RESUMO
AIMS: Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. METHODS: Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. RESULTS: Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild-type cells. Within 1 week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. CONCLUSIONS: Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.
Assuntos
Cardiomiopatias , Desmina , Músculos , Animais , Cardiomiopatias/genética , Desmina/genética , Humanos , Camundongos , Músculo Esquelético/patologia , Músculos/patologia , Mutação , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
AIMS: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). METHODS: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. RESULTS: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. CONCLUSIONS: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.
Assuntos
Proteômica , Paraplegia Espástica Hereditária , Animais , Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Mutação , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismoRESUMO
Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.
Assuntos
Cardiomiopatias , Desmina , Hexoquinase , Aminoácidos/metabolismo , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Citratos/metabolismo , Creatina Quinase Mitocondrial/metabolismo , Desmina/genética , Desmina/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Fosforilação Oxidativa , ProteômicaRESUMO
BACKGROUND: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype. METHODS: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin. RESULTS: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype. CONCLUSIONS: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.
Assuntos
Cardiomiopatias/genética , Cardiomiopatias/terapia , Desmina/genética , Miocárdio/patologia , Índice de Gravidade de Doença , Adolescente , Animais , Cateterismo Cardíaco/métodos , Cardiomiopatias/diagnóstico por imagem , Desmina/metabolismo , Técnicas de Introdução de Genes/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/ultraestrutura , Marca-Passo ArtificialRESUMO
NEW FINDINGS: What is the central question of this study? While muscle fibre atrophy in response to immobilisation has been extensively examined, intramuscular connective tissue, particularly endomysium, has been largely neglected: does endomysium content of the soleus muscle increase during bed rest? What is the main finding and its importance? Absolute endomysium content did not change, and previous studies reporting an increase are explicable by muscle fibre atrophy. It must be expected that even a relative connective tissue accumulation will lead to an increase in muscle stiffness. ABSTRACT: Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness.
Assuntos
Fibras Musculares Esqueléticas , Músculo Esquelético , Animais , Repouso em Cama , Humanos , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/patologia , MiocárdioRESUMO
Muscle biomechanics relies on active motor protein assembly and passive strain transmission through cytoskeletal structures. The desmin filament network aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness. As the effect of R349P desmin on axial biomechanics in fully differentiated single muscle fibers is unknown, we used our MyoRobot to compare passive visco-elasticity and active contractile biomechanics in single fibers from fast- and slow-twitch muscles from adult to senile mice, hetero- or homozygous for the R349P desmin mutation with wild type littermates. We demonstrate that R349P desmin presence predominantly increased axial stiffness in both muscle types with a pre-aged phenotype over wild type fibers. Axial viscosity and Ca2+-mediated force were largely unaffected. Mutant single fibers showed tendencies towards faster unloaded shortening over wild type fibers. Effects of aging seen in the wild type appeared earlier in the mutant desmin fibers. Our single-fiber experiments, free of extracellular matrix, suggest that compromised muscle biomechanics is not exclusively attributed to fibrosis but also originates from an impaired intermediate filament network.
Assuntos
Envelhecimento/genética , Desmina/genética , Fibras Musculares Esqueléticas/química , Miofibrilas/genética , Envelhecimento/fisiologia , Animais , Fenômenos Biomecânicos , Cálcio/metabolismo , Citoesqueleto/química , Citoesqueleto/genética , Desmina/química , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Filamentos Intermediários/química , Filamentos Intermediários/genética , Camundongos , Contração Muscular/genética , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Mutação/genética , Miofibrilas/químicaRESUMO
Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only â¼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.
Assuntos
Endocitose/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Actinas/metabolismo , Animais , Axônios/patologia , Cálcio/metabolismo , Proteínas de Transporte , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Oligonucleotídeos Antissenso , Fenótipo , Terminações Pré-Sinápticas/metabolismo , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Transmissão Sináptica/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Heterozygous missense mutations in the human VCP gene cause inclusion body myopathy associated with Paget disease of bone and fronto-temporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). The exact molecular mechanisms by which VCP mutations cause disease manifestation in different tissues are incompletely understood. In the present study, we report the comprehensive analysis of a newly generated R155C VCP knock-in mouse model, which expresses the ortholog of the second most frequently occurring human pathogenic VCP mutation. Heterozygous R155C VCP knock-in mice showed decreased plasma lactate, serum albumin and total protein concentrations, platelet numbers, and liver to body weight ratios, and increased oxygen consumption and CD8+/Ly6C + T-cell fractions, but none of the typical human IBMPFD or ALS pathologies. Breeding of heterozygous mice did not yield in the generation of homozygous R155C VCP knock-in animals. Immunoblotting showed identical total VCP protein levels in human IBMPFD and murine R155C VCP knock-in tissues as compared to wild-type controls. However, while in human IBMPFD skeletal muscle tissue 70% of the total VCP mRNA was derived from the mutant allele, in R155C VCP knock-in mice only 5% and 7% mutant mRNA were detected in skeletal muscle and brain tissue, respectively. The lack of any obvious IBMPFD or ALS pathology could thus be a consequence of the very low expression of mutant VCP. We conclude that the increased and decreased fractions of the R155C mutant VCP mRNA in man and mice, respectively, are due to missense mutation-induced, divergent alterations in the biological half-life of the human and murine mutant mRNAs. Furthermore, our work suggests that therapy approaches lowering the expression of the mutant VCP mRNA below a critical threshold may ameliorate the intrinsic disease pathology.
