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Mutations in the DES gene, which encodes the intermediate filament desmin, lead to desminopathy, a rare disease characterized by skeletal muscle weakness and different forms of cardiomyopathies associated with cardiac conduction defects and arrhythmias. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying the DES p.R406W mutation, and employed CRISPR/Cas9 to rectify the mutation in the patient's hiPSC line and introduced the mutation in an hiPSC line from a control individual unrelated to the patient. These hiPSC lines represent useful models for delving into the mechanisms of desminopathy and developing new therapeutic approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The intermediate filament (IF) cytoskeleton supports cellular structural integrity, particularly in response to mechanical stress. The most abundant IF proteins in mature cardiomyocytes are desmin and lamins. The desmin network tethers the contractile apparatus and organelles to the nuclear envelope and the sarcolemma, while lamins, as components of the nuclear lamina, provide structural stability to the nucleus and the genome. Mutations in desmin or A-type lamins typically result in cardiomyopathies and recent studies emphasized the synergistic roles of desmin and lamins in the maintenance of nuclear integrity in cardiac myocytes. Here we explore the emerging roles of the interdependent relationship between desmin and lamins in providing resilience to nuclear structure while transducing extracellular mechanical cues into the nucleus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Arrhythmogenic cardiomyopathy (AC) is a heritable myocardial disorder and a major cause of sudden cardiac death. It is typically caused by mutations in desmosomal genes. Desmin gene (DES) variants have been previously reported in AC but with insufficient evidence to support their pathogenicity., Methods: We aimed to assess a large AC patient cohort for DES mutations and describe a unique phenotype associated with a recurring variant in three families. A cohort of 138 probands with a diagnosis of AC and no identifiable desmosomal gene mutations were prospectively screened by whole-exome sequencing., Results: A single DES variant (p.Leu115Ile, c.343C>A) was identified in 3 index patients (2%). We assessed the clinical phenotypes within their families and confirmed cosegregation. One carrier required heart transplantation, 2 died suddenly, and 1 died of noncardiac causes. All cases had right- and left-ventricular (LV) involvement. LV late gadolinium enhancement was present in all, and circumferential subepicardial distribution was confirmed on histology. A significant burden of ventricular arrhythmias was noted. Desmin aggregates were not observed macroscopically, but analysis of the desmin filament formation in transfected cardiomyocytes derived from induced pluripotent stem cells, and SW13 cells revealed cytoplasmic aggregation of mutant desmin. Atomic force microscopy revealed that the mutant form accumulates into short protofilaments and small fibrous aggregates., Conclusions: DES p.Leu115Ile leads to disruption of the desmin filament network and causes a malignant biventricular form of AC, characterized by LV dysfunction and a circumferential subepicardial distribution of myocardial fibrosis., (Copyright © 2020 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Background: Desmin is the major intermediate filament (IF) protein in human heart and skeletal muscle. So-called 'desminopathies' are disorders due to pathogenic variants in the DES gene and are associated with skeletal myopathies and/or various types of cardiomyopathies. So far, only a limited number of DES pathogenic variants have been identified and functionally characterized., Methods and Results: Using a Sanger- and next generation sequencing (NGS) approach in patients with various types of cardiomyopathies, we identified two novel, non-synonymous missense DES variants: p.(Ile402Thr) and p.(Glu410Lys). Mutation carriers developed dilated (DCM) or arrhythmogenic cardiomyopathy (ACM), and cardiac conduction disease, leading to spare out the exercise-induced polymorphic ventricular tachycardia; we moved this variant to data in brief. To investigate the functional impact of these four DES variants, transfection experiments using SW-13 and H9c2 cells with native and mutant desmin were performed and filament assembly was analyzed by confocal microscopy. The DES_p.(Ile402Thr) and DES_p.(Glu410Lys) cells showed filament assembly defects forming cytoplasmic desmin aggregates. Furthermore, immunohistochemical and ultrastructural analysis of myocardial tissue from mutation carriers with the DES_p.(Glu410Lys) pathogenic variant supported the in vitro results., Conclusions: Our in vitro results supported the classification of DES_p.(Ile402Thr) and DES_p.(Glu410Lys) as novel pathogenic variants and demonstrated that the cardiac phenotypes associated with DES variants are diverse and cell culture experiments improve in silico analysis and genetic counseling because the pathogenicity of a variant can be clarified., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Desmin, being a major intermediate filament of muscle cells, contributes to stabilization and positioning of mitochondria. Desmin mutations have been reported in conjunction with skeletal myopathies accompanied by mitochondrial dysfunction. Depending on the ability to promote intracellular aggregates formation, mutations can be considered aggregate-prone or non-aggregate-prone. The aim of the present study was to describe how expression of different desmin mutant isoforms effects mitochondria and contributes to the development of myocyte dysfunction. To achieve this goal, two non-aggregate-prone (Des S12F and Des A213V) and four aggregate-prone (Des L345P, Des A357P, Des L370P, Des D399Y) desmin mutations were expressed in skeletal muscle cells. We showed that all evaluated mutations affected the morphology of mitochondrial network, suppressed parameters of mitochondrial respiration, diminished mitochondrial membrane potential, increased ADP/ATP ratio, and enhanced mitochondrial DNA (mtDNA) release. mtDNA was partially secreted through exosomes as demonstrated by GW4869 treatment. Dysfunction of mitochondria was observed regardless the type of mutation: aggregate-prone or non-aggregate-prone. However, expression of aggregate-prone mutations resulted in more prominent phenotype. Thus, in this comparative study of six pathogenic desmin mutations that cause skeletal myopathy development, we confirmed a role of mitochondrial dysfunction and mtDNA release in the pathogenesis of desmin myopathies, regardless of the aggregation capacity of the mutated desmin., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: Mechanoelectric feedback (MEF) was related to malignant arrhythmias in heart failure (HF). Desmin is a cytoskeleton protein and could be involved in MEF as a mechanoelectrical transducer. In this study, we will discuss the role of desmin alterations in mechanical electrical feedback in heart failure and its mechanisms., Methods: We used both an in vivo rat model and an in vitro cardiomyocyte model to address this issue. For the in vivo experiments, we establish a sham group, an HF group, streptomycin (SM) group, and an MDL-28170 group. The occurrence of ventricular arrhythmias (VA) was recorded in each group. For the in vitro cardiomyocyte model, we established an NC group, a si-desmin group, and a si-desmin + NBD IKK group. The expression of desmin, IKKβ, p-IKKβ, IKBα, p-NF-κB, and SERCA2 were detected in both in vivo and in vitro experiments. The content of Ca 2+ in cytoplasm and sarcoplasmic were detected by confocal imaging in vitro experiments., Results: An increased number of VAs were found in the HF group. SM and MDL-28170 can reduce desmin breakdown and the number of VAs in heart failure. The knockdown of desmin in the cardiomyocyte can activate the NF-κB pathway, decrease the level of SERCA2, and result in abnormal distribution of Ca 2+ . While treatment with NF-κB inhibitor can elevate the level of SERCA2 and alleviate the abnormal distribution of Ca 2+ ., Significance: Overall, desmin may participate in MEF through the NF-κB pathway. This study provides a potential therapeutic target for VA in HF., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Joubert syndrome (JBTS) is a predominantly autosomal recessive neurodevelopmental disorder that presents with characteristic malformations of the cerebellar vermis, superior cerebellar peduncles and midbrain in humans. Accompanying these malformations are a heterogeneous set of clinical symptoms, which frequently include deficits in motor and muscle function, such as hypotonia (low muscle tone) and ataxia (clumsiness). These symptoms are attributed to improper development of the hindbrain, but no direct evidence has been reported linking these in JBTS. Here, we describe muscle developmental defects in a mouse with a targeted deletion of the Abelson helper integration site 1 gene, Ahi1, one of the genes known to cause JBTS in humans. While FVB/NJ Ahi1 -/- mice display no gross malformations of the cerebellum, deficits are observed in several measures of motor function, strength, and body development. Specifically, Ahi1 -/- mice show delayed physical development, delays in surface reflex righting as neonates, and reductions in grip strength and spontaneous locomotor activity as adults. Additionally, Ahi1 -/- mice showed evidence of muscle-specific contributions to this phenotype, such as reductions in 1) myoblast differentiation potential in vitro, 2) muscle desmin expression, and 3) overall muscle mass, myonuclear domain, and muscle fiber cross-sectional area. Together, these data suggest that loss of Ahi1 may cause abnormalities in the differentiation of myoblasts to mature muscle cells. Moreover, Ahi1 loss impacts muscle development directly, outside of any indirect impact of cerebellar malformations, revealing a novel myogenic cause for hypotonia in JBTS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: This study aimed to investigate the correlation between pancreatic stellate cell activation, matrix metallopeptidase 2 (MMP2) expression and lymph node metastasis in pancreatic carcinoma., Methods: Alpha-smooth muscle actin (ACTA2), Desmin (DES) and MMP2 were detected in 40 pancreatic carcinoma patients and 10 cases of normal pancreas tissues using immunohistochemistry. Then MMP2 and ACTA2 expression profiles in pancreatic cancer were obtained from UCSC (University of California, Santa Cruz) and SurvExpress., Results: A total of 67.5% and 55.0% of cases positively expressed ACTA2 and DES in pancreatic carcinoma, respectively. MMP2 in pancreatic carcinoma was expressed in 55.0% of cases, and there were significant differences between the lymph node metastasis group and the lymph node nonmetastasis group, as well as invasion and noninvasion to the peripheral tissue group (P < 0.01). High throughput sequencing databases verified that ACTA2 and MMP2 gene expression were both upregulated in pancreatic carcinoma tissues., Conclusions: The coexpression of ACTA2 and DES was related to the expression of MMP2, and positively correlated with lymph node metastasis. Activation of pancreatic stellate cells may promote the expression of MMP2 and enhance the invasion and metastasis of pancreatic carcinoma., (Copyright © 2018. Published by Elsevier Inc.)

