Desmin Aggregates Research Articles

Desmin, Mechanics and Myofibrillar Myopathies

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.

N-Acetyl-L-Cysteine Prevents Stress-Induced Desmin Aggregation in Cellular Models of Desminopathy

Mutations within the human desmin gene are responsible for a subcategory of myofibrillar myopathies called desminopathies. However, a single inherited mutation can produce different phenotypes within a family, suggesting that environmental factors influence disease states. Although several mouse models have been used to investigate organ-specific desminopathies, a more general mechanistic perspective is required to advance our knowledge toward patient treatment. To improve our understanding of disease pathology, we have developed cellular models to observe desmin behaviour in early stages of disease pathology, e.g., upon formation of cytoplasmic desmin aggregates, within an isogenic background. We cloned the wildtype and three mutant desmin cDNAs using a Tet-On Advanced® expression system in C2C12 cells. Mutations were selected based on positioning within desmin and capacity to form aggregates in transient experiments, as follows: DesS46Y (head domain; low aggregation), DesD399Y (central rod domain; high aggregation), and DesS460I (tail domain; moderate aggregation). Introduction of these proteins into a C2C12 background permitted us to compare between desmin variants as well as to determine the role of external stress on aggregation. Three different types of stress, likely encountered during muscle activity, were introduced to the cell models—thermal (heat shock), redox-associated (H2O2 and cadmium chloride), and mechanical (stretching) stresses—after which aggregation was measured. Cells containing variant DesD399Y were more sensitive to stress, leading to marked cytoplasmic perinuclear aggregations. We then evaluated the capacity of biochemical compounds to prevent this aggregation, applying dexamethasone (an inducer of heat shock proteins), fisetin or N-acetyl-L-cysteine (antioxidants) before stress induction. Interestingly, N-acetyl-L-cysteine pre-treatment prevented DesD399Y aggregation during most stress. N-acetyl-L-cysteine has recently been described as a promising antioxidant in myopathies linked to selenoprotein N or ryanodin receptor defects. Our findings indicate that this drug warrants further study in animal models to speed its potential development as a therapy for DesD399Y-linked desminopathies.

P.5.10 Clinical and ultrastructural changes in transportinopathy

Muscle histopathological, ultrastructural and genetic features of a large Italian-Spanish family with autosomal dominant LGMD, previously mapped to 7q32.1–32.2 (LGMD1F) were studied in 3 biopsies. We collected the clinical history in 19 of 60 patients; muscle biopsy histopathology was investigated in one pair of affected patients (mother 1 biopsy, her daughter 2 consecutive biopsies at 9 and 22 years). We observed that the age of onset varied from 2 to 35 years, and occurred either in upper or in the lower girdle; in 14 cases there was hypotrophy both in proximal upper and in lower extremities in calf muscles. The severity was not increased in successive generations. Unreported clinical findings were arachnodactyly, dysphagia and dysarthria. Moreover, we noticed a discrepancy between the clinical severity and muscle biopsy involvement: the daughter has a more severe clinical course, the first biopsy had only type 1 fiber atrophy while increased fiber atrophy was observed in the second biopsy. The mother has a compromised muscle histopathology (more muscle fiber variation, and autophagic changes by acid phosphatase stain). An abnormal sarcomeric assembly is the cause of progressive atrophy and myofiber loss. Electron microscopy revealed accumulation of myofibrillar bodies in muscle fibers. Accumulation of desmin and myotilin and p62-positive aggregates was observed. A defect in transportin-3 gene has been found to be the cause of this disease, which represents a new mechanism of dominant myopathy. Our morphological and ultrastructural data seems to suggest a phenotype similar to myofibrillar disease; however, autophagosomes were also present. It is possible that SR protein cannot migrate or be transported in- and out-of the nuclear membrane.

