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Patrick Vicart - One of the best experts on this subject based on the ideXlab platform.
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Desmin mutation in the c terminal domain impairs traction force generation in myoblasts
Biophysical Journal, 2016Co-Authors: Elisabeth E. Charrier, Atef Asnacios, Patrick Vicart, Rachel Milloud, Richard De Mets, Martial Balland, Florence Delort, Olivier Cardoso, Sabrina Batonnetpichon, Sylvie HénonAbstract:The cytoskeleton plays a key role in the ability of cells to both resist mechanical stress and generate force, but the precise involvement of intermediate filaments in these processes remains unclear. We focus here on Desmin, a type III intermediate filament, which is specifically expressed in muscle cells and serves as a skeletal muscle differentiation marker. By using several complementary experimental techniques, we have investigated the impact of overexpressing Desmin and expressing a mutant Desmin on the passive and active mechanical properties of C2C12 myoblasts. We first show that the overexpression of wild-type-Desmin increases the overall rigidity of the cells, whereas the expression of a mutated E413K Desmin does not. This mutation in the Desmin gene is one of those leading to Desminopathies, a subgroup of myopathies associated with progressive muscular weakness that are characterized by the presence of Desmin aggregates and a disorganization of sarcomeres. We show that the expression of this mutant Desmin in C2C12 myoblasts induces Desmin network disorganization, Desmin aggregate formation, and a small decrease in the number and total length of stress fibers. We finally demonstrate that expression of the E413K mutant Desmin also alters the traction forces generation of single myoblasts lacking organized sarcomeres.
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Desmin, Mechanics and Myofibrillar Myopathies
Biophysical Journal, 2014Co-Authors: Elisabeth E. Charrier, Atef Asnacios, Sabrina Batonnet-pichon, Patrick Vicart, Sylvie HénonAbstract:The cytoskeleton plays a central role in transmitting and generating mechanical forces through the cell. It is composed of three interconnected networks, actin, microtubules and intermediate filaments (IF). Desmin belongs to the type III IF, specifically expressed in muscles. Desmin is essential to maintain the integrity and functioning of muscles. More than fifty mutations have been identified in the gene encoding Desmin leading to rare diseases belonging to MyoFibrillar Myopathy group (MFM). These pathologies are mainly characterized by aggregates formation in muscle tissue, associated with misorganizations of the contractile apparatus. Moreover patients progressively develop muscle weakness. Currently, pathophysiology and molecular defects of MFMs remain largely unknown, and no treatments are available.In this context, the aim of our study is to clarify whether Desmin mutations implicated in MFMs plays a role at early stage of expression, by impairing the properties of pre-muscular cells, the myoblasts. First we have studied the formation of Desmin aggregates in living myoblasts over-expressing for 24h wild-type (WT) or different mutant Desmins. We show that each mutant has a specific impact on the Desmin network organization. Second we have performed mechanical measurements on C2C12 cells, focusing on the E413K mutant, which induces a large Desmin network disorganization associated with important aggregate formation: we have compared the mechanical properties of WT-cells, C2C12 over-expressing Desmin-WT-GFP and C2C12 overexpressing mutated Desmin E413K-GFP. Visco-elastic properties of cells have been evaluated by using two custom-made set-ups, optical tweezers and a single-cell rheometer: we show that the 3 cells types share the same visco-elastic behaviour. Finally, we have investigated the impact of mutated Desmin on the contractility of myoblasts, and we demonstrate that E413K-mutation significantly decreases cell contraction abilities specifically for cells with Desmin aggregates, while aggregates of WT-Desmin do no induce the same effect.