In this study, we aimed to assess the differential toxic impact, induced by furan exposure, on the liver and kidney tissues by estimating reactive oxygen species (ROS) level, total antioxidant capacity (TAC), oxidative damage, and the tissue injury markers in a male rat model. To explain such impacts, 20 rats were assigned into two groups: a control group, where rats were administered corn oil as a vehicle, and a furan-administered group, where furan was orally administered to rats at a dose of 16 mg/kg b wt/day (five days per week over eight weeks). The transcriptional levels of intermediate filament proteins (desmin, vimentin, nestin, and connexin 43) were assessed by using quantitative real-time polymerase chain reaction (PCR), and the cell proliferation markers (proliferating cell nuclear antigen [PCNA] and proliferation-associated nuclear antigen [Ki-67]) were recognized by immunohistochemical analysis. Furthermore, the ultrastructural changes of liver and kidney were monitored using electron microscopy. Our findings showed that furan exposure could induce hepatic and renal damage to different extents. Furan can increase the ROS content, oxidative damage indices, and liver tissue injury indices but not kidney injury indices. Furthermore, it decreases the TAC in the serum of exposed rats. In addition, furan exposure was associated with changes in the mRNA expression pattern of intermediate filament proteins in both kidney and liver tissues. Moreover, furan enhances the expression of PCNA and Ki-67 in the liver tissues but not in the kidney tissues. The ultrastructure evaluation revealed the incidence of glomerular podocyte degeneration and hepatocyte injury. These results conclusively demonstrate that the deleterious effects of furan are caused by promoting fibrosis and hepatocyte proliferation in liver tissues and triggering podocyte injury in the kidney tissues., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The majority of hereditary and acquired myopathies are clinically characterized by progressive muscle weakness. We hypothesized that ongoing derangement of skeletal muscle cytoarchitecture at the single fiber level may precede and be responsible for the progressive muscle weakness. Here, we analyzed the effects of aging in wild-type (wt) and heterozygous (het) and homozygous (hom) R349P desmin knock-in mice. The latter harbor the ortholog of the most frequently encountered human R350P desmin missense mutation. We quantitatively analyzed the subcellular cytoarchitecture of fast- and slow-twitch muscles from young, intermediate, and aged wt as well as desminopathy mice. We recorded multiphoton second harmonic generation and nuclear fluorescence signals in single muscle fibers to compare aging-related effects in all genotypes. The analysis of wt mice revealed that the myofibrillar cytoarchitecture remained stable with aging in fast-twitch muscles, whereas slow-twitch muscle fibers displayed structural derangements during aging. In contrast, the myofibrillar cytoarchitecture and nuclear density were severely compromised in fast- and slow-twitch muscle fibers of hom R349P desmin mice at all ages. Het mice only showed a clear degradation in their fiber structure in fast-twitch muscles from the adult to the presenescent age bin. Our study documents distinct signs of normal and R349P mutant desmin-related remodeling of the 3D myofibrillar architecture during aging, which provides a structural basis for the progressive muscle weakness., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Human iPSC line was generated from patient-specific adipose tissue-derived mesenchymal multipotent stromal cells carrying desmin (DES) gene heterozygous splice site mutation using non-integrative reprogramming method. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai viruses. iPSCs were characterized by sequencing, karyotype analysis, STR analysis, immunocytochemistry, RT-PCR and teratoma formation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Pancreatic stellate cells (PSCs) play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells, immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis, inflammation and immunity., Methods: The presence of cytoskeletal proteins, integrin α5β1 or TLR4, was determined by immunocytochemistry while the production of desmin, collagen I, MMP-1, MMP-2, TIMP-2, or CD14 was evaluated by Western blotting. The levels of desmin, collagen I, IL-1 and IL-6 mRNA were determined by real-time quantitative PCR. The secretion of cytokines was detected by ELISA. Cell function was assessed using adhesion, migration, or proliferation assays., Results: A stable activated rat PSC cell line (designated as RP-2) was established by RSV promoter/enhancer-driven SV40 large T antigen expression. RP-2 cells retained typical PSC properties, exhibited a myofibroblast-like phenotype and persistently produced desmin. The cells produced collagen I protein, matrix metalloproteinases and inhibitors thereof. RP-2 cells demonstrated typical PSC functions, including proliferation, adherence, and migration, the latter two of which occurred in response to fibronectin and were mediated by integrin α5β1. TLR4 and its response genes including proinflammatory cytokines (IL-1, IL-6, TNF-alpha) and chemotactic cytokines (MCP-1, MIP-1α, Rantes) were produced by RP-2 cells and activated by LPS. LPS-induced IL-1 or IL-6 mRNA expression in this cell line was fully blocked with MyD88 inhibitor., Conclusion: RP-2 cells provide a novel tool for analyzing the properties and functions of PSCs in the pathogenesis of fibrosis, inflammation and immunity in the pancreas.

