RESUMEN
Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Unión Neuromuscular , Transmisión Sináptica , Animales , Unión Neuromuscular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Factores Despolimerizantes de la Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Actinas/metabolismo , Sarcómeros/metabolismo , Técnicas de Silenciamiento del Gen , Citoesqueleto de Actina/metabolismo , Miopatías Nemalínicas/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/patologíaRESUMEN
Actin is a highly expressed protein in eukaryotic cells and is essential for numerous cellular processes. In particular, efficient striated muscle contraction is dependent upon the precise regulation of actin-based thin filament structure and function. Alterations in the lengths of actin-thin filaments can lead to the development of myopathies. Leiomodins and tropomodulins are members of an actin-binding protein family that fine-tune thin filament lengths, and their dysfunction is implicated in muscle diseases. An Lmod3 mutation [G326R] was previously identified in patients with nemaline myopathy (NM), a severe skeletal muscle disorder; this residue is conserved among Lmod and Tmod isoforms and resides within their homologous leucine-rich repeat (LRR) domain. We mutated this glycine to arginine in Lmod and Tmod to determine the physiological function of this residue and domain. This G-to-R substitution disrupts Lmod and Tmod's LRR domain structure, altering their binding interface with actin and destroying their abilities to regulate thin filament lengths. Additionally, this mutation renders Lmod3 nonfunctional in vivo. We found that one single amino acid is essential for folding of Lmod and Tmod LRR domains, and thus is essential for the opposing actin-regulatory functions of Lmod (filament elongation) and Tmod (filament shortening), revealing a mechanism underlying the development of NM.
Asunto(s)
Actinas , Miopatías Nemalínicas , Humanos , Actinas/metabolismo , Tropomodulina/genética , Tropomodulina/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Proteínas Musculares/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Sarcómeros/genética , Sarcómeros/metabolismo , Mutación , Músculo Esquelético/metabolismoRESUMEN
The TNNT1 gene encoding the slow skeletal muscle TnT has been identified as a causative gene for nemaline myopathy. TNNT1 nemaline myopathy is mainly characterized by neonatal-onset muscle weakness, pectus carinatum and respiratory insufficiency. Herein, we report on a Chinese girl with TNNT1 nemaline myopathy with mild clinical phenotypes without thoracic deformities or decreased respiratory function. Muscle biopsy showed moderate to marked type 1 fiber atrophy and nemaline rods. Next-generation sequencing identified the compound heterozygous c. 587dupA (p. D196Efs*41) and c. 387+5G>A mutations in the TNNT1 gene according to the transcript NM_003283.4. RNA sequencing revealed complete exon 9 skipping caused by the c. 387+5G>A mutation. Through quantitative PCR, we found that both the truncation c. 587dupA (p. D196Efs*41) and the splicing c. 387+5G>A mutations triggered nonsense-mediated mRNA decay (NMD). Western blotting showed the residual amount of the truncated TNNT1 protein by deletion of exon 9, which may ameliorate the disease to some extent.
Asunto(s)
Miopatías Nemalínicas , Humanos , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Músculo Esquelético/patología , Mutación , Debilidad Muscular/genética , Exones/genéticaRESUMEN
Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function.
Asunto(s)
Proteínas Musculares , Miopatías Nemalínicas , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Animales , Modelos Animales de Enfermedad , Ratones , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Sarcómeros/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Nemaline myopathy (NM) is a congenital skeletal muscle disorder that typically results in muscle weakness and the presence of rod-like structures (nemaline bodies) in the sarcoplasma and/or in the nuclei of myofibres. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 1-month-old male with severe NM caused by a homozygous recessive mutation in the ACTA1 gene (c.121C > T, p.Arg39Ter). The iPSC lines demonstrated typical morphology, expressed pluripotency markers, exhibited trilineage differentiation potential and displayed a normal karyotype. These isogenic lines represent a potential resource to investigate and model recessive ACTA1 disease in a human context.
Asunto(s)
Células Madre Pluripotentes Inducidas , Miopatías Nemalínicas , Actinas/genética , Actinas/metabolismo , Homocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Masculino , Músculo Esquelético/metabolismo , Mutación , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismoRESUMEN
Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle-specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize 2 potentially novel TPM2 variants and 1 recurring variant that we identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that our myogenic assays can predict the clinical severity of TPM2 variants.
Asunto(s)
Miopatías Nemalínicas , Miopatías Estructurales Congénitas , Animales , Humanos , Ratones , Desarrollo de Músculos/genética , Miopatías Nemalínicas/metabolismo , Miopatías Estructurales Congénitas/metabolismo , Tropomiosina/genética , Pez CebraRESUMEN
In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.