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Viral-mediated expression of Desmin mutants to create mouse models of myofibrillar myopathy
Skeletal Muscle, 2013Co-Authors: Pierre Joanne, Patrick Vicart, Oussama Chourbagi, Christophe Hourdé, Arnaud Ferry, Gillian Butler-browne, Julie Dumonceaux, Onnik AgbulutAbstract:Background The clinical features of myofibrillar myopathies display a wide phenotypic heterogeneity. To this date, no studies have evaluated this parameter due to the absence of pertinent animal models. By studying two mutants of Desmin, which induce subtle phenotypic differences in patients, we address this issue using an animal model based on the use of adeno-associated virus (AAV) vectors carrying mutated Desmin cDNA. Methods After preparation of the vectors, they were injected directly into the tibialis anterior muscles of C57BL/6 mice to allow expression of wild-type (WT) or mutated (R406W or E413K) Desmin. Measurements of maximal force were carried out on the muscle in situ and then the injected muscles were analyzed to determine the structural consequences of the Desmin mutations on muscle structure (microscopic observations, histology and immunohistochemistry). Results Injection of AAV carrying WT Desmin results in the expression of exogenous Desmin in 98% of the muscle fibers without any pathological or functional perturbations. Exogenous WT and endogenous Desmin are co-localized and no differences were observed compared to non-injected muscle. Expression of Desmin mutants in mouse muscles induce morphological changes of muscle fibers (irregular shape and size) and the appearance of Desmin accumulations around the nuclei (for R406W) or in subsarcolemmal regions of fibers (for E413K). These accumulations seem to occur and disrupt the Z-line, and a strong regeneration was observed in muscle expressing the R406W Desmin, which is not the case for E413K. Moreover, both mutants of Desmin studied here induce a decrease in muscle force generation capacity. Conclusions In this study we show that AAV-mediated expression of Desmin mutants in mouse muscles recapitulate the aggregation features, the decrease in contractile function and the morphological changes observed in patients with myofibrillar myopathy. More importantly, our results suggest that the R406W Desmin mutant induces a robust muscle regeneration, which is not the case for the E413K mutant. This difference could help to explain the phenotypic differences observed in patients. Our results highlight the heterogeneous pathogenic mechanisms between different Desmin mutants and open the way for new advances in the study of myofibrillar myopathies.
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Desmin mutations in the terminal consensus motif prevent synemin-Desmin heteropolymer filament assembly.
Experimental cell research, 2011Co-Authors: Oussama Chourbagi, Patrick Vicart, Francine Bruston, Marianna Carinci, Zhigang Xue, Denise Paulin, Onnik AgbulutAbstract:Disorganization of the Desmin network is associated with cardiac and skeletal myopathies characterized by accumulation of Desmin-containing aggregates in the cells. Multiple associations of intermediate filament proteins form a network to increase mechanical and functional stability. Synemin is a Desmin-associated type VI intermediate filament protein. Neither its impact on Desmin network nor how it integrates into Desmin filament is yet elucidated. To gain more insight into the molecular basis of these processes, we coexpressed synemin with different Desmin mutants in ex vivo models. The screening of fourteen Desmin mutants showed that synemin with Desmin mutants revealed two behaviors. Firstly, synemin was co-localized in Desmin aggregates and its coexpression decreased the number of cells containing aggregates. Secondly, synemin was excluded from the aggregates, then synemin had no effect on Desmin network organization. Among fourteen Desmin mutants, there were only three mutants, p.E401K, p.R406W and p.E413K, in which synemin was not found in aggregates. This behavior was correlated to the abnormal salt-bridges of Desmin-dimer as seen in silico constructs. Moreover, Desmin constructs in silico and published results in literature have predicted that the salt-bridges absence in the Desmin filament building prevent longitudinal annealing and/or radial compaction. These results suggest that the state of Desmin-filament assembly is crucial for synemin anchorage and consequently might involve mechanical and functional stability of the cytoskeletal network.
