Desmin Mutations Research Articles

5162Novel insights into desminopathy in the era of next generation sequencing

Abstract Aims The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. This disease of intermediate filaments causes not only a contractile dysfunction in cardiomyopathy and skeletal myopathy, but also a secondary mitochondrial dysfunction. We aimed to describe prevalence, phenotypic expression and mitochondrial function in desmin mutation carriers identified in a large cohort of patients with unexplained cardiomyopathy. Methods and results A representative cohort of 327 Czech patients with unexplained cardiomyopathy underwent whole exome sequencing. The cohort consisted of cases with familial and sporadic dilated cardiomyopathy (81%), left ventricular noncompaction cardiomyopathy (LVNC) (13%), and less frequently of restrictive (3%) or arrhythmogenic cardiomyopathy (3%). Clinical and laboratory data of desmin mutation carriers were collected. Morphology, desmin expression and mitochondrial function were studied in available myocardial and skeletal muscle specimens. Rare, conserved and possibly pathogenic desmin variants were identified in 6 (1.8%) probands. Two desmin mutations previously classified as variants of uncertain significance (p.K43E, p.S57L), one novel desmin variant (p.A210D) and two known pathogenic desmin mutations (p.R406W, p.R454W) were in affected individuals associated with characteristic pathological desmin aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel desmin variant p.Q364H had decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal biopsy and presented with familial left ventricular non-compaction cardiomyopathy, a novel phenotype of desminopathy. Assessment of mitochondrial function in four carriers of desmin mutations with fresh-frozen skeletal and/or myocardial muscle specimens confirmed decreased metabolic capacity of mitochondrial respiratory chain complexes, which was in case of myocardial succinate respiration more profound than in end-stage heart failure of other etiologies. Genetic testing corrected an inappropriate clinical diagnosis in two probands previously diagnosed with mitochondrial disease and inflammatory cardiomyopathy. During a median follow-up of 56 months, 5 (83%) probands developed end-stage heart failure. Conclusions Desminopathy is a rare cause of cardiomyopathy and/or skeletal muscle myopathy with a pleomorphic clinical presentation and poor prognosis. The presence of desminopathy should be considered also in individuals with left ventricular non-compaction cardiomyopathy, and in the differential diagnosis of mitochondrial diseases and inflammatory cardiomyopathy. Acknowledgement/Funding Research grant of the Ministry of Health, Czech Republic [MZ 15-27682A], No. 00023001 (IKEM, Prague, CZ), the Czech Science Foundation [14-36804G].

Alterations of redox dynamics and desmin post-translational modifications in skeletal muscle models of desminopathies

Desminopathies are a type of myofibrillar myopathy resulting from mutations in DES, encoding the intermediate filament protein desmin. They display heterogeneous phenotypes, suggesting environment influences. Patient muscle proteins show oxidative features linking oxidative stress, protein aggregation, and abnormal protein deposition. To improve understanding of redox balance in desminopathies, we further developed cellular models of four pathological mutants localized in 2B helical domain (the most important region for desmin polymerization) to explore desmin behavior upon oxidative stress. We show that the mutations desQ389P and desD399Y share common stress-induced aggregates, desR406W presents more scattered cytoplasmic aggregative pattern, and pretreatment with N-acetyl-l-cysteine (NAC), an antioxidant molecule, prevents all type of aggregation. Mutants desD399Y and desR406W had delayed oxidation kinetics following H2O2 stress prevented by NAC pretreatment. Further, we used AAV-injected mouse models to confirm in vivo effects of N-acetyl-l-cysteine. AAV-desD399Y-injected muscles displayed similar physio-pathological characteristics as observed in patients. However, after 2 months of NAC treatment, they did not have reduced aggregates. Finally, in both models, stress induced some post-translational modifications changing Isoelectric Point, such as potential hyperphosphorylations, and/or molecular weight of human desmin by proteolysis. However, each mutant presented its own pattern that seemed to be post-aggregative. In conclusion, our results indicate that individual desmin mutations have unique pathological molecular mechanisms partly linked to alteration of redox homeostasis. Integrating these mutant-specific behaviors will be important when considering future therapeutics.