Assuntos
Esclerose Lateral Amiotrófica/genética , Demência Frontotemporal/genética , Genes Letais , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação , Miosite de Corpos de Inclusão/genética , Osteíte Deformante/genética , Proteína com Valosina/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Encéfalo/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Modelos Animais de Doenças , Feminino , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , Osteíte Deformante/metabolismo , Osteíte Deformante/patologia , Transdução de Sinais , Especificidade da Espécie , Proteína com Valosina/metabolismoRESUMO
Mutations of the human VCP gene, which encodes the V: alosin C: ontaining P: rotein (synonyms: p97, TER ATPase), are associated with various multi-systemic protein aggregation diseases. We report on a patient with progressive myopathy and incipient cognitive deficits. A diagnostic muscle biopsy revealed an inclusion body myopathy with protein aggregates. Magnetic resonance imaging and F18-positron-emission-tomography disclosed a fronto-temporal atrophy and glucose hypometabolism of the frontal and temporal lobes, respectively. Based on the clinical findings, a genetic analysis was performed which revealed a heterozygous c.277C>T (p.Arg93Cys) mutation of the VCP gene, thus confirming the diagnosis of IBMPFD (I: nclusion B: ody M: yopathie with P: aget Disease of the Bones and F: ronto-temporal D: ementia).
Assuntos
Demência Frontotemporal/complicações , Demência Frontotemporal/genética , Distrofia Muscular do Cíngulo dos Membros/complicações , Distrofia Muscular do Cíngulo dos Membros/genética , Miosite de Corpos de Inclusão/complicações , Miosite de Corpos de Inclusão/genética , Osteíte Deformante/complicações , Osteíte Deformante/genética , Proteína com Valosina/genética , Idoso , Atrofia , Biópsia , Demência Frontotemporal/diagnóstico por imagem , Glucose/metabolismo , Humanos , Imageamento por Ressonância Magnética , Masculino , Músculos/patologia , Distrofia Muscular do Cíngulo dos Membros/diagnóstico por imagem , Mutação , Miosite de Corpos de Inclusão/diagnóstico por imagem , Osteíte Deformante/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/metabolismoRESUMO
BACKGROUND: During aging a mosaic of normal cells and cells with mitochondrial deficiency develops in various tissues including the heart. Whether this contributes to higher susceptibility for arrhythmia following myocardial infarction (MI) is unknown. METHODS AND RESULTS: Myocardial cryoinfarction was performed in 12-month-old transgenic mice with accelerated accumulation of deletions in mitochondrial DNA. Occurrence and pathogenesis of arrhythmia was investigated after two weeks. Holter-ECG recordings revealed higher rates of premature ventricular complexes (incidence > 10/24 h: 100% vs. 20%; p = 0.048) and more severe spontaneous arrhythmia during stress test in mutant mice with MI as compared to control mice with MI. Mice with mitochondrial dysfunction exhibited longer spontaneous AV-blocks (467 ± 26 ms vs. 377 ± 24 ms; p = 0.013), an increased probability for induction of ventricular tachycardia during in vivo electrophysiological investigation (22% vs. 9%; p = 0.044), and a reduced conduction velocity in the infarct borderzone (38.5 ± 0.5 cm/s vs. 55.3 ± 0.9 cm/s; p = 0.001). Furthermore, mutant mice exhibited a significant reduction of the phospho-Cx43/Cx43 ratio in right (0.59 ± 0.04 vs. 0.85 ± 0.01; p = 0.027) and left ventricular myocardium (0.72 ± 0.01 vs. 0.86 ± 0.02; p = 0.023). CONCLUSIONS: Aging-related cardiac mosaic respiratory chain dysfunction facilitates the occurrence of spontaneous and inducible cardiac arrhythmia after myocardial infarction and is associated with slowing of electrical impulse propagation in the infarct borderzone.