Maintenance of the highly organized striated muscle tissue requires a cell-wide dynamic network that through interactions with all vital cell structures, provides an effective mechanochemical integrator of morphology and function, absolutely necessary for intra-cellular and intercellular coordination of all muscle functions. A good candidate for such a system is the desmin intermediate filament cytoskeletal network. Human desmin mutations and post-translational modifications cause disturbance of this network, thus leading to loss of function of both desmin and its binding partners, as well as potential toxic effects of the formed aggregates. Both loss of normal function and gain of toxic function are linked to mitochondrial defects, cardiomyocyte death, muscle degeneration and development of skeletal myopathy and cardiomyopathy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Desmin, being a major intermediate filament of mature muscle cell, interacts with mitochondria within the cell and participates in mitochondria proper localization. The goal of the present study was to assess the effect of aggregate-prone and non-aggregate-prone desmin mutations on mitochondrial calcium uptake. Primary murine satellite cells were transduced with lentiviruses carrying desmin in wild type or mutant form, and were induced to differentiate into myotubes. Four mutations resulting in different degree of desmin aggregates formation were analyzed. Tail domain mutation Asp399Tyr has the mildest impact on desmin filament polymerization, rod domain mutation Ala357Pro causes formation of large aggregates composed of filamentous material, and Leu345Pro and Leu370Pro are considered to be the most severest in their impact on desmin polymerization and structure. For mitochondrial calcium measurement cells were loaded with rhod 2-AM. We found that aggregate-prone mutations significantly decreased [Ca(2+)]mit, whereas non-aggregate-prone mutations did not decrease [Ca(2+)]mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca(2+)]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca(2+)]mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Primary muscle cell model systems from farm animals are widely used to acquire knowledge about muscle development, muscle pathologies, overweight issues and tissue regeneration. The morphological properties of a bovine primary muscle cell model system, in addition to cell proliferation and differentiation features, were characterized using immunocytochemistry, western blotting and real-time PCR. We observed a reorganization of the Golgi complex in differentiated cells. The Golgi complex transformed to a highly fragmented network of small stacks of cisternae positioned throughout the myotubes as well as around the nucleus. Different extracellular matrix (ECM) components were used as surface coatings in order to improve cell culture conditions. Our experiments demonstrated improved proliferation and early differentiation for cells grown on surface coatings containing a mixture of both glycosaminoglycans (GAGs) and fibrous proteins. We suggest that GAGs and fibrous proteins mixed together into a composite biomaterial can mimic a natural ECM, and this could improve myogenesis for in vitro cell cultures., (© 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Objective: This study investigates the effects of noradrenaline (NA) on cytoskeletal protein expression of vascular smooth muscle cells (VSMCs)., Methods: VSMCs were isolated from rat aortic tissue and cultured. The cultured VSMCs were divided into 4 experimental groups: (1) control group, (2) NA treatment group, (3) starvation group, and (4) NA treatment+starvation group. The expression of cytoskeletal protein (smooth muscle α-actin, β-tubulin and desmin) was evaluated by (i) Coomassie blue staining, (ii) immunofluorescent staining, and (iii) RT-PCR and Western Blot., Results: NA treatment significantly downregulated the expression of SM α-actin, β-tubulin and desmin (P<0.05). The serum starvation did not affect the expression of cytoskeletal protein (SM α-actin, β-tubulin and desmin), but when the cells were treated with the combination of NA and serum starvation, the expression of SM α-actin, β-tubulin and desmin were down-regulated than those of the serum starvation group (P<0.05)., Conclusion: These results suggested that NA might play a key role in regulating the cytoskeletal protein expression of VSMCs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Myofibrillar myopathies (MFMs) are rare, inherited or sporadic, progressive neuromuscular disorders with considerable clinical and genetic heterogeneity. MFMs are defined morphologically by foci of myofibril dissolution that begins at the Z-disk, accumulation of myofibrillar degradation products, and ectopic expression of a large number of proteins including desmin. To date, mutations in six genes are known to cause MFMs, accounting for approximately half of the MFM patients identified. The causative genes encode mainly sarcomeric Z-disk(-related) proteins: desmin, αB-crystallin, myotilin, Z-band alternatively spliced PDZ motif containing protein (ZASP), filamin C and the antiapoptotic BCL2-associated athanogene 3 (Bag3). Although in most MFM patients the disease presents in adulthood and evolves slowly, some patients with desminopathy, αB-crystallinopathy or Bag3opathies have an infantile or juvenile disease onset. Cardiac involvement is very common in desminopathies and can sometimes be the initial or only symptom of the disease. Respiratory symptoms are noted during childhood in αB-crystallinopathies. Early severe cardiac and respiratory involvement is seen in Bag3opathies. Optical microscopic and immunohistochemical features are similar in MFMs; however, ultrastructural findings can be useful to differentiate between the distinct MFM subtypes. No curative treatment for MFMs is currently available. Careful follow-up, especially of cardiac and respiratory function, is important., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Muscle-targeted gene therapy using insulin genes has the potential to provide an inexpensive, low maintenance alternative or adjunctive treatment method for canine diabetes mellitus. A canine skeletal muscle cell line was established through primary culture, as well as through transdifferentiation of canine fibroblasts after infection with a myo-differentiation gene containing adenovirus vector. A novel mutant furin-cleavable canine preproinsulin gene insert (cppI4) was designed and created through de novo gene synthesis. Various cell lines, including the generated canine muscle cell line, were transfected with nonviral plasmids containing cppI4. Insulin and desmin immunostaining were used to prove insulin production by muscle cells and specific canine insulin ELISA to prove mature insulin secretion into the medium. The canine myoblast cultures proved positive on desmin immunostaining. All cells tolerated transfection with cppI4-containing plasmid, and double immunostaining for insulin and desmin proved present in the canine cells. Canine insulin ELISA assessment of medium of cppI4-transfected murine myoblasts and canine myoblast and fibroblast mixture proved presence of mature fully processed canine insulin, 24 and 48 h after transfection. The present study provides proof of principle that canine muscle cells can be induced to produce and secrete canine insulin on transfection with nonviral plasmid DNA containing a novel mutant canine preproinsulin gene that produces furin-cleavable canine preproinsulin. This technology could be developed to provide an alternative canine diabetes mellitus treatment option or to provide a constant source for background insulin, as well as C-peptide, alongside current treatment options., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Heterozygous mutations of the human desmin gene on chromosome 2q35 cause hereditary and sporadic myopathies and cardiomyopathies. The expression of mutant desmin brings about partial disruption of the extra sarcomeric desmin cytoskeleton and abnormal protein aggregation in the sarcoplasm of striated muscle cells. The precise molecular pathways and sequential steps that lead from a desmin gene defect to progressive muscle damage are still unclear. We tested whether mutant desmin changes the biomechanical properties and the intrinsic mechanical stress response of primary cultured myoblasts derived from a patient carrying a heterozygous R350P desmin mutation. Compared to wildtype controls, undifferentiated mutant desmin myoblasts revealed increased cell death and substrate detachment in response to cyclic stretch on flexible membranes. Moreover, magnetic tweezer microrheometry of myoblasts using fibronectin-coated beads showed increased stiffness of diseased cells. Our findings provide the first evidence that altered mechanical properties may contribute to the progressive striated muscle pathology in desminopathies. We postulate that the expression of mutant desmin leads to increased mechanical stiffness, which results in excessive mechanical stress in response to strain and consecutively to increased mechanical vulnerability and damage of muscle cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