The MyoRobot technology discloses a premature biomechanical decay of skeletal muscle fiber bundles derived from R349P desminopathy mice

Mutations in the Des gene coding for the muscle-specific intermediate filament protein desmin lead to myopathies and cardiomyopathies. We previously generated a R349P desmin knock-in mouse strain as a patient-mimicking model for the corresponding most frequent human desmin mutation R350P. Since nothing is known about the age-dependent changes in the biomechanics of affected muscles, we investigated the passive and active biomechanics of small fiber bundles from young (17–23 wks), adult (25–45 wks) and aged (>60 wks) heterozygous and homozygous R349P desmin knock-in mice in comparison to wild-type littermates. We used a novel automated biomechatronics platform, the MyoRobot, to perform coherent quantitative recordings of passive (resting length-tension curves, visco-elasticity) and active (caffeine-induced force transients, pCa-force, ‘slack-tests’) parameters to determine age-dependent effects of the R349P desmin mutation in slow-twitch soleus and fast-twitch extensor digitorum longus small fiber bundles. We demonstrate that active force properties are not affected by this mutation while passive steady-state elasticity is vastly altered in R349P desmin fiber bundles compatible with a pre-aged phenotype exhibiting stiffer muscle preparations. Visco-elasticity on the other hand, was not altered. Our study represents the first systematic age-related characterization of small muscle fiber bundle preparation biomechanics in conjunction with inherited desminopathy.

Desmin is Critical to the Nuclear Architecture of Cardiomyocytes

Long-term changes in mechanical stress on the heart are known to induce pathological remodeling, but the signaling pathways underlying the conversion of mechanical stress into transcriptional changes have not been established. Previous reports indicate that strain in the myocyte is transmitted to the nucleus at least in part by desmin, and it has been suggested that desmin may interact with the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. Further, both gain and loss of function mutations in desmin are associated with the development of cardiomyopathy. Taken together, these observations raise questions about the role that desmin may play in myocyte sensing of and the transcriptional response to mechanical stress. To assess the role desmin plays in nuclear structure and transcriptional regulation, we acutely knocked down desmin using shRNA in adult cardiomyocytes. Initial fluorescence images indicate that desmin knockdown leads to a decrease in nuclear volume and large rearrangements (wrinkling) of the nuclear lamina. Electron microscopy on nuclei in these myocytes reveals dramatic nuclear structural changes following desmin knockdown. The disruptions of the lamina accompany large scale inward folding of the nuclear membrane, more than tripling membrane concave area. Additionally, the electron-dense chromatin regions typically associated with perilaminar heterochromatic domains expand to larger areas reaching, in many cases, hundreds of nanometers deeper into the interior of the nucleus. We also note substantial transcriptional and epigenetic changes resulting from desmin knockdown. These results suggest that desmin plays a critical role in maintaining nuclear architecture, and could form a link between mechanical stimuli acting on cardiomyocytes and their transcriptional results in the nucleus.

Molecular insights into cardiomyopathies associated with desmin (DES) mutations.

Increasing usage of next-generation sequencing techniques pushed during the last decade cardiogenetic diagnostics leading to the identification of a huge number of genetic variants in about 170 genes associated with cardiomyopathies, channelopathies, or syndromes with cardiac involvement. Because of the biochemical and cellular complexity, it is challenging to understand the clinical meaning or even the relevant pathomechanisms of the majority of genetic sequence variants. However, detailed knowledge about the associated molecular pathomechanism is essential for the development of efficient therapeutic strategies in future and genetic counseling. Mutations in DES, encoding the muscle-specific intermediate filament protein desmin, have been identified in different kinds of cardiac and skeletal myopathies. Here, we review the functions of desmin in health and disease with a focus on cardiomyopathies. In addition, we will summarize the genetic and clinical literature about DES mutations and will explain relevant cell and animal models. Moreover, we discuss upcoming perspectives and consequences of novel experimental approaches like genome editing technology, which might open a novel research field contributing to the development of efficient and mutation-specific treatment options.