Assuntos
Envelhecimento , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Mitocôndrias Cardíacas , Doenças Mitocondriais/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças Mitocondriais/complicações , Infarto do Miocárdio/complicaçõesRESUMO
The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro)anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and transparent TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, Triptychon (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ligação Competitiva , Proteínas de Ligação a DNA/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica de Plantas , Microscopia de Fluorescência , Modelos Biológicos , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Transformação Genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Desmina/genética , Filamentos Intermediários/patologia , Mitocôndrias/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/genética , Animais , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Desmina/metabolismo , Humanos , Filamentos Intermediários/genética , Camundongos Transgênicos , Mitocôndrias/patologia , Doenças Musculares/patologia , Mutação/genéticaRESUMO
Myofibrillar myopathies (MFM) are progressive diseases of human heart and skeletal muscle with a severe impact on life quality and expectancy of affected patients. Although recently several disease genes for myofibrillar myopathies could be identified, today most genetic causes and particularly the associated mechanisms and signaling events that lead from the mutation to the disease phenotype are still mostly unknown. To assess whether the zebrafish is a suitable model system to validate MFM candidate genes using targeted antisense-mediated knock-down strategies, we here specifically inactivated known human MFM disease genes and evaluated the resulting muscular and cardiac phenotypes functionally and structurally. Consistently, targeted ablation of MFM genes in zebrafish led to compromised skeletal muscle function mostly due to myofibrillar degeneration as well as severe heart failure. Similar to what was shown in MFM patients, MFM gene-deficient zebrafish showed pronounced gene-specific phenotypic and structural differences. In summary, our results indicate that the zebrafish is a suitable model to functionally and structurally evaluate novel MFM disease genes in vivo.
Assuntos
Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologiaRESUMO
Protein turnover and quality control by the proteasome is of paramount importance for cell homeostasis. Dysfunction of the proteasome is associated with aging processes and human diseases such as neurodegeneration, cardiomyopathy, and cancer. The regulation, i.e. activation and inhibition of this fundamentally important protein degradation system, is still widely unexplored. We demonstrate here that the evolutionarily highly conserved type II triple-A ATPase VCP and the proteasome inhibitor PSMF1/PI31 interact directly, and antagonistically regulate proteasomal activity. Our data provide novel insights into the regulation of proteasomal activity.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Ciclo Celular/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas/fisiologia , Biopolímeros , Humanos , Proteína com ValosinaRESUMO
Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive and sporadic forms of protein aggregation myopathies and cardiomyopathies. We generated R349P desmin knock-in mice, which harbor the ortholog of the most frequently occurring human desmin missense mutation R350P. These mice develop age-dependent desmin-positive protein aggregation pathology, skeletal muscle weakness, dilated cardiomyopathy, as well as cardiac arrhythmias and conduction defects. For the first time, we report the expression level and subcellular distribution of mutant versus wild-type desmin in our mouse model as well as in skeletal muscle specimens derived from human R350P desminopathies. Furthermore, we demonstrate that the missense-mutant desmin inflicts changes of the subcellular localization and turnover of desmin itself and of direct desmin-binding partners. Our findings unveil a novel principle of pathogenesis, in which not the presence of protein aggregates, but disruption of the extrasarcomeric intermediate filament network leads to increased mechanical vulnerability of muscle fibers. These structural defects elicited at the myofiber level finally impact the entire organ and subsequently cause myopathy and cardiomyopathy.