Circulation Research “In This Issue” Anthology

<i>Circulation Research</i> “In This Issue” Anthology

Functional characterization of desmin mutant p.P419S

Recently, Hedberg et al1 identified a DES mutation (p.P419S) in a Swedish family, suffering from myofibrillar myopathy (MFM) in combination with arrhythmogenic right ventricular cardiomyopathy (ARVC), by next-generation sequencing. Originally, a linkage analysis indicated that the genetic defect is located on chromosome 10q22.3 in this family.2 The analysis of muscle biopsies of affected patients demonstrated an aggregation of desmin and further proteins.

Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.

Desmin mutations in the terminal consensus motif prevent synemin-desmin heteropolymer filament assembly

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.

P2.19 Desmin-related myopathies: Effects of a cyclic mechanical stress on the aggregation of desmin in transfected myoblasts

P2.19 Desmin-related myopathies: Effects of a cyclic mechanical stress on the aggregation of desmin in transfected myoblasts

QTc Prolongation and Family History of Sudden Death in a Patient with Desmin Cardiomyopathy

This case report describes a pregnant female patient who presented with new-onset congestive heart failure symptoms and prolonged QTc, with strong family history of sudden death. Endomyocardial biopsy and genetic testing revealed myocardial desmin accumulation and a previously described mutation in the DES (desmin) gene, as well as variants in two LQT genes, SCN5A and KCNH2. The case highlights the phenotypic variability for a particular desmin genotype, and the possible interaction of desminopathy with LQT variants not independently associated with large differences in current properties or QT prolongation from wild type.

Adenosine regulation of microtubule dynamics in cardiac hypertrophy

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.

Desmin tail mutation linked to human dilated cardiomyopathy promotes cleavage of the N-terminus and abolishes z-disc localization

A missense mutation (Ile 451 to Met) at the tail domain of the muscle specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. Most of the desmin mutations (usually at the central rod domain) have as result the intracytoplasmic accumulation of desmin and other proteins aggregates in muscle cells. These aggregates are a major histological hallmark of desminopathies. Surprisingly most of the desmin tail domain mutants have the ability to form proper filaments on their own, so we hypothesize that filament-forming mutations will critically interfere with the binding of the filaments to z-disc or intercalated disc components, or proteins that are part of muscle-specific signaling cascades.

Proteasome malfunction increases autophagosomes in cardiomyocytes and mouse hearts

Background:Autophagy and the ubiquitin‐proteasome system (UPS) are two main routes for intracellular protein degradation. The effect of proteasome malfunction on autophagy in cardiomyocytes and the heart remains to be demonstrated.Methods &amp; results:Proteasomal function in cultured neonatal ventricular myocytes (NRVMs) or mouse hearts is manipulated using pharmacological and/or genetic means. The resultant effects on autophagy are assessed by two methods: LC3‐II/LC‐I ratio measured by western blot analysis; autophagy index measured via confocal microscopic analysis of GFP‐LC3 distribution. Cardiac‐restricted expression of a 7‐amino‐acid deletion mutation of the desmin gene (D7‐des) has been linked to human desminopathy which develops aberrant desmin aggregation and severe proteasome malfunction in heart muscle cells. D7‐des increases LC3‐II/LC3‐I ratio and punctate GFP‐LC3 in mouse heart as early as 2 month. The increased autophagosomes is also detected in mouse heart 12 hours after an intravenous injection of proteasome inhibitor MG262 (5umol/kg). In NRVMs, proteasome inhibition by MG132 (1uM) increases the LC3‐II/LC3‐I ratio and autophagic flux measured as the difference of autophagosomes in the absence and presence of lysosomal protease inhibitors.Conclusions:Pharmacological and/or genetic proteasome inhibition increase autophagosomes in both NRVMs and mouse hearts.

Cardiomiopatia restritiva por depósito de desmina

Restrictive cardiomyopathies may have different etiologies, among which we can point out storage diseases by accumulation of different materials such as desmin. Desminopathies are uncommon diseases that progress with conduction abnormalities, peripheral myopathies, and ventricular dysfunction. The present report describes a patient with complete atrioventricular block as the initial event; he later developed skeletal muscle alterations and heart failure. The investigation led to the diagnosis of restrictive cardiomyopathy due to desmin accumulation.

