous network from the existing amorphous form in the growing adult cardiac myocytes in vitro. Later, desmin became organized in various forms. In addition to the presence of a periodic array of desmin in the Z-line regions as observed in cardiac myocytes in vivo, longitudinally and transversely oriented strands of desmin were observed along the length of myofibrils in cardiac myocytes in vitro. These desmin strands and transverse perodicities formed a complex interwoven network, interlacing myofibrils of cells. Desmin and agr;-actinin were organized in ribbon- or aponeuroses-like structures that appeared as sheet-like, supportive structures for the cell body. The cellular cytoplasmic processes containing myofibrils were supported by desmin bars. The complex desmin network, desmin bars, transverse strands and ribbons or aponeuroses were observed in in vitro cardiac myocytes in contrast to in vivo cardiac myocytes. The functional implication of desmin, as indicated by in vivo studies, required more information concerning the organization of desmin for its supportive function, and is addressed in the present study. The elaborate organization of desmin provides evidence for its supportive function for the maintenance of the structural integrity and function of cardiac muscle cells.     

Lectin binding and desmin staining during bupivicaine-induced necrosis and regeneration in rat skeletal muscle

The processes of bupivicaine-induced necrosis and regeneration in rat skeletal muscle have been studied with a panel of biotinylated lectins and by immunohistochemical staining for desmin. The results indicate that the binding of lectins to the periphery of muscle fibres is not altered in necrotic and regenerating fibres at the light microscopic level although abnormal cytoplasmic staining by lectins occurs early in necrosis and is also present during regeneration. Desmin staining is lost at an early stage of necrosis and is present at an early stage of regeneration. Desmin staining in combination with morphometrical assessment of the rate of fusion and growth of regenerating fibres provides a powerful method for evaluating the processes of muscle regeneration in experimental situations.     

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. Hum Mutat 18:388–396, 2001. © 2001 Wiley‐Liss, Inc.     

Desminopathies in muscle disease

A recently identified class of myopathies is produced by abnormal desmin, and is characterized by a disorganization of the desmin filament network, the accumulation of insoluble desmin-containing aggregates, and destructive changes in the sarcomeric organization of striated muscles. The desmin filaments interact with various other cytoskeletal proteins. The distinct clinical phenotypes are heterogeneous, with progressive skeletal myopathy, cardiomyopathy, and respiratory insufficiency as the most prominent features. Most of the desmin mutations are autosomal dominant. Identification of the causal genetic mutations shows that the desmin gene is not the only gene implicated in desminopathies; other genes encoding desmin-associated proteins, such as alpha-B-crystallin, and synemin may also be involved. Patients with mutations in their alpha-B-crystallin gene, which produce similar skeletal and cardiac myopathies, also have opaque lenses. Knockout mice have helped to reveal the fundamental role of desmin filaments in cell architecture, sarcomere alignment, myofibril organization, and the distribution of mitochondria. Transgenic mice, which accumulate aggregates of desmin and associated proteins in their muscles, show that the loss of desmin intermediate function as a result of mutations in desmin itself, or in the desmin-associated constituents, is important for disease progression.     

The identification of myogenic cells in skeletal muscle, with emphasis on the use of tritiated thymidine autoradiography and desmin antibodies

The identification of myogenic precursor cells (mpc) is a key factor in determining the early events in the myogenesis and regeneration of skeletal muscle. Although satellite cells have long been established as the providers of myoblastic cells, very little is really known (apart from their anatomical location in relation to muscle fibres and their ability to migrate) about the precise role of satellite cells in myogenesis. Numerous techniques for labelling mpc have been devised, but none of these has proven to be completely reliable in firmly establishing the origin of myogenic cells. The use of tritiated thymidine to label DNA in proliferating mpc (which are not specifically distinguishable at the time) and the subsequent location of their labelled progeny in myotube nuclei has revealed a great deal of data on the timing of myogenesis, but not about the nature of mpc themselves. DNA synthesis can also be detected by antibodies to the thymidine analogue, bromodeoxyuridine, and also by antibody staining for proliferating nuclear cell antigen. Like tritiated thymidine, these other markers are not specific for muscle but are general markers for DNA synthesis. In situ hybridisation of various muscle-specific genetic markers and their products has been informative, as has immunolabelling of myogenin, MyoD1 and desmin. Desmin labelling has been particularly instructive in identifying mpc because it is one of the first muscle-specific proteins to be produced in mpc. This review covers some of the techniques mentioned above and their usefulness in determining the early events in myogenesis.     