Assuntos
Desmina/genética , Desmina/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Miocárdio/patologia , Animais , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Escherichia coli , Técnicas de Introdução de Genes , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Camundongos Transgênicos , Debilidade Muscular/patologia , Debilidade Muscular/fisiopatologia , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Mutação de Sentido Incorreto , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMO
BACKGROUND: Coronin proteins are known as regulators of actin-based cellular processes, and some of them are associated with the malignant progression of human cancer. Here, we show that expression of coronin 2A is up-regulated in human colon carcinoma. METHODS: This study included 26 human colon tumour specimens and 9 normal controls. Expression and localisation of coronin 2A was studied by immunohistochemistry, immunofluorescence imaging, cell fractionation, and immunoblotting. Functional roles of coronin 2A were analysed by over-expression and knock-down of the protein. Protein interactions were studied by co-immunoprecipitation and pull-down experiments, mass spectrometry analyses, and in vitro kinase and methylation assays. RESULTS: Histopathological investigation revealed that the expression of coronin 2A in colon tumour cells is up-regulated during the adenoma-adenocarcinoma progression. At the subcellular level, coronin 2A localised to multiple compartments, i.e. F-actin stress fibres, the front of lamellipodia, focal adhesions, and the nuclei. Over-expression of coronin 2A led to a reduction of F-actin stress fibres and elevated cell migration velocity. We identified two novel direct coronin 2A interaction partners. The interaction of coronin 2A with MAPK14 (mitogen activated protein kinase 14 or MAP kinase p38α) led to phosphorylation of coronin 2A and also to activation of the MAPK14 pathway. Moreover, coronin 2A interacted with PRMT5 (protein arginine N-methyltransferase 5), which modulates the sensitivity of tumour cells to TRAIL-induced cell death. CONCLUSIONS: We show that increased expression of coronin 2A is associated with the malignant phenotype of human colon carcinoma. Moreover, we linked coronin 2A to MAPK14 and PRMT5 signalling pathways involved in tumour progression.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Transdução de Sinais , Adenocarcinoma/patologia , Adenoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Fosforilação , Transporte Proteico , Proteína-Arginina N-Metiltransferases/metabolismo , Pseudópodes/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fibras de Estresse/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
Desmina , Fibras Musculares Esqueléticas , Animais , Desmina/metabolismo , Desmina/genética , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Técnicas de Introdução de Genes , Prótons , Mitocôndrias/metabolismo , Distrofias Musculares , CardiomiopatiasRESUMO
The intermediate filament protein desmin is an essential component of the extra-sarcomeric cytoskeleton in muscle cells. This three-dimensional filamentous framework exerts central roles in the structural and functional alignment and anchorage of myofibrils, the positioning of cell organelles and signaling events. Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive, and sporadic myopathies and/or cardiomyopathies with marked phenotypic variability. The disease onset ranges from childhood to late adulthood. The clinical course is progressive and no specific treatment is currently available for this severely disabling disease. The muscle pathology is characterized by desmin-positive protein aggregates and degenerative changes of the myofibrillar apparatus. The molecular pathophysiology of desminopathies is a complex, multilevel issue. In addition to direct effects on the formation and maintenance of the extra-sarcomeric intermediate filament network, mutant desmin affects essential protein interactions, cell signaling cascades, mitochondrial functions, and protein quality control mechanisms. This review summarizes the currently available data on the epidemiology, clinical phenotypes, myopathology, and genetics of desminopathies. In addition, this work provides an overview on the expression, filament formation processes, biomechanical properties, post-translational modifications, interaction partners, subcellular localization, and functions of wild-type and mutant desmin as well as desmin-related cell and animal models.
Assuntos
Desmina/metabolismo , Filamentos Intermediários/metabolismo , Mutação/genética , Animais , Cardiomiopatias/epidemiologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Desmina/genética , Modelos Animais de Doenças , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/patologiaRESUMO
To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat - a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments.
Assuntos
Ausência de Peso , Rotação , Técnicas de Cultura de Células , Fibras Musculares EsqueléticasRESUMO
PURPOSE: Chronic exposure to hypoxia can induce muscle wasting in unaccustomed individuals. Detailed assessment of the effects of hypoxia on muscle tissue adaptation in elite mountaineers has not been performed. This study aims to assess muscle volume after exposure to normobaric hypoxia. METHODS: Two professional mountaineers (A and B) participated in a 35-d intervention of graded normobaric hypoxia with the aim of 14 d exposure to 8% oxygen corresponding to 7112-m altitude. Volume of the shank, thigh, and hip muscles was assessed by magnetic resonance imaging pre- and postintervention. Dietary intake and physical activity were monitored throughout the study from food images and accelerometry analysis, together with blood analysis and anthropometric measurements. RESULTS: Hypoxia reduced total leg muscle volume by 3.3% ± 6.0% in A and by 9.4% ± 7.3% in B. A lost 288 g and B 642 g of muscle mass, whereas dietary intake only declined by ~23% in the last intervention week. Arterial oxygen saturation declined from 95% and 86% to 77% and 72% in A and B, respectively. In hypoxia, participants could not maintain their physical activity levels. Notably, muscle loss varied substantially across muscle groups amounting to 5.4% ± 3.0%, 8.3% ± 5.2%, and 4.1% ± 8.6% for hip, thigh, and shank muscles, respectively. CONCLUSIONS: Our results indicate that hypoxia and resultant reductions in physical activity and caloric intake lead to substantial loss of muscle mass that was accentuated in proximal muscle as opposed to distal muscles. Surprisingly, thigh muscle wasting during this intervention is comparable with that observed during strict 56-d bed rest.