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restrictive cardiomyopathy with atrioventricular conduction block resulting from a Desmin mutation
International Journal of Cardiology, 2007Co-Authors: Piotr Pruszczyk, Patrick Vicart, Bertrand Goudeau, Nyamkhishig Sambuughin, Anna Kosterapruszczyk, Aleksey Shatunov, Agnieszka Draminska, Kazuyo Takeda, Sergei V StrelkovAbstract:Background: According to the predominant view, Desmin mutations cause dilated cardiomyopathy (DCM). We evaluated a family with restrictive cardiomyopathy (RCM) associated with a novel Desmin mutation and reviewed recent reports regarding the frequency of RCM in patients with Desmin myopathy. Methods: Cardiovascular examination was performed in three affected and five at-risk members of a family from Poland, histopathologic study of skeletal muscle biopsy was done in a single patient, and functional analysis of mutant Desmin protein was carried out in cultured cells. Results: Cardiovascular assessment led to the diagnosis of RCM in affected family members. Histopathological study of skeletal muscle biopsy revealed features characteristic of Desmin myopathy. A novel Desmin E413K mutation was identified in each affected family member, but not unrelated controls. The pathogenicity of the E413K mutation was confirmed in transfected cell cultures showing inability of mutant Desmin to form a cellular filamentous network or support a pre-existing network formed by other intermediate filaments. Three-dimensional modeling and electrostatic calculations indicated that the E413K mutation located in a functionally unique domain of Desmin molecule potentially disrupts intramolecular interactions. Analysis of previously reported observations indicates that RCM in Desminopathy patients may be as frequent as DCM. Conclusions: A novel E413K mutation in Desmin caused autosomal dominant RCM rather than DCM. The location of the E413K mutation at a highly conserved end of the α-helical rod domain may be related to the phenotypic differences from the previously described DCM-associated Desmin mutations. Functional and structural analyses of mutant Desmin allowed to identify likely pathogenic mechanisms.
Yassemi Capetanaki - One of the best experts on this subject based on the ideXlab platform.
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Amelioration of Desmin network defects by αB-crystallin overexpression confers cardioprotection in a mouse model of dilated cardiomyopathy caused by LMNA gene mutation
Journal of Molecular and Cellular Cardiology, 2018Co-Authors: Zoi Galata, Ismini Kloukina, Ioanna Kostavasili, Aimilia Varela, Constantinos Davos, Manousos Makridakis, Gisѐle Bonne, Yassemi CapetanakiAbstract:The link between the cytoplasmic Desmin intermediate filaments and those of nuclear lamins serves as a major integrator point for the intracellular communication between the nucleus and the cytoplasm in cardiac muscle. We investigated the involvement of Desmin in the cardiomyopathy caused by the lamin A/C gene mutation using the LmnaH222P/H222P mouse model of the disease. We demonstrate that in these mouse hearts Desmin loses its normal Z disk and intercalated disc localization and presents aggregate formation along with mislocalization of basic intercalated disc protein components, as well as severe structural abnormalities of the intercalated discs and mitochondria. To address the extent by which the observed Desmin network defects contribute to the progression of LmnaH222P/H222P cardiomyopathy, we investigated the consequences of Desmin-targeted approaches for the disease treatment. We showed that cardiac-specific overexpression of the small heat shock protein αΒ-Crystallin confers cardioprotection in LmnaH222P/H222P mice by ameliorating Desmin network defects and by attenuating the Desmin-dependent mislocalization of basic intercalated disc protein components. In addition, αΒ-Crystallin overexpression rescues the intercalated disc, mitochondrial and nuclear defects of LmnaH222P/H222P hearts, as well as the abnormal activation of ERK1/2. Consistent with that, by generating the LmnaH222P/H222PDes+/- mice, we showed that the genetically decreased endogenous Desmin levels have cardioprotective effects in LmnaH222P/H222P hearts since less Desmin is available to form dysfunctional aggregates. In conclusion, our results demonstrate that Desmin network disruption, disorganization of intercalated discs and mitochondrial defects are a major mechanism contributing to the progression of this LMNA cardiomyopathy and can be ameliorated by αΒ-Crystallin overexpression.
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Desmin mediates tnf α induced aggregate formation and intercalated disk reorganization in heart failure
Journal of Cell Biology, 2008Co-Authors: P Panagopoulou, Derek J Milner, Constantinos H Davos, Emily Varela, Jo Ann Cameron, Douglas L Mann, Yassemi CapetanakiAbstract:We explored the involvement of the muscle-specific intermediate filament protein Desmin in the model of tumor necrosis factor α (TNF-α)–induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-α in the heart (α–myosin heavy chain promoter-driven secretable TNF-α [MHCsTNF]), Desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with heat shock protein 25 and ubiquitin. Additionally, other ID proteins such as desmoplakin and β-catenin show similar localization changes in a Desmin-dependent fashion. To address underlying mechanisms, we examined whether Desmin is a substrate for caspase-6 in vivo as well as the implications of Desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a Desmin mutant (D263E) and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E Desmin, desmoplakin, and β-catenin largely retain their proper ID localization. Importantly, D263E Desmin expression attenuated cardiomyocyte apoptosis, prevented left ventricular wall thinning, and improved the function of MHCsTNF hearts.