As the specific composition of lipids is essential for the maintenance of membrane integrity, enzyme function, ion channels, and membrane receptors, an alteration in lipid composition or metabolism may be one of the crucial changes occurring during skeletal and cardiac myopathies. Although the inheritance (autosomal dominant, autosomal recessive, and X-linked traits) and underlying/defining mutations causing these myopathies are known, the contribution of lipid homeostasis in the progression of these diseases needs to be established. The purpose of this review is to present the current knowledge relating to lipid changes in inherited skeletal muscle disorders, such as Duchenne/Becker muscular dystrophy, myotonic muscular dystrophy, limb-girdle myopathic dystrophies, desminopathies, rostrocaudal muscular dystrophy, and Dunnigan-type familial lipodystrophy. The lipid modifications in familial hypertrophic and dilated cardiomyopathies, as well as Barth syndrome and several other cardiac disorders associated with abnormal lipid storage, are discussed. Information on lipid alterations occurring in these myopathies will aid in the design of improved methods of screening and therapy in children and young adults with or without a family history of genetic diseases.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main etiological agents of Hand, Foot and Mouth Disease (HFMD), a common disease among children and had caused several outbreaks in the Asia-Pacific region. Although being genetically close to each other, EV71 infection can cause serious and fatal neurological complications like encephalitis, myocarditis, acute flaccid paralysis (AFP) and aseptic meningitis, but not in CA16 infections. In this study, the cellular response of host cells infected with EV71 and CA16 was characterized and compared by 2-dimensional proteome analyses. A total of 16 proteins were identified to be differentially expressed in EV71 and CA16-infected host cells. Desmin and HSP27, both indirectly regulate the contraction of muscle cells, were significantly downregulated as a result of EV71 infection, suggesting a link to acute flaccid paralysis. The ability of EV71 to evade host immune system may be due to the downregulation of MHC-I synthesis proteins like protein disulfide isomerase A3 and calreticulin. Proteins such as nucleophosmin, nuclear ribonucleoprotein C, and eukaryotic translation initiation factor 2 were all downregulated significantly, suggesting the rapid shutting down of host translation machinery by EV71. These findings provide insight into the nature of high virulent EV71 infection as compared to CA16., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background: Mutations in the gene encoding desmin (DES), an intermediate filament protein, underlie a heterogeneous phenotype, which is referred to as desmin-related myopathy (DRM). Right ventricular involvement including an arrhythmogenic right ventricular cardiomyopathy (ARVC)(-like) phenotype has occasionally been described in DES mutation-carrying patients., Objective: To determine the effects of a DES missense mutation on the structure of different intercalated disk proteins, to evaluate right ventricular involvement in DES mutation carriers, and to establish the role of DES mutations in ARVC(-like) phenotypes., Methods: We evaluated the clinical phenotype in two families carrying two different DES mutations. One family was diagnosed with DRM, with an ARVC(-like) phenotype in one patient, while the other family presented with a severe biventricular cardiomyopathy. Additional immunohistochemistry of desmosomal proteins was performed in myocardial tissue from two patients of the last family. The DES gene was screened for mutations in 50 ARVC(-like) patients., Results: Except for two different DES mutations (p.N342D and p.R454W) in two families with DRM and severe biventricular cardiomyopathy, respectively, we did not find additional DES mutations in ARVC(-like) patients. In addition to desmin aggregates, immunohistochemistry demonstrated a decreased amount of desmoplakin and plakophilin-2 at the intercalated disk in p.R454W mutation carriers., Conclusions: We confirmed that either an ARVC-like phenotype or a severe cardiomyopathy with right ventricular involvement are possible, yet infrequent, cardiac phenotypes in DRM. Moreover, we demonstrated that the DES mutation p.R454W affects the localization of desmoplakin and plakophilin-2 at the intercalated disk, suggesting a link between desmosomal cardiomyopathies (mainly affecting the right ventricle) and cardiomyopathies caused by DES mutations., (Copyright 2010 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-alpha-helical carboxy-terminal domain ("tail"), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin "tail" mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l(p). Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the "tail" mutants (l(p)=1.0-2.0 microm) and the wild-type desmin (l(p)=1.1+/-0.5 microm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered. In order to explore the importance of the "tail" domain as such for filament network properties, we employed a "tail"-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the "tail" domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background: Desmin-related myopathy is a clinically heterogenous group of disorders encompassing myopathies, cardiomyopathies, conduction disease, and combinations of these disorders. Mutations in the gene encoding desmin (DES), a major intermediate filament protein, can underlie this phenotype., Objective: The purpose of this study was to investigate the clinical and pathologic characteristics of 27 patients from five families with an identical mutation in the head domain region (p.S13F) of desmin., Methods/results: All 27 carriers or obligate carriers of a p.S13F DES founder mutation demonstrated a fully penetrant yet variable phenotype. All patients demonstrated cardiac involvement characterized by high-grade AV block at young ages and important right ventricular (RV) involvement. RV predominance was demonstrated by the presence of right bundle branch block in 10 patients (sometimes as a first manifestation) and by RV heart failure in 6 patients, including 2 patients who fulfilled the diagnostic criteria for arrhythmogenic RV cardiomyopathy. Because of this clinical overlap with desmosome cardiomyopathies, we also studied the organization of the intercalated disks, particularly the distribution of desmosomal proteins. Normal amounts of the major desmosomal proteins were found, but the intercalated disks were more convoluted and elongated and had a zigzag appearance., Conclusion: In this largest series to date of individuals with a single head domain DES mutation, patients show a variable yet predominantly cardiologic phenotype characterized by conduction disease at an early age and RV involvement including right bundle branch block and/or RV tachycardias and arrhythmogenic RV cardiomyopathy phenocopies. A localized effect of desmin on the structure of the cardiac intercalated disks might contribute to disease pathogenesis.