Asunto(s)
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miofibrillas/ultraestructura , Actinas/química , Actinas/metabolismo , Animales , Tomografía con Microscopio Electrónico , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Proteínas Musculares/genética , Mutación , Miocardio/química , Miocardio/metabolismo , Miocardio/ultraestructura , Miofibrillas/química , Miofibrillas/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Miosinas/química , Miosinas/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Músculos Psoas/química , Músculos Psoas/metabolismo , Músculos Psoas/ultraestructura , Sarcómeros/química , Sarcómeros/metabolismo , Sarcómeros/ultraestructuraRESUMEN
OBJECTIVE: To define the proteomic profile of sporadic late-onset nemaline myopathy (SLONM) and explore its pathogenesis. METHODS: We performed mass spectrometry on laser-dissected frozen muscle samples from five patients with SLONM, three of whom with an associated monoclonal protein (MP), and four controls, to determine the proteomic profile of SLONM. Furthermore, we assessed the role of the MP by evaluating the expression of the immunoglobulin light chain variable regions (IGVL). RESULTS: There were 294 differentially expressed proteins: 272 upregulated and 22 downregulated. Among the top 100 upregulated proteins, the most common categories were: nuclear or nucleic acid metabolism (24%), extracellular matrix and basal lamina (17%), immune response (13%), and actin dynamics (8%). Downregulated proteins consisted mostly of contractile proteins. Among upregulated proteins, there were 65 with a role related to the immune system, including eight proteins involved in major histocompatibility complex 1 (MHC1) and antigen processing, 15 in MHCII complex and phagocytosis, and 23 in B and/or T-cell function. Among nine upregulated immunoglobulin proteins, there were two IGVL genes. However, these were also detected in SLONM cases without an MP, with no evidence of clonally dominant immunoglobulin deposition. In muscle sections from SLONM patients, nemaline rods tended to accumulate in atrophic fibers with marked rarefaction of the myofibrils. Increased MHC1 reactivity was present in fibers containing nemaline rods as well as adjacent nonatrophic fibers. CONCLUSION: Our findings suggest that aberrant immune activation is present in SLONM, but do not support a direct causal relationship between the MP and SLONM.
Asunto(s)
Miopatías Nemalínicas , Humanos , Inmunoglobulinas , Músculos/patología , Miopatías Nemalínicas/complicaciones , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , ProteómicaRESUMEN
Sporadic late-onset nemaline myopathy (SLONM) associated with monoclonal protein (MP) is a rare disease with an aggressive, and often fatal course. Whether SLONM + MP represents a malignancy or dysimmune disease remains unclear. Currently, two main approaches are used to treat SLONM + MP: nonchemotherapy-based treatment (immunosuppression, intravenous immunoglobulins, plasmapheresis and plasma exchange) or chemotherapy with or without autologous stem cell transplantation. Due to the rare occurrence of the disease, the best treatment modality is unknown. We analyzed treatment and outcomes in a large cohort of 53 patients with SLONM + MP: four our own patients and 49 cases from published literature. Neurological improvement in the nonchemotherapy group (N = 25) was observed in 52% of patients: 8% reached marked improvement, 8% moderate response, 36% mild response; none reached complete remission (CR). In the chemotherapy group (N = 28), neurological improvement was seen in 86% of patients: 46% reached CR, 25% marked response, 11% moderate response and 4% mild response. The best neurological improvement correlated with deep hematological remission. Mean time to best response in the chemotherapy group was 8 months versus 21 months in the nonchemotherapy group (P < .001). Overall survival was higher in patients in the chemotherapy group. A chemotherapy approach should be the preferred treatment for patients with SLOMN + MP with the goal to reach complete hematologic remission. Based on the clinical, morphological peculiarities, aggressive disease course and superior clinical benefits of chemotherapy over nonchemotherapy, SLONM + MP should be considered as a hematological malignancy with the presence of MP of clinical rather than undetermined significance.
Asunto(s)
Quimioterapia/métodos , Inmunoglobulinas Intravenosas/administración & dosificación , Proteínas de Mieloma/metabolismo , Miopatías Nemalínicas/tratamiento farmacológico , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Esquema de Medicación , Femenino , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Masculino , Persona de Mediana Edad , Miopatías Nemalínicas/metabolismo , Miopatías Nemalínicas/terapia , Inducción de Remisión , Trasplante Autólogo , Resultado del TratamientoRESUMEN
Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.