Cardiac cell modelling of myofibrillar myopathy by AAV-mediated transduction of desmin mutations in human iPSC-derived and neonatal rat cardiomyocytes

Myofibrillar myopathy (MFM) is a group of severe cytokeletal proteins known to be caused by mutations in genes encoding. Among the myofibrillar proteins that are accumulated within the muscle fibers of affected patients, they appear to be responsible for the structural integrity of striated muscle. Mutations in the gene lead not only to skeletal muscle myopathies but also to cardiomyopathies, however the cardiac pathological consequences caused by them remain poorly understood. The objective of this work is to examine the effects of MFM-induced mutations in the structural organization of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) as identified by troponinT immunostaining, and rat neonatal cardiomyocytes. We propose an in vitro cardiac disease-based cellular model based on adeno-associated virus (AAV)-mediated transduction of wild-type (WT) and mutated desmin in fusion with a c-myc tag to distinguish between endogenous and transgenic desmin proteins. Our preliminary results demonstrated that cardiomyocytes were successfully transduced by AAV vectors and expressed exogenous WT desmin as revealed by c-myc immunostaining. For better recapitulating the in vivo anisotropic cardiomyocyte organization, we have developed micro-patterned culture plates made by microcontact printing where thin strips of gelatin have been deposited on top of glass. Culture of both hiPSC-derived and rat neonatal cardiomyocytes on this system improved their sarcomere alignment. The analysis based on immunostaining of protein-markers to examine structural organization in AAV-transduced cardiomyocytes is in progress. Our results indicate that the proposed in vitro model is suitable for the study of the structural defects of desmin-mutated cardiomyocytes.

The desmin network is a determinant of the cytoplasmic stiffness of myoblasts.

The mechanical properties of cells are essential to maintain their proper functions, and mainly rely on their cytoskeleton. A lot of attention has been paid to actin filaments, demonstrating their central role in the cells mechanical properties, but much less is known about the participation of intermediate filament (IF) networks. Indeed the contribution of IFs, such as vimentin, keratins and lamins, to cell mechanics has only been assessed recently. We study here the involvement of desmin, an IF specifically expressed in muscle cells, in the rheology of immature muscle cells. Desmin can carry mutations responsible for a class of muscle pathologies named desminopathies. In this study, using three types of cell rheometers, we assess the consequences of expressing wild-type (WT) or mutated desmin on the rheological properties of single myoblasts. We find that the mechanical properties of the cell cortex are not correlated to the quantity, nor the quality of desmin expressed. On the contrary, the overall cell stiffness increases when the amount of WT or mutated desmin polymerised in cytoplasmic networks increases. However, myoblasts become softer when the desmin network is partially depleted by the formation of aggregates induced by the expression of a desmin mutant. We demonstrate that desmin plays a negligible role in the mechanical properties of the cell cortex but is a determinant of the overall cell stiffness. More particularly, desmin participates to the cytoplasm viscoelasticity. Desminopathies are associated with muscular weaknesses attributed to a disorganisation of the structure of striated muscle that impairs the active force generation. The present study evidences for the first time the key role of desmin in the rheological properties of myoblasts, raising the hypothesis that desmin mutations could also alter the passive mechanical properties of muscles, thus participating to the lack of force build up in muscle tissue.