Background/aims: To reveal the microRNA (miRNA) expression profile and related roles in rat HSCs during activation., Methods: miRNA expression profiling was analyzed in quiescent and in culture-activated HSCs by microarray. The differentially expressed miRNAs, as verified by RT-PCR, were subjected to gene ontology (GO) analysis. Furthermore, the effects of miR-16 and miR-15b on the apoptosis of activated HSCs were investigated by Hoechst 33258, TUNEL staining and annexin-V/PI labeling flow cytometry. The underlying mechanism related to Bcl-2 and caspases was assessed., Results: The upregulated and downregulated miRNAs in activated HSCs were 12 miRNAs and 9 miRNAs, respectively. The differential expression of miR-16, -15b, -122, -138, -143, and -140 was validated. High-enrichment GOs containing apoptosis-related targeted genes and miRNA-gene networks characterized by Bcl-2, which was targeted by the miR-15/16 family, uncovered the critical role of miR-16 and miR-15b in apoptosis. Restoring the intracellular miRNAs by miR-16 and miR-15b administration greatly reduced Bcl-2, and increased the expression of caspases 3, 8, and 9. Significantly elevated rates of apoptosis were then induced in activated HSCs., Conclusions: The activation of HSCs relate to 21 miRNAs. Among these, mir-15b and miR-16 may be essential for apoptosis by targeting Bcl-2 and the caspase signaling pathway.

Mutations in the intermediate filament (IF) protein desmin cause severe forms of myofibrillar myopathy characterized by partial aggregation of the extrasarcomeric desmin cytoskeleton and structural disorganization of myofibrils. In contrast to prior expectations, we showed that some of the known disease-causing mutations, such as DesA360P, DesQ389P and DesD399Y, are assembly-competent and do allow formation of bona fide IFs in vitro and in vivo. We also previously demonstrated that atomic force microscopy can be employed to measure the tensile properties of single desmin IFs. Using the same approach on filaments formed by the aforementioned mutant desmins, we now observed two different nanomechanical behaviors: DesA360P exhibited tensile properties similar to that of wild-type desmin IFs, whereas DesQ389P and DesD399Y exhibited local variations in their tensile properties along the filament length. Based on these findings, we hypothesize that DesQ389P and DesD399Y may cause muscle disease by altering the specific biophysical properties of the desmin filaments, thereby compromising both its mechanosensing and mechanotransduction ability.

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, alpha-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2(+) cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin(-) cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin(-) cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.