Asunto(s)
Proteínas Musculares/genética , Mutación Missense , Miofibrillas/metabolismo , Miopatías Nemalínicas/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animales , Heterocigoto , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Miofibrillas/patología , Miofibrillas/ultraestructura , Miopatías Nemalínicas/metabolismo , Sarcómeros/metabolismo , Sarcómeros/patología , Sarcómeros/ultraestructura , Tropomiosina/química , Tropomiosina/metabolismo , Troponina/química , Troponina/metabolismo , Difracción de Rayos XRESUMEN
The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.
Asunto(s)
Proteínas Musculares/metabolismo , Relajación Muscular , Miopatías Nemalínicas/metabolismo , Sarcómeros/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Humanos , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/patología , Sarcómeros/patología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Respiratory failure due to diaphragm dysfunction is considered a main cause of death in nemaline myopathy (NM) and we studied both isometric force and isotonic shortening of diaphragm muscle in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was found in nebulin-deficient intact muscle that is frequency dependent, with the largest deficits at low-intermediate stimulation frequencies (e.g., a deficit of 72% at a stimulation frequency of 20 Hz). The effect of the fast skeletal muscle troponin activator (FSTA) tirasemtiv on force was examined. Tirasemtiv had a negligible effect at maximal stimulation frequencies, but greatly reduced the force deficit of the diaphragm at sub-maximal stimulation levels with an effect that was largest in Neb cKO diaphragm. As a result, the force deficit of Neb cKO diaphragm fell (from 72% to 29% at 20 Hz). Similar effects were found in in vivo experiments on the nerve-stimulated gastrocnemius muscle complex. Load-clamp experiments on diaphragm muscle showed that tirasemtiv increased the shortening velocity, and reduced the deficit in mechanical power by 33%. Thus, tirasemtiv significantly improves muscle function in a mouse model of nebulin-based nemaline myopathy.
Asunto(s)
Diafragma/fisiología , Imidazoles/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miopatías Nemalínicas/metabolismo , Pirazinas/metabolismo , Troponina/metabolismo , Animales , Transportador de Cobre 1/genética , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular , Proteínas Musculares/genéticaRESUMEN
Nemaline myopathy-4 (NEM4) is a very rare inherited muscle disorder caused by a heterozygous mutation in tropomyosin-2 (TPM2) gene. We established an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a 3-month-old girl with NEM4 carrying a heterozygous mutation (c.397C>T (p.R133W)) in TPM2 gene. This iPSC line showed a normal karyotype, expressed pluripotency markers, showed differentiation potential and harbored the original mutation of c.397C>T in the TPM2 gene.
Asunto(s)
Línea Celular/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miopatías Nemalínicas/genética , Tropomiosina/genética , Diferenciación Celular , Línea Celular/citología , Femenino , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/citología , Lactante , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Mutación , Miopatías Nemalínicas/metabolismo , Miopatías Nemalínicas/fisiopatología , Tropomiosina/metabolismoRESUMEN
Nemaline myopathies are a heterogenous group of congenital myopathies caused by de novo, dominantly or recessively inherited mutations in at least twelve genes. The genes encoding skeletal α-actin (ACTA1) and nebulin (NEB) are the commonest genetic cause. Most patients have congenital onset characterized by muscle weakness and hypotonia, but the spectrum of clinical phenotypes is broad, ranging from severe neonatal presentations to onset of a milder disorder in childhood. Most patients with adult onset have an autoimmune-related myopathy with a progressive course. The wide application of massively parallel sequencing methods is increasing the number of known causative genes and broadening the range of clinical phenotypes. Nemaline myopathies are identified by the presence of structures that are rod-like or ovoid in shape with electron microscopy, and with light microscopy stain red with the modified Gömöri trichrome technique. These rods or nemaline bodies are derived from Z lines (also known as Z discs or Z disks) and have a similar lattice structure and protein content. Their shape in patients with mutations in KLHL40 and LMOD3 is distinctive and can be useful for diagnosis. The number and distribution of nemaline bodies varies between fibres and different muscles but does not correlate with severity or prognosis. Additional pathological features such as caps, cores and fibre type disproportion are associated with the same genes as those known to cause the presence of rods. Animal models are advancing the understanding of the effects of various mutations in different genes and paving the way for the development of therapies, which at present only manage symptoms and are aimed at maintaining muscle strength, joint mobility, ambulation, respiration and independence in the activities of daily living.
Asunto(s)
Mutación , Miopatías Nemalínicas , Actinas/genética , Actinas/metabolismo , Edad de Inicio , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Miopatías Nemalínicas/patología , Sarcómeros/genética , Sarcómeros/metabolismo , Sarcómeros/