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structural and functional roles of Desmin in mouse skeletal muscle during passive deformation
Biophysical Journal, 2004Co-Authors: Sameer B Shah, Noah Weisleder, Yassemi Capetanaki, Jennifer Davis, Ioanna Kostavassili, Andrew D Mcculloch, Evelyn Ralston, Richard L. LieberAbstract:Mechanical interactions between Desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and Desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein α-actinin, even at very high strains and stresses. These data indicate that Desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and Desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to Desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires Desmin. Passive mechanical analysis revealed that Desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of α-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of Desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of Desmin during passive fiber loading.
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Cardiomyocyte-specific Desmin rescue of Desmin null cardiomyopathy excludes vascular involvement.
Journal of molecular and cellular cardiology, 2004Co-Authors: Noah Weisleder, Elisavet Soumaka, Shahrzad Abbasi, Heinrich Taegtmeyer, Yassemi CapetanakiAbstract:Mice deficient in Desmin, the muscle-specific member of the intermediate filament gene family, display defects in all muscle types and particularly in the myocardium. Desmin null hearts develop cardiomyocyte hypertrophy and dilated cardiomyopathy (DCM) characterized by extensive myocyte cell death, calcific fibrosis and multiple ultrastructural defects. Several lines of evidence suggest impaired vascular function in Desmin null animals. To determine whether altered capillary function or an intrinsic cardiomyocyte defect is responsible for Desmin null DCM, transgenic mice were generated to rescue Desmin expression specifically to cardiomyocytes. Desmin rescue mice display a wild-type cardiac phenotype with no fibrosis or calcification in the myocardium and normalization of coronary flow. Cardiomyocyte ultrastructure is also restored to normal. Markers of hypertrophy upregulated in Desmin null hearts return to wild-type levels in Desmin rescue mice. Working hearts were perfused to assess coronary flow and cardiac power. Restoration of a wild-type cardiac phenotype in a Desmin null background by expression of Desmin specifically within cardiomyocyte indicates that defects in the Desmin null heart are due to an intrinsic cardiomyocytes defect rather than compromised coronary circulation.
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caspase proteolysis of Desmin produces a dominant negative inhibitor of intermediate filaments and promotes apoptosis
Journal of Biological Chemistry, 2003Co-Authors: Feng Chen, Yassemi Capetanaki, Roger Chang, Marcus Trivedi, Vincent L CrynsAbstract:Abstract Caspase cleavage of key cytoskeletal proteins, including several intermediate filament proteins, triggers the dramatic disassembly of the cytoskeleton that characterizes apoptosis. Here we describe the muscle-specific intermediate filament protein Desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD↓M264) by caspase-6 in vitro and in myogenic cells undergoing apoptosis. We demonstrate that caspase cleavage of Desmin at Asp263 has important functional consequences, including the production of an amino-terminal cleavage product, N-Desmin, which is unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-Desmin functions as a dominant-negative inhibitor of filament assembly, both for Desmin and the structurally related intermediate filament protein vimentin. We also show that stable expression of a caspase cleavage-resistant Desmin D263E mutant partially protects cells from tumor necrosis factor-α-induced apoptosis. Taken together, these results indicate that caspase proteolysis of Desmin at Asp263produces a dominant-negative inhibitor of intermediate filaments and actively participates in the execution of apoptosis. In addition, these findings provide further evidence that the intermediate filament cytoskeleton has been targeted systematically for degradation during apoptosis.
Gloria M Conover - One of the best experts on this subject based on the ideXlab platform.