Desmin contributes to structural integrity and function of the myocardium but its function seems to be redundant in early cardiomyogenesis in the desmin null mouse model. To test the hypothesis that desmin also plays a supportive role in cardiomyogenic commitment and early differentiation of cardiomyocytes we investigated cardiomyogenesis in embryoid bodies expressing different desmin alleles. Constitutive expression of desmin and increased synthesis during mesoderm formation led to the up-regulation of brachyury and nkx2.5 genes, accelerated early cardiomyogenesis and resulted in the development of large, proliferating, highly interconnected, and synchronously beating cardiomyocyte clusters, whereas desmin null cardiomyocytes featured an opposite phenotype. In contrast, constitutive expression of amino-terminally truncated desmin(Delta1-48) interfered with the beginning of cardiomyogenesis, caused down-regulation of mesodermal and myocardial transcription factors, and hampered myofibrillogenesis and survival of cardiomyocytes. These results provide first evidence that a type III intermediate filament protein takes part in regulating the differentiation of mesoderm to cardiomyocytes at the very beginning of cardiomyogenesis.

Desmin contributes to the stability of the myocardium and its amino-terminal domain influences intermediate filament formation and interacts with a variety of proteins and DNAs. Specific serine residues located in this domain are reversibly phosphorylated in a cell cycle and developmental stage-dependent manner as has been demonstrated also for other cytoplasmic type III intermediate filament proteins. Although absence of desmin apparently does not affect cardiomyogenesis, homozygous deletion of the amino-terminal domain of desmin severely inhibited in vitro cardiomyogenesis. To demonstrate the significance of phosphorylation of this domain in cardiomyogenic commitment and differentiation, we inhibited phosphorylation of serine residues 6, 7, and 8 by mutation to alanine, and investigated early cardiomyogenesis in heterozygous embryoid bodies. As control, serine residues 31 and 32, which are not phosphorylated by kinases mutating serine residues 6, 7, and 8, were mutated to alanine in a second set. Desmin(S6,7,8A) interfered with cardiomyogenesis and myofibrillogenesis in a dominant negative fashion, whereas desmin(S31,32A) produced only a mild phenotype. Desmin(S6,7,8A) led to the down-regulation of the transcription factor genes brachyury, goosecoid, nkx2.5, and mef2C and increased apoptosis of presumptive mesoderm and differentiating cardiomyocytes. Surviving cardiomyocytes which were few in number had no myofibrils. Demonstration that some but not any mutant desmin interfered with the very beginning of cardiomyogenesis suggests an important function of temporarily phosphorylated serine residues 6, 7, and 8 in the amino-terminal domain of desmin in cardiomyogenic commitment and differentiation.

Desmin-related myopathy is characterised by skeletal muscle weakness often combined with cardiac involvement. Mutations in the desmin gene have been described as a cause of desmin-related myopathy (OMIM 601419). We report here on two distantly related Dutch families with autosomal dominant inheritance of desmin-related myopathy affecting 15 family members. A highly heterogeneous clinical picture is apparent, varying from isolated dilated cardiomyopathy to a more generalised skeletal myopathy and mild respiratory problems. Morphological analysis of muscle biopsies revealed intracytoplasmic desmin aggregates (desmin and p62 staining). In both families we identified an identical novel pathogenic heterozygous missense mutation, S13F, in the 'head' domain of the desmin gene which cosegregates with the disease phenotype. This is the 5th reported missense mutation located at the 'head' domain of the desmin gene and the first reported Dutch family with desmin-related myopathy. This article illustrates the importance of analysing the desmin gene in patients with (familial) cardiac conduction disease, dilated cardiomyopathy and/or a progressive skeletal myopathy resembling limb-girdle muscular dystrophy.

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 alpha-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.

Background/aims: Hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. However, their origin is still unknown. We tested the hypothesis that bone marrow (BM) contributes to the population of HSCs., Methods: Chimeric mice transplanted with donor BM from collagen alpha1(I)-GFP+ reporter mice were subjected to the bile duct ligation (BDL)-induced liver injury., Results: In response to injury, BM-derived collagen-expressing GFP+ cells were detected in liver tissues of chimeric mice. However, these cells were not activated HSCs in that they did not express alpha-smooth muscle actin or desmin and could not be isolated with the HSC fraction. Meanwhile, the majority of these BM-derived cells co-expressed collagen-GFP+ and CD45+, suggesting that these cells represent a unique population of fibrocytes. Consistent with their lymphoid origin, the number of GFP+CD45+ fibrocytes found in BM and spleen of chimeric mice increased in response to injury. Fibrocytes cultured in the presence of TGF-beta1 differentiated into SMA+desmin+ collagen-producing myofibroblasts, potentially contributing to liver fibrosis., Conclusions: In response to the BDL-induced liver injury: (i) HSCs do not originate in the BM; (ii) collagen-producing fibrocytes are recruited from the BM to damaged liver.

It has been documented that mutations in the human desmin gene lead to a severe type of myofibrillar myopathy, termed more specifically desminopathy, which affects cardiac and skeletal as well as smooth muscle. We showed recently that 14 recombinant versions of these disease-causing desmin variants, all involving single amino acid substitutions in the alpha-helical rod domain, interfere with in vitro filament formation at distinct stages of the assembly process. We now provide mechanistic details of how these mutations affect the filament assembly process by employing analytical ultracentrifugation, time-lapse electron microscopy of negatively stained and glycerol-sprayed/low-angle rotary metal-shadowed samples, quantitative scanning transmission electron microscopy, and viscometric studies. In particular, the soluble assembly intermediates of two of the mutated proteins exhibit unusually high s-values, compatible with octamers and other higher-order complexes. Moreover, several of the six filament-forming mutant variants deviated considerably from wild-type desmin with respect to their filament diameters and mass-per-length values. In the heteropolymeric situation with wild-type desmin, four of the mutant variants caused a pronounced "hyper-assembly", when assayed by viscometry. This indicates that the various mutations may cause abortion of filament formation by the mutant protein at distinct stages, and that some of them interfere severely with the assembly of wild-type desmin. Taken together, our findings provide novel insights into the basic intermediate filament assembly mechanisms and offer clues as to how amino acid changes within the desmin rod domain may interfere with the normal structural organization of the muscle cytoskeleton, eventually leading to desminopathy.