Expression of mutant ubiquitin (UBB+1) and p62 in myotilinopathies and desminopathies

Protein aggregates in muscle cells are the morphological hallmark of myofibrillar myopathies, including myotilinopathies and desminopathies. The aim of the present study is to analyse the expression of mutant ubiquitin (UBB+1), an aberrant form of ubiquitin which accumulates in certain disorders characterized by intracellular aggregates of proteins, and p62, a multimeric signal protein which plays an active role in aggregate formation, in muscle biopsies from patients suffering from myotilinopathy and desminopathy in order to gain understanding of the mechanisms leading to protein aggregation in these disorders. Single immunohistochemistry, and single- and double-labelling immunofluorescence and confocal microscopy for UBB+1 and p62, has been performed in muscle biopsies from patients suffering from myotilinopathy and desminopathy. Strong UBB+1 immunoreactivity, colocalizing with myotilin aggregates, was found in muscle fibres in myotilinopathies. UBB+1 accumulation, colocalizing with desmin aggregates, also occurs in desminopathies. In addition, strong p62 immunoreactivity colocalizing with myotilin aggregates was observed in myotilinopathies. Similarly, p62 immunoreactivity colocalizing with desmin aggregates was found in desminopathies. The present findings suggest that accumulation of protein aggregates in myotilinopathies and in desminopathies may be related with UBB+1/abnormal protein complexes which are resistant to proteasome degradation. Furthermore, these observations suggest a relationship between the presence of p62 and the formation of inclusions in different subtypes of myofibrillar myopathies.

Two related Dutch families with a clinically variable presentation of cardioskeletal myopathy caused by a novel S13F mutation in the desmin gene

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.

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absent.

TNF-a induces desmin modification, aggregation and loss of intercalated disc localization

TNF-a induces desmin modification, aggregation and loss of intercalated disc localization

Myopathy causing αB‐crystallin Mutants have Distinct Properties in Myocytes

αB-crystallin is a small heat shock protein which is ubiquitously expressed and at high levels in muscle and eye lens. αB-crys consists of an N-terminal domain that contains the phosphorylated serines; a highly conserved “crystallin” domain, from a. a. 64 to 150; and a flexible c-terminal domain, which is involved in chaperone function and solubility. One mutation in the crystallin domain, R120G, causes a dominant desmin-related myopathy. The affected patients have desmin accumulation in skeletal and cardiac tissue, with progressive weakness, and develop cataracts. Recently, two mutations in αB-crys have been shown to cause progressive myopathy with myofibrillar degeneration at the z-disk. These mutations truncate the c-terminal domain of the protein. Adenovirus expressing wild-type human αB-crys (WT) as a cfp fusion protein, and the three mutated versions of crystallin: R120G-cfp, 464delCT-cfp, and Q151X-cfp were expressed in cardiomyocytes. Percentage of cells with aggregates, assessed by microscopy, are: R120G, 42.2; Q151X, 21.3; 464delCT, 20.2; WT, 11.0; GFP, 6.9. By IE focusing they all have different pIs from WT. Only the c-terminal mutants caused a downregulation of endogenous crystallin by immunoanalysis. We conclude the αB-crys mutants have unique and functionally significant properties in myocytes. This work was supported by the American Heart Association and the Falk Foundation.

Endogenous laminin is required for human airway smooth muscle cell maturation

BackgroundAirway smooth muscle (ASM) contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM) components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells.MethodsHuman ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured.ResultsMyocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP) significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype.ConclusionWhile earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the first time that endogenously expressed laminin is required for ASM maturation to the contractile phenotype. As endogenously expressed laminin chains α2, β1 and γ1 are uniquely increased during myocyte maturation, these laminin chains may be key in this process. Thus, human ASM maturation appears to involve regulated endogenous expression of a select set of laminin chains that are essential for accumulation of contractile phenotype myocytes.

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

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