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αB-crystallin is a sensor for assembly intermediates and for the subunit topology of Desmin intermediate filaments
Cell Stress and Chaperones, 2017Co-Authors: Sarika Sharma, Gloria M Conover, Harald Herrmann, Jayne L. Elliott, Ming Der Perng, Roy A. QuinlanAbstract:Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein Desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for Desmin on CRYAB have been determined, Desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to Desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of Desmin filaments, we show that the binding of CRYAB to Desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of Desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated Desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the Desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of Desmin-CRYAB myopathies.
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nebulin binding impedes mutant Desmin filament assembly
Molecular Biology of the Cell, 2013Co-Authors: Laura K Baker, David C Gillis, Sarika Sharma, Andy Ambrus, Harald Herrmann, Gloria M ConoverAbstract:Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of Desmin to sarcomeres and their role in Desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of Desmin and three Desminopathy-causing mutant Desmin variants carrying mutations in the head, rod, or tail domains of Desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of Desmin. We discovered that, although nebulin M160–164 bound to both Desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type Desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant Desmin E245D relative to wild-type Desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant Desmin slows Desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming Desmin mutants, the molecular etiology of Desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.
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the Desmin coil 1b mutation k190a impairs nebulin z disc assembly and destabilizes actin thin filaments
Journal of Cell Science, 2011Co-Authors: Gloria M Conover, Carol C GregorioAbstract:Desmin intermediate filaments intimately surround myofibrils in vertebrate muscle forming a mesh-like filament network. Desmin attaches to sarcomeres through its high-affinity association with nebulin, a giant F-actin binding protein that co-extends along the length of actin thin filaments. Here, we further investigated the functional significance of the association of Desmin and nebulin in cultured primary myocytes to address the hypothesis that this association is key in integrating myofibrils to the intermediate filament network. Surprisingly, we identified eight peptides along the length of Desmin that are capable of binding to C-terminal modules 160–170 in nebulin. In this study, we identified a targeted mutation (K190A) in the Desmin coil 1B region that results in its reduced binding with the nebulin C-terminal modules. Using immunofluorescence microscopy and quantitative analysis, we demonstrate that expression of the mutant Desmin K190A in primary myocytes results in a significant reduction in assembled endogenous nebulin and Desmin at the Z-disc. Non-uniform actin filaments were markedly prevalent in myocytes expressing GFP-tagged Desmin K190A, suggesting that the near-crystalline organization of actin filaments in striated muscle depends on a stable interaction between Desmin and nebulin. All together, these data are consistent with a model in which Z-disc-associated nebulin interacts with Desmin through multiple sites to provide efficient stability to satisfy the dynamic contractile activity of myocytes.
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a myopathy linked Desmin mutation perturbs striated muscle actin filament architecture
Molecular Biology of the Cell, 2008Co-Authors: Gloria M Conover, Syerra N Henderson, Carol C GregorioAbstract:Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a Desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of Desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of Desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this Desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the Desmin-E245D mutant in myocytes displaces endogenous Desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of Desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the Desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for Desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the Desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated Desmin mutations.
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a myopathy linked Desmin mutation perturbs striated muscle actin filament architecture
Molecular Biology of the Cell, 2008Co-Authors: Gloria M Conover, Syerra N Henderson, Carol C GregorioAbstract:Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a Desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of Desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of Desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this Desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the Desmin-E245D mutant in myocytes displaces endogenous Desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of Desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the Desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for Desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the Desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated Desmin mutations.
Harald Herrmann - One of the best experts on this subject based on the ideXlab platform.