Previous studies demonstrated that impaired left ventricular (LV) relaxation in cardiac allografts limits exercise tolerance post-transplant despite preserved systolic ejection fraction (EF). This study tested in human cardiac allografts whether the isovolumic relaxation time (IVRT), which provides the basis for most of diastolic LV filling, relates with gene expression of regulatory proteins of calcium homeostasis or cardiac matrix proteins. Gene expression was studied in 31 heart transplant recipients (25 male, 6 female) 13-83 months post-transplant with LVEF >50%, LV end-diastolic pressure <20 mmHg, normal LV mass index and without allograft rejection or significant cardiac pathology. IVRT related with the other diastolic parameters e-wave velocity (r = -0.46; p = 0.01), e/a-wave ratio (r = -0.5; p < 0.01) but not with heart frequency (r = -0.16; p = 0.4). No relation of IVRT was observed for immunosuppression, mean rejection grade or other medication. IVRT was not related with gene expression of desmin, collagen I, phospholamban, the Na+-Ca2+ exchanger, the ryanodine receptor or interstitial fibrosis but correlated inversely with SERCA2a (r = -0.48; p = 0.02). Prolonged IVRT is associated with decreased SERCA2a expression in cardiac allografts without significant other pathology. Similar observations in non-transplanted patients with diastolic failure suggest that decreased SERCA2a expression is an important common pathomechanism.

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.

Ten Spanish patients from six unrelated families diagnosed with desmin-related myopathy (DRM) were studied. The pattern of DRM inheritance was autosomal dominant in three families, autosomal recessive in one, and there was no family history in two cases. The disease onset was in early adulthood. Cardiac myopathy was the initial presentation in two patients, respiratory insufficiency in one, and lower limb weakness in all others. Cardiac involvement was observed in four patients. Lens opacities were found in four. CK level was normal or slightly elevated, and electrophysiological examination was consistent with myopathy. Muscle biopsies identified intracytoplasmic desmin-immunoreactive inclusions. In addition to desmin, synemin, actin, gelsolin, ubiquitin, alphaB-crystallin and amyloid betaA4 were also present in the deposits. Ultrastructural examination revealed areas of myofibrillary disruption, abnormal electron-dense structures and accumulations of granulofilamentous material. A missense R406W mutation and a novel single amino acid deletion in the desmin gene were identified in two patients; the other patients did not show mutations in desmin, synemin, syncoilin or alphaB-crystallin genes. Analysis of 10 Spanish DRM cases illustrates a wide clinical, myopathological and genetic spectrum of DRM, reinforcing the need for further exploration of genetic causes for this group of disorders.

Several studies have already demonstrated that micro- and milli-calpains (CAPN 1-CAPN 2), calcium-dependent intracellular cysteine-proteases are involved in many biological phenomenon including muscle growth and development. More particularly, recent studies have demonstrated that milli-calpain is implicated in myoblast fusion. Moreover, in primary muscle cells, these proteases do not appear simultaneously throughout muscle cell differentiation. Because micro- and milli-calpains do not have the same intracellular localization, it appears likely that these two calcium-dependent proteases have different biological roles during muscle cell differentiation. The goal of this study is to determine the role of micro-calpain. We therefore, have developed a muscle cell line in which micro-calpain is over-expressed, using the inducible Tet Regulated Expression System. The outcome is observed by following the behavior of different proteins, considered to be potential substrates of the protease. The present study shows important decreases in the expression level of ezrin (68%), vimentin (64%) and caveolin 3 (76%) whereas many other cytoskeletal proteins remain remarkably stable. Concerning the myogenic transcription factors, only the level of myogenin decreased (59%) after the over-expression of micro-calpain. Ultra structural studies have shown that the myofibrils formed near the cell periphery are normally oriented, lying along the longitudinal axis. This regularity is lost progressively towards the cell center where the cytoskeleton presented an increasing disorganization. All these results indicate that micro-calpain is involved in regulation pathway of myogenesis via at least its action on ezrin, vimentin, caveolin 3 and myogenin, a muscle transcription factor.

Mice lacking the intermediate filament protein desmin demonstrate abnormal mitochondria behavior, disruption of muscle architecture, and myocardial degeneration with extensive calcium deposits and fibrosis. These abnormalities are associated with cardiomyocyte hypertrophy, cardiac chamber dilation and eventually with heart failure. In an effort to elucidate the molecular mechanisms leading to the observed pathogenesis, we have analyzed gene expression changes in cardiac tissue using differential display polymerase chain reaction and cDNA atlas array methods. The most substantial changes were found in genes coding the small extracellular matrix proteins osteopontin and decorin that are dramatically induced in the desmin-null myocardium. We further analyzed their expression pattern both at the RNA and protein levels and we compared their spatial expression with the onset of calcification. Extensive osteopontin localization is observed by immunohistochemistry in the desmin-null myocardium in areas with massive myocyte death, as well as in hypercellular regions with variable degrees of calcification and fibrosis. Osteopontin is consistently co-localized with calcified deposits, which progressively are transformed to psammoma bodies surrounded by decorin, especially in the right ventricle. These data together with the observed up-regulation of transforming growth factor-beta1 and angiotensin-converting enzyme, could explain the extensive fibrosis and dystrophic calcification observed in the heart of desmin-null mice, potentially crucial events leading to heart failure.

Cell architecture is largely based on the interaction of cytoskeletal proteins, which include intermediate filaments (IF), microfilaments, microtubules, as well as their type-specific membrane-attachment structures and associated proteins. In order to further our understanding of IF proteins and to address the fundamental issue whether different IF perform unique functions in different tissues, we expressed a desmin transgene in the basal epidermis of mice. Ectopic expression of desmin led to the formation of an additional, keratin-independent IF cytoskeleton and did not interfere with the keratin-desmosome interaction. We show that ectopic expression of a type III IF protein in basal keratinocytes did not interfere with the normal epidermal architecture and the program of terminal differentiation. This demonstrated that keratinocytes suffered no obvious detrimental effects from extra desmin filaments in their cytoplasm. In addition, we asked whether stable expression of desmin could rescue K5 null mice, which served as a model for severe EBS. Transgenic mice ectopically expressing desmin in the basal layer were mated with K5 heterozygous deficient animals to generate desmin rescue mice and analysed. In summary, our study support the notion that the different IF like desmin or keratins composing a IF network in vivo are central to cytoskeletal architecture and design in cells.