P6291Molecular and cellular characterization of a novel desmin mutation identified in a patient with a cardiac and skeletal myopathy

P6291Molecular and cellular characterization of a novel desmin mutation identified in a patient with a cardiac and skeletal myopathy

Distinct fibrosis pattern in desmosomal and phospholamban mutation carriers in hereditary cardiomyopathies

Desmosomal and phospholamban (PLN) mutations are associated with arrhythmogenic cardiomyopathy. Ultimately, most cardiomyopathic hearts develop significant cardiac fibrosis. To compare the fibrosis patterns of desmosomal and p. Arg14del PLN-associated cardiomyopathies with the pattern in hearts with other hereditary cardiomyopathies. A midventricular transversal slice was obtained from hearts of 30 patients with a cardiomyopathy with a known underlying mutation and from 8 controls. Fibrosis and fatty changes were quantitatively analyzed using digital microscopy. Hearts from patients with desmosomal mutations (n=6) showed fibrosis and fibrofatty replacement in the left ventricular (LV) outer myocardium, mainly in the posterolateral wall, and in the right ventricle. A similar phenotype, but with significantly more severe fibrotic changes in the LV, was found in the PLN mutation group (n = 8). Cardiomyopathies associated with lamin A/C (n= 5), sarcomeric (n = 8), and desmin (n = 3) mutations all showed a different pattern from that of the desmosomal and PLN mutation carriers. The posterolateral LV wall appeared to be the most discriminative area with fibrosis andfatty changes predominantly at the outer compact myocardium in 13 of 14 hearts with desmosomal and PLN mutations (93%), in 0 of 13 hearts with lamin A/C and sarcomeric mutations (0%), and in 1 of 3 desminopathic hearts (33%) (P<.001). Desmosomal- and PLN-associated cardiomyopathies have a fibrosis pattern distinct from the patterns in other hereditary cardiomyopathies. The posterolateral LV wall appeared to be the most discriminative region between mutation groups. These results may provide a roadmap for cardiac imaging interpretation and may help in further unraveling disease mechanisms.

Exome Sequencing Identifies a Novel DES Mutation (R227C) in a Chinese Dilated Cardiomyopathy Family

Objectives: Dilated cardiomyopathy (DCM) is a common disease in the clinic, and it is the leading cause of heart failure and sudden cardiac death. Previous studies have proven that genetic factors play a crucial role in the occurrence of DCM; more than 50 disease genes including desmin (DES) have been identified to be associated with DCM. At present, most DES mutations are reported in desmin-related myofibrilla myopathy patients, but variants leading to isolated DCM are rarely reported. Methods: We applied whole-exome sequencing and cardiomyopathy-related gene filtering strategies to discover the genetic factors in a Chinese DCM family. Results: A novel mutation (c.679 C>T /p.R227C) in exon 3 of DES was identified and cosegregated with the affected members of a Chinese family with isolated DCM phenotypes (left ventricle and left atrial diameters). Conclusion: This mutation leads to a substitution of arginine by cysteine and it is predicted to be deleterious by bioinformatics programs. Our study not only contributes to the genetic diagnosis and counseling of families with DCM, but it also further proves that DES mutations may lead to isolated DCM and provides a new case for the study of the relationship between DES mutations and DCM.

Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.

EP 40. Gynecomastia – A sign of a neuromuscular disease?