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Dual functional states of R406W-Desmin assembly complexes cause cardiomyopathy with severe intercalated disc derangement in humans and in knock-in mice
Circulation, 2020Co-Authors: Harald Herrmann, Eva Cabet, Nicolas Chevalier, Julia Moosmann, Dorothea Schultheis, Jan Haas, Mirjam Schowalter, Carolin Berwanger, Veronika Weyerer, Abbas AgaimyAbstract:Background: Mutations in the human Desmin gene cause myopathies and cardiomyopathies. Aim of this study was to elucidate molecular mechanisms initiated by the heterozygous R406W-Desmin mutation in the development of a severe and early-onset cardiac phenotype. Methods: We report an adolescent patient, who underwent cardiac transplantation due to restrictive cardiomyopathy caused by a heterozygous R406W-Desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-Desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of Desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious impact of the mutation on heart tissue, we further generated and analyzed R405W-Desmin knock-in mice harboring the orthologous form of the human R406W-Desmin. Results: Microscopic analysis of the explanted heart revealed Desmin aggregates and the absence of Desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-Desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-Desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type Desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-Desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathological results from the patient are recapitulated in the hearts from R405W-Desmin knock-in mice of both genotypes. Moreover, while heterozygous knock-in mice have a normal life span, homozygous animals die at three months of age due to a smooth muscle-related gastrointestinal phenotype. Conclusions: We demonstrate that R406W-Desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant Desmin assembly complexes underlie the uncoupling of Desmin filaments from intercalated discs and their structural disorganization.
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αB-crystallin is a sensor for assembly intermediates and for the subunit topology of Desmin intermediate filaments
Cell Stress and Chaperones, 2017Co-Authors: Sarika Sharma, Gloria M Conover, Harald Herrmann, Jayne L. Elliott, Ming Der Perng, Roy A. QuinlanAbstract:Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein Desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for Desmin on CRYAB have been determined, Desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to Desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of Desmin filaments, we show that the binding of CRYAB to Desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of Desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated Desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the Desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of Desmin-CRYAB myopathies.
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nebulin binding impedes mutant Desmin filament assembly
Molecular Biology of the Cell, 2013Co-Authors: Laura K Baker, David C Gillis, Sarika Sharma, Andy Ambrus, Harald Herrmann, Gloria M ConoverAbstract:Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of Desmin to sarcomeres and their role in Desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of Desmin and three Desminopathy-causing mutant Desmin variants carrying mutations in the head, rod, or tail domains of Desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of Desmin. We discovered that, although nebulin M160–164 bound to both Desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type Desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant Desmin E245D relative to wild-type Desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant Desmin slows Desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming Desmin mutants, the molecular etiology of Desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.
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forced expression of Desmin and Desmin mutants in cultured cells impact of myopathic missense mutations in the central coiled coil domain on network formation
Experimental Cell Research, 2006Co-Authors: Harald Bar, Anna Kostareva, Gunnar Sjoberg, Thomas Sejersen, Hugo A Katus, Harald HerrmannAbstract:Abstract We recently demonstrated that inherited disease-causing mutations clustered in the α-helical coiled-coil “rod” domain of the muscle-specific intermediate filament (IF) protein Desmin display a wide range of inhibitory effects on regular in vitro assembly. In these studies, we showed that individual mutations exhibited phenotypes that were not, with respect to the severity of interference, predictable by our current knowledge of the structural design of IF proteins. Moreover, the behavior of some mutated proteins in a standard tissue culture cell expression system was found to be even more complex. Here, we systematically investigate the behavior of these disease mutants in four different cell types: three not containing Desmin or the related IF protein vimentin and the standard fibroblast line 3T3, which has an extensive vimentin system. The ability of the mutants to form filaments in the vimentin-free cells varies considerably, and only the mutants forming IFs in vitro generate extended filamentous networks. Furthermore, these latter mutants integrate into the 3T3 vimentin network but all the others do not. Instead, they cause the endogenous network of 3T3 vimentin to reorganize into perinuclear bundles. In addition, most of these assembly-deficient mutant Desmins completely segregate from the vimentin system. Instead, the small round to fibrillar particles formed distribute independently throughout the cytoplasm as well as between the collapsed vimentin filament arrays in the perinuclear area.