Introduction of nonselectable mutations into the genome of embryonic stem cells by homologous recombination allows to investigate the function of genes at the molecular level and has been achieved, however, at very low efficiencies by the Hit and Run, Tag and Exchange, and Double Replacement strategies. Comparing those strategies at a single locus with vectors derived from a single fragment of the desmin gene led to the improvement of two strategies by employing a new selection cassette and modified selection procedures. Modified strategies resulted in the introduction of nonselectable point-mutations in 53% of the Hit and Run derived embryonic stem cell clones and in 0.7% of the Tag and Exchange clones. Efficiency of intrachromosomal recombination at Hit alleles outscored replacement-type recombination at the tagged alleles making the modified Hit and Run strategy the method of choice for the efficient introduction of nonselectable point mutations into the genome of embryonic stem cells.

In the shark Scyliorhinus stellaris we have biochemically identified and cDNA-cloned orthologs of human vimentin and desmin, SstV and SstD, as deduced from immunoblotting and sequence alignment with teleost, frog and human vimentin and desmin, respectively. This allowed us to further clarify the relationship of previously identified lower vertebrate intermediate filament proteins to mammalian vimentin and desmin. Immunofluorescence microscopy with antibodies H5 and VIM13.2 showed vimentin expression in shark eye and brain and absence in epithelia, which resembles the situation in higher vertebrates. In addition, SstV is expressed in many mesenchymal cell types which corresponds to the case in terrestrial vertebrates but strongly differs from teleosts. Surprisingly, shark interstitial cells, including fibroblasts, express neither SstV nor keratins but other as yet unidentified intermediate filament proteins as deduced from their reactivity with antibody IFA. In vitro assembly studies of recombinant SstV revealed a temperature optimum for uncompromised filament assembly of 15 degrees C. At 18 degrees C, but more pronounced at 21 degrees C and 24 degrees C, which is notably above the animal's inherent preferred environmental temperature, both, SstV and SstD assemble into thick and inflexible fibers. Thus, environmental temperature apparently is, as a general principle, a driving force for the fine tuning of protein primary structure and eventually 3D structure.

Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer.

Background: In chronic renal disease, the decrease of excretory renal function closely correlates with the extent of interstitial fibrosis. A common feature of interstitial fibrosis is the occurrence of myofibroblasts, which are regarded as the predominant cells in matrix synthesis. We studied the transformation of renal fibroblasts into myofibroblasts in vitro as a model to elucidate the mechanisms underlying this process., Methods: Primary cultures of freshly isolated rat inner medullary fibroblasts were established as reported previously. mRNA expression of myofibroblast markers and interstitial collagens was examined by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Phenotypic expression was investigated by immunolabeling. Endogenous transforming growth factor-beta (TGF-beta) production was inhibited by addition of a neutralizing antibody or by TGF-beta 1 antisense oligodeoxynucleotides (ODNs)., Results: Initially and during the first 36 hours, primary culture cells expressed neither alpha-smooth muscle actin nor desmin mRNA. From day 2 of primary culture, we observed a strong increase in these mRNAs, as evaluated by RT-PCR, followed by the phenotypic expression of these myofibroblast markers. Collagen type I mRNA was first detectable from day 4 of primary culture and showed a strong increase in its expression level during the following days, with phenotypic expression predominantly in myofibroblasts. The transformation rate of fibroblasts to myofibroblasts largely decreased in cocultures with collecting duct cells. This effect could be reversed by reducing the seeding density. We examined in this system the effect of TGF-beta 1 to define further its putative fibrogenic activity. However, neither the addition of exogenous TGF-beta 1 nor the inhibition of endogenous TGF-beta production showed any significant effect on fibroblast transformation, suggesting that this cytokine exerts its effects at other levels or requires a cofactor., Conclusions: We present a novel model to examine the de novo expression of myofibroblast markers and collagen type I in freshly isolated fibroblasts under defined conditions in primary culture. This model could provide a strategy for the molecular characterization of myofibroblast formation and the phenomena associated with this process.

We have investigated the physiological role of desmin in skeletal muscle by measuring isometric tension generated in skinned fibres and intact skeletal muscles from desmin knock-out (DES-KO) mice. About 80% of skinned single extensor digitorum longus (EDL) fibres from adult DES-KO mice generated tensions close to that of wild-type (WT) controls. Weights and maximum tensions of intact EDL but not of soleus (SOL) muscles were lowered in DES-KO mice. Repeated contractions with stretch did not affect subsequent isometric tension in EDL muscles of DES-KO mice. Tension during high frequency fatigue (HFF) declined faster and this deficiency was compensated in DES-KO EDL muscles by 5 mM caffeine which had no influence on HFF in WT EDL. Furthermore, caffeine evoked twitch potentiation was higher in DES-KO than in WT muscles. We conclude that desmin is not essential for acute tensile strength but rather for optimal activation of intact myofibres during E-C coupling., (Copyright 2000 Academic Press.)

The desmin gene encodes an intermediate filament protein that is present in skeletal, cardiac, and smooth muscle cells. This study shows that the 4-kb upstream region of the murine desmin promoter directs expression of a lacZ reporter gene throughout the heart from E7.5 and in skeletal muscle and vascular smooth muscle cells from E9. 5. The distal fragment (-4005/-2495) is active in arterial smooth muscle cells but not in venous smooth muscle cells or in the heart in vivo. It contains a CArG/octamer overlapping element (designated CArG4) that can bind the serum response factor (SRF) and an Oct-like factor. The desmin distal fragment can replace a SM22alpha regulatory region (-445/-126) that contains two CArG boxes, to cis-activate a minimal (-125/+65) SM22alpha promoter fragment in arterial smooth muscle cells of transgenic embryos. lacZ expression was abolished when mutations were introduced into the desmin CArG4 element that abolished the binding of SRF and/or Oct-like factor. These data suggest that a new type of combined CArG/octamer element plays a prominent role in the regulation of the desmin gene in arterial smooth muscle cells, and SRF and Oct-like factor could cooperate to drive specific expression in these cells., (Copyright 2000 Academic Press.)