Objective The aim of a neurological examination is to narrow down by signs and symptoms which components of the neurological system are affected. In neuromuscular diseases non-neurological signs such as gynecomastia can be a valuable hint. Gynecomastia is defined as excess glandular growth of breast tissue in males, pathologically associated e.g. with testicular neoplasms or gonadal dysfunction [1,2]. However there are also neuromuscular diseases such as spinal and bulbar muscular atrophy, myofibrillar myopathies and thyrotoxic hypokalemic periodic paralysispresenting with gynecomastia, we herby want to summarize. Methods A systematic PubMed literature search was carried out by application of the search terms “spinal and bulbar muscular atrophy”, “Kennedy’s disease”, “gynecomastia neuromuscular disease”, “myopathy gynecomastia”. Additionally, references in these articles were searched for further papers suitable to be included. Results Spinal and bulbar muscular atrophy (Kennedy’s disease, SBMA) is a slowly progressive adult onset neurodegenerative disorder, characterized by proximal muscle weakness and atrophy of the extremity and bulbar muscles, due to loss of lower motor neurons in the spinal cord and brainstem. It is caused by the expansion of a CAG repeat in the androgen receptor gene on the X Chromosome [3–6]. The length of the CAG expansion correlates inversely with age of disease onset and disease severity [7]. Up to 78% of patients present with gynecomastia due to partial androgen insensitivity [5,8]. Desminopathies and Alpha-B crystallinopathies belong to the myofibrillar myopathies (MFM), a group of neuromuscular disorders, characterized by desmin-positive protein aggregations in muscle fibres and myofibrillar degeneration. They are caused by mutations in genes encoding for extramyofibrillar proteins (desmin (DES) and αB-crystallin (CRYAB)) [9,10]. Desminopathiesdue to a variety of human desmin gene mutations on chromosome 2q35 cause different myopathy phenotypes, cardiac diseases and respiratory insufficiency [11,12]. DES mutation (R350P) leads to a variability of clinical manifestation ranging from scapuloperoneal, limb girdle and distal phenotypes. Gynecomastia can be observed [13]. Alpha-B crystallinopathy due to mutations in the CRYAB gene presents with proximal and distal limb muscle weakness often associated with neck, trunk, and palatopharyngeal muscle involvement, cardiomyopathy and pulmonary insufficiency. Alpha-B Crystallin is a chaperone stabilizing desmin molecule by preventing its aggregation [14,12]. Missense mutation p.Gly154Ser of CRYAB gene is associated with a late-onset distal vacuolar myopathy with protein aggregates. Mild gynecomastia can be present [15]. Thyrotoxic, hypokalemic periodic paralysis (THPP): Periodic paralysis associated with hyperthyroidism and hypokalemia is an uncommon disorder reported primarily in Asian males [1]. It is characterized by muscle paralysis and hypokalemia. Proximal muscles are more severely affected than distal muscles [17,18] Uni- or bilateral gynecomastia can be a symptom even prior to the onset of paralysis [18,19]. Conclusion There is a limited range of neuromuscular disease associated with gynecomastia. Looking carefully at your patient whilst examination and noticing symptoms other than neurological symptoms can speed up to find the correct diagnosis, especially SBMA. Finally, most important, breast cancer has to be excluded, as there is an incidence of up to 2% in men.

Mutant desmin substantially perturbs mitochondrial morphology, function and maintenance in skeletal muscle tissue.

Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1592-7) contains supplementary material, which is available to authorized users.

DesR349P Mutation Results in Ultrastructural Disruptions and Compromise of Skeletal Muscle Biomechanics Already at Preclinical Stages in Young Mice before the Onset of Protein Aggregation

Mutations in the extra-sarcomeric protein desmin give rise to protein aggregate myopathies that interfere with cellular function. The R350P mutation is the most common human desminopathy. A murine DesR349P model of human DesR350P desminopathy was previously engineered. In this study, we followed the hypothesis of whether muscle cytoarchitecture and biomechanical properties are already altered in preclinical stages of R349P desminopathy. We applied non-linear second harmonic generation (SHG) and 2-photon fluorescence morphometry analysis to single fibres of fast- and slow-twitch muscle to analyze sarcomeric architecture and nuclear morphology. We found a vast disruption of the lateral sarcomere lattice (indicated by large ‘vernier’ densities) and myofibrillar angular deviations (indicated by cosine angle sums) in single DesR349P fibres. Homozygous fibres were more severely affected than heterozygous. The former showed a marked nuclear pathology with spheric nuclei, increased nuclear number and density. For biomechanics, we assessed active and passive properties. Axial stiffness of DesR349P muscle was much increased, both in fibre and myofibrillar homozygous bundles. Lateral stiffness of the membrane complex in myoblasts was increased in cells heterozygous for DesR349P while homozygous were similar to wt. Caffeine-induced force was compromised in heterozygous fibre bundles but increased in homozygous bundles. This was explained by a marked shift of myofibrillar Ca2+ sensitivity: heterozygous bundles being less sensitive but homozygous more sensitive to Ca2+. This points towards a specific compensatory mechanism in homozygous mutation to cope with otherwise compromised force production already at preclinical stages of desminopathy. Our findings provide a very first complete picture to explain compromised force in heterozygous DesR350P patients: increased muscle stiffness, higher susceptibility towards stretch-induced injury, disrupted myofibrillar alignment and compromised active force production.