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Pathogenic effects of a novel heterozygous R350P Desmin mutation on the assembly of Desmin intermediate filaments in vivo and in vitro
Human molecular genetics, 2005Co-Authors: Harald Bar, Harald Herrmann, Patrick Vicart, Dirk Fischer, Bertrand Goudeau, Rudolf A. Kley, Christoph S. Clemen, Matthias Vorgerd, Rolf SchröderAbstract:Mutations of the human Desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Here, we report the pathogenic effects of a novel heterozygous R350P Desmin missense mutation, which resides in the evolutionary highly conserved coil 2B domain of the alpha-helical coiled-coil Desmin rod domain, on the assembly of Desmin intermediate filaments (IF) in cultured cells and in vitro. By transfection experiments, we show that R350P Desmin is incapable of de novo formation of a Desmin IF network in vimentin-free BMGE+H, MCF7 and SW13 cells and that it disrupts the endogenous vimentin cytoskeleton in 3T3 fibroblast cells. Hence, transfected cells displayed abnormal cytoplasmic protein aggregates reminiscent of Desmin-positive protein deposits seen in the immunohistochemical and ultrastructural analysis of skeletal muscle derived from the index patient of the affected family. To study the functional effects of the R350P Desmin mutation at the protein level, we performed in vitro assembly studies with wild-type (WT) and mutant Desmin protein. Our analysis revealed that the in vitro assembly process of R350P Desmin is already disturbed at the unit length filament level and that further association reactions generate huge, tightly packed protein aggregates. On assessing the pathogenic effects of R350P Desmin in various mixtures with WT Desmin, we show that a ratio of 1 : 3 (R350P Desmin/WT Desmin) is sufficient to effectively block the normal polymerization process of Desmin IFs. Our findings indicate that the heterozygous R350P Desmin mutation exerts a dominant negative effect on the ordered lateral arrangement of Desmin subunits. This disturbance of the lateral packing taking place in the first phase of assembly is ultimately leading to abnormal protein aggregation.
Denise Paulin - One of the best experts on this subject based on the ideXlab platform.
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Posttranslational modifications of Desmin and their implication in biological processes and pathologies
Histochemistry and Cell Biology, 2014Co-Authors: Daniel L. Winter, Denise Paulin, Mathias Mericskay, Zhenlin LiAbstract:Desmin, the muscle-specific intermediate filament, is involved in myofibrillar myopathies, dilated cardiomyopathy and muscle wasting. Desmin is the target of posttranslational modifications (PTMs) such as phosphorylation, ADP-ribosylation and ubiquitylation as well as nonenzymatic modifications such as glycation, oxidation and nitration. Several PTM target residues and their corresponding modifying enzymes have been discovered in human and nonhuman Desmin. The major effect of phosphorylation and ADP-ribosylation is the disassembly of Desmin filaments, while ubiquitylation of Desmin leads to its degradation. The regulation of the Desmin filament network by phosphorylation and ADP-ribosylation was found to be implicated in several major biological processes such as myogenesis, myoblast fusion, muscle contraction, muscle atrophy, cell division and possibly Desmin interactions with its binding partners. Phosphorylation of Desmin is also implicated in many forms of Desmin-related myopathies (Desminopathies). In this review, we summarize the findings on Desmin PTMs and their implication in biological processes and pathologies, and discuss the current knowledge on the regulation of the Desmin network by PTMs. We conclude that the Desmin filament network can be seen as an intricate scaffold for muscle cell structure and biological processes and that its dynamics can be affected by PTMs. There are now precise tools to investigate PTMs and visualize cellular structures that have been underexploited in the study of Desminopathies. Future studies should focus on these aspects.
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Desmin mutations in the terminal consensus motif prevent synemin-Desmin heteropolymer filament assembly.
Experimental cell research, 2011Co-Authors: Oussama Chourbagi, Patrick Vicart, Francine Bruston, Marianna Carinci, Zhigang Xue, Denise Paulin, Onnik AgbulutAbstract:Disorganization of the Desmin network is associated with cardiac and skeletal myopathies characterized by accumulation of Desmin-containing aggregates in the cells. Multiple associations of intermediate filament proteins form a network to increase mechanical and functional stability. Synemin is a Desmin-associated type VI intermediate filament protein. Neither its impact on Desmin network nor how it integrates into Desmin filament is yet elucidated. To gain more insight into the molecular basis of these processes, we coexpressed synemin with different Desmin mutants in ex vivo models. The screening of fourteen Desmin mutants showed that synemin with Desmin mutants revealed two behaviors. Firstly, synemin was co-localized in Desmin aggregates and its coexpression decreased the number of cells containing aggregates. Secondly, synemin was excluded from the aggregates, then synemin had no effect on Desmin network organization. Among fourteen Desmin mutants, there were only three mutants, p.E401K, p.R406W and p.E413K, in which synemin was not found in aggregates. This behavior was correlated to the abnormal salt-bridges of Desmin-dimer as seen in silico constructs. Moreover, Desmin constructs in silico and published results in literature have predicted that the salt-bridges absence in the Desmin filament building prevent longitudinal annealing and/or radial compaction. These results suggest that the state of Desmin-filament assembly is crucial for synemin anchorage and consequently might involve mechanical and functional stability of the cytoskeletal network.