How much mutant protein is needed to cause a protein aggregate myopathy in vivo? Lessons from an exceptional desminopathy

Myofibrillar myopathies are caused by mutations in desmin, B‐crystallin, myotilin, ZASP, and filamin genes. Since the vast majority of myofibrillar myopathy causing mutations are heterozygous single amino acid substitutions or small in‐frame deletions, the pathogenic role of mutant versus wild‐type protein cannot be assessed in human skeletal muscle by standard immunodetection techniques. We report on an exceptional desminopathy due to a heterozygous c.735G>C mutation. Immunoblotting detected full‐length 53desmin and a truncated 50variant in skeletal muscle from three affected patients of two different families. RT‐PCR identified three desmin mRNA species encoding for wild‐type and two mutant proteins, p.Glu245Asp and p.Asp214_Glu245del. Since previous functional studies on the p.Glu245Asp mutant showed biological properties identical to wild‐type desmin, the truncated p.Asp214_Glu245del desmin is the disease‐causing mutant. Semiquantitative RT‐PCR established a fraction of the truncated desmin mRNA species in a range from 24% to 37%. Initial quantification of corresponding desmin proteins in the muscle biopsy of the index patient of one family indicated a fraction of only 10% of the truncated species. However, serial analyses of different sections from each muscle biopsy revealed a high intra‐ and interindividual variability of the truncated desmin protein level within a range from 5% to 43%. Desmin assembly studies in vitro have established clear‐cut pathogenic ratios of mutant versus wild‐type proteins. However, our findings point out a far more complex situation in human skeletal muscle. The heterogeneously distributed mutation load within and between individual specimens, which reflects local differences in the expression and/or turnover of the mutant protein in different areas containing multiple myonuclear domains, renders it impossible to define an exact pathogenic threshold of a specific mutant in vivo. © 2008 Wiley‐Liss, Inc.     

Carp,a cardiac ankyrin-repeated protein,and its new homologue,Arpp, are differentially expressed in heart,skeletal muscle,and rhabdomyosarcomas

Arpp, a protein containing an ankyrin repeat domain, PEST sequence, and proline-rich region, is a novel ankyrin-repeated protein highly homologous to Carp, which is proposed to be the putative genetic marker for cardiac hypertrophy. In this study, we comparatively analyzed expression of Arpp and Carp protein in skeletal and cardiac muscles and rhabdomyosarcomas (RMSs). In adult skeletal muscle, Arpp was preferentially expressed in the nucleus and cytoplasm of type I fibers, whereas Carp was barely detectable in skeletal muscle. On the other hand, in adult cardiac muscle, interestingly, Arpp was expressed in ventricles mostly, whereas Carp was expressed throughout the atrium and ventricle. Furthermore, although Carp was identified in fetal heart at 11 developmental weeks, Arpp was very low or undetectable in these fetal hearts. These results suggest that Arpp and Carp are differentially expressed and function in both skeletal and cardiac muscle of fetus and adult. We found that Arpp expression was induced during the differentiation of C2C12 cells in vitro, suggesting that Arpp-expression may be associated with the differentiation stage during myogenesis. Both Arpp and Carp were found to be expressed in all of the RMS cases studied. Because the expression patterns of Arpp in RMS were different from those of muscle actin or desmin, Arpp may be detectable in RMS cases that do not express other existing RMS markers.     

Desmin splice variants causing cardiac and skeletal myopathy

Desmin myopathy is a hereditary or sporadic cardiac and skeletal myopathy characterised by intracytoplasmic accumulation of desmin reactive deposits in muscle cells. We have characterised novel splice site mutations in the gene desmin resulting in deletion of the entire exon 3 during the pre-mRNA splicing. Sequencing of cDNA and genomic DNA identified a heterozygous de novo A to G change at the +3 position of the splice donor site of intron 3 (IVS3+3A→G) in a patient with sporadic skeletal and cardiac myopathy. A G to A transition at the highly conserved -1 nucleotide position of intron 2 affecting the splice acceptor site (IVS2-1G→A) was found in an unrelated patient with a similar phenotype. Expression of genomic DNA fragments carrying the IVS3+3A→G and IVS2-1G→A mutations confirmed that these mutations cause exon 3 deletion. Aberrant splicing leads to an in frame deletion of 32 complete codons and is predicted to result in mutant desmin lacking 32 amino acids from the 1B segment of the alpha helical rod. Functional analysis of the mutant desmin in SW13 (vim-) cells showed aggregation of abnormal coarse clumps of desmin positive material dispersed throughout the cytoplasm. This is the first report on the pathogenic potentials of splice site mutations in the desmin gene.


Keywords: cardiac and skeletal myopathy; desmin splice site mutations; expression study; genotype-phenotype correlation     

Contractile and electrical responses of vagus-innervated frog sartorius muscles

1. Frog sartorius muscles were transplanted to the thoracic region and re-innervated by the gastric vagus nerve. Contractile responses of the re-innervated muscles were studied. Micro-electrodes were used to measure electrical properties of the muscle fibre membrane. Histological studies of the sartorius and vagus nerves and re-innervated muscles were also carried out.2. Autonomic nerve fibres of the gastric vagus form functional connexions with the skeletal muscle fibres. Such vagus-innervated muscle fibres do not atrophy.3. Neither the contractile nor the passive electrical properties of the muscle fibres are altered by vagal innervation.4. Synaptic transmission is quantal in nature and describable by a Poisson distribution as at normal sartorius junctions. The muscle fibres, however, do show extensive multiple-innervation, and unlike normal sartorius junctions, vagus-muscle junctions have a low quantal content and show a long-lasting facilitation.5. Properties of the vagus nerve fibres apparently are not altered by synapsing with skeletal muscle fibres. They remain small diameter and have high threshold for electrical stimulation.6. The ability of these nerve and muscle fibres to influence each other is rather limited in the adult amphibian.     

Transverse stiffness of myofibrils of skeletal and cardiac muscles studied by atomic force microscopy

The transverse stiffness of single myofibrils of skeletal and cardiac muscles was examined by atomic force microscopy. The microscopic images of both skeletal and cardiac myofibrils in a rigor state showed periodical striation patterns separated by Z-bands, which is characteristic of striated muscle fibers. However, sarcomere patterns were hardly distinguishable in the stiffness distributions of the relaxed myofibrils of skeletal and cardiac muscles. Myofibrils in a rigor state were significantly stiff compared with those in a relaxed state, and in each state, cardiac myofibrils were significantly stiffer compared with skeletal myofibrils. By proteolytic digestions of sarcomere components of myofibrils, it was suggested that cardiac myofibrils are laterally stiffer than skeletal myofibrils because Z-bands, connectin (titin) filament networks, and other components of sarcomere structures for the former myofibrils are stronger than those for the latter.     

Lymphocyte transformation test with liver-specific protein and phytohaemagglutinin in patients with liver disease.

(1) A new antibody has been found by immunofluorescence which reacts with cardiac conducting tissue using ox heart false tendons. It was detected in eight out of ninety-three cases of idiopathic heart block (8-6%), in one out of twenty-two cases of secondary heart block (4-5%) and in seven of 165 normal controls (4.2%), in titres varying from 1:10 to 1:40. Previous authors had indicated that this tissue might contain unique antigens. (2) Sera reacting with type I fibres in skeletal muscle (red zebra) were found to be of two varieties, one of which stained conducting fibres diffusely while it gave minimal staining of cardiac muscle; the other reacting with myofibrils in Purkinje cells and heart. (3) Some sera with high titre smooth muscle antibodies (SMA) reacted with conducting tissue together with skeletal and cardiac muscle, suggesting that the four tissues have at least one antigen in common. (4) Known non-organ specific antibodies behaved as expected on beef conducting fibres: striational fluorescence of myasthenia gravis sera reacted with the same patterns on Purkinje myofibrils; AMA and ANA produced IFL in expected locations; ribosomal antibodies reacted strongly, while LKM and reticulin antibodies showed no reactivity. (5) Although the incidence of specific Purkinje fibre antibodies was not significantly raised in idiopathic heart block, the clinical associations suggest that some cases might be related to autoimmunity possibly involving cell-mediated mechanisms as in polymyositis.     

Congenital nemaline myopathy with dilated cardiomyopathy: an autopsy study

A 3-year-old boy with congenital nemaline myopathy had generalized muscle weakness and hypotonia since birth. He developed cardiac symptoms at 2 years of age and died from congestive heart failure. At autopsy, the heart was markedly dilated, involving both ventricles. Rod bodies were recognized not only in skeletal muscles but in cardiac muscles on light and electron microscopy. Desmin and alpha-actinin, which constitute Z-line protein, were shown to localize in the rod structures in both skeletal and myocardial cells by immunohistochemistry. Seven cases of nemaline myopathy with cardiomyopathy have been reported in the literature. All of these patients were over 20 years of age, and the condition appeared mostly in the adult onset and the asymptomatic forms. This is the first infantile case of congenital nemaline myopathy which showed dilated cardiomyopathy with a fatal outcome.     

Autopsy case of desminopathy involving skeletal and cardiac muscle

Desminopathy is a familial or sporadic skeletal and cardiac muscular dystrophy caused by mutation in the desmin gene. Desmin-reactive deposits in the affected muscles are the morphological hallmarks of this disease. Herein is reported an autopsy case of a 57-year-old Japanese man with adult-onset skeletal muscle weakness and atrioventricular (A-V) conducting block, with a missense A337P mutation in exon 5 of the desmin gene. Disease onset occurred when the patient was 45 years old. The initial presentation was lower limb weakness, and the weakness progressed to the upper limbs. When the patient was 51 years old, a cardiac pacemaker was implanted due to complete A-V block. When the patient was 53 years old, respiratory insufficiency occurred due to weakness of respiratory muscles, and the patient died at the age of 57 years. On autopsy, intrasarcoplasmic desmin-immunoreactive deposits were identified in the skeletal and cardiac muscle, and abnormal accumulations of granulofilamentous material were identified at the ultrastructural level. In the cardiac conducting system, calcification was observed at the bundle of His, and sporadic calcium deposits were observed at the left and right bundle branches.