Myofibrillar myopathies: State of the art, present and future challenges

Myofibrillar myopathies (MFM) have been described in the mid-1990s as a group of diseases sharing common histological features, including an abnormal accumulation of intrasarcoplasmic proteins, the presence of vacuoles and a disorganization of the intermyofibrillar network beginning at the Z-disk. The boundaries of this concept are still uncertain, and whereas six genes (DES, CRYAB, LDB3/ZASP, MYOT, FLNC and BAG3) are now classically considered as responsible for MFM, other entities such as FHL1 myopathy or Hereditary Myopathy with Early Respiratory Failure linked to mutations of titin can now as well be included in this group. The diagnosis of MFM is not always easy; as histological lesions can be focal, and muscle biopsy may be disappointing; this has led to a growing importance of muscle imaging, and the selectivity of muscle involvement has now been described in several disorders. Due to the rarity of these myopathies, if some clinical patterns (such as distal myopathy associated with cardiomyopathy due to desmin mutations) are now well known, surprises remain possible and should lead to systematic testing of the known genes in case of a typical histological presentation. In this paper, we aim at reviewing the data acquired on the six main genes listed above as well as presenting the experience from two French reference centres, Paris and Marseilles.

Loss-of-Function SCN5A Mutations Associated With Sinus Node Dysfunction, Atrial Arrhythmias, and Poor Pacemaker Capture.

Cardiac device implantation can be complicated by inability to adequately place leads because of significant lead capture issues. This study sought to determine whether there are genetic bases that underlie poor lead capture. Retrospective review of all patients with structurally normal hearts who underwent new device implantation at Texas Children's Hospital between 2009 and 2014 was performed. Patients with inability to capture at 10 V or a final capture threshold ≥3 V at 0.4 ms during implant were analyzed. Among a total of 136 patients (median age, 13 years; range, 3 days to 46 years), 11 patients (8.1%) who underwent dual chamber device implantation had elevated thresholds in the atria (4), ventricle (3), or both chambers (4; atrial-lead threshold, 4.7±4.3 versus 0.7±0.3 V; ventricular-lead, 3.0±3.3 versus 0.7±0.3 V). All 11 patients presented with sinus node dysfunction and 10 had atrial arrhythmias. At implant, inability to find atrial capture was seen in 4 patients. Three demonstrated intermittent complete loss of ventricular capture after implantation: 1 has recurrent syncope, 2 eventually died. Genetic testing performed in 10 demonstrated 7 patients with 6 distinct SCN5A mutations, all predicted to be severe loss-of-function mutations by bioinformatic analyses. In the remaining patients, although putative pathogenic mutations were not found, multiple SCN5A polymorphisms were identified in 2 and a desmin mutation in 1. This study suggests that significant capture issues at implant may be because of loss-of-function SCN5A mutations, providing new insights into SCN5A function. Recognition of this association may be critical for planning device implantation strategies and patient follow-up.

Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies

Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation.

Two desmin gene mutations associated with myofibrillar myopathies in Polish families.

Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (DES) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two DES mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on DES expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.

Aggregate-prone desmin mutations impair mitochondrial calcium uptake in primary myotubes

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 [Ca2+]mit, whereas non-aggregate-prone mutations did not decrease [Ca2+]mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca2+]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca2+]mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions.

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.