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Cardiac conduction disturbances and differential effects on atrial and ventricular electrophysiological properties in Desmin deficient mice.
Journal of Interventional Cardiac Electrophysiology, 2010Co-Authors: Jan W. Schrickel, Denise Paulin, Zhenlin Li, Florian Stöckigt, Wieslaw Krzyzak, Indra Lübkemeier, Bernd K. Fleischmann, Philipp Sasse, Markus Linhart, Thorsten LewalterAbstract:Purpose Desmin mutations in humans cause Desmin-related cardiomyopathy, resulting in heart failure, atrial and ventricular arrhythmias, and sudden cardiac death. The intermediate filament Desmin is strongly expressed in striated muscle cells and in Purkinje fibers of the ventricular conduction system. The aim of the present study was to characterize electrophysiological cardiac properties in a Desmin-deficient mouse model.
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Desmin a major intermediate filament protein essential for the structural integrity and function of muscle
Experimental Cell Research, 2004Co-Authors: Denise PaulinAbstract:Desmin is a muscle-specific protein and a key subunit of the intermediate filament in cardiac, skeletal and smooth muscles. Desmin filaments are mainly located at the periphery of Z-disk of striated muscles and at the dense bodies of smooth muscle cells, and they have been postulated to play a critical role in the maintenance of structural and mechanical integrity of the contractile apparatus in muscle tissues. This review summarizes the findings in the regulation of the Desmin gene and function of the Desmin protein. The expression of Desmin gene is regulated by a combination of different transcription control regions in muscle cells. The results from mice deficient in Desmin reveal the fundamental role of Desmin filaments in cell architecture, force transmission and mitochondrial function. Mice lacking Desmin postnatally develop a dilated cardiomyopathy, a skeletal myopathy and smooth muscle defects. Some of Desmin-related myopathies are attributable to a missense mutations and deletions in the Desmin gene. Other Desmin-related myopathies, in which the Desmin gene is not mutated, could be related to mutations of the genes encoding the proteins that interact with Desmin.
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Human Desmin gene: cDNA sequence, regional localization and exclusion of the locus in a familial Desmin-related myopathy.
Human genetics, 1996Co-Authors: Patrick Vicart, Jean-marie Dupret, Jamilé Hazan, Gabor Gyapay, Rajagopal Krishnamoorthy, Jean Weissenbach, Michel Fardeau, Denise PaulinAbstract:Desmin is a muscle-specific intermediate filament that is encoded by a gene assigned to human chromosome 2q35. Desmin-related myopathies are inherited disorders characterized by an intrasarcoplasmic accumulation of Desmin. Recently, the knockout of the Desmin gene was shown to generate a myopathic syndrome in transgenic mice, suggesting that functional abnormality of Desmin may generate similar clinical symptoms in mouse and human. To determine the potential role of the Desmin gene in a well-defined Desmin-related myopathy (autosomal dominant form of Fardeau), human Desmin cDNAs obtained from affected and unaffected individuals were cloned, sequenced and compared. No obvious mutation was detected. A BssHII restriction fragment length polymorphism (RFLP) was identified in exon 6 of the Desmin gene. This RFLP was associated with a previously identified EcoRV RFLP in exon 4 to generate a tetra-allelic system, which was tested for linkage to the Desmin-related myopathy in three families. The human Desmin gene was localized within an 11-cM interval on chromosome 2q using a panel of radiation hybrids. This 11-cM region was clearly excluded by linkage analysis in the three Desmin-related myopathy families using a set of highly polymorphic microsatellite markers. These results suggest that the Desmin gene is not primarily involved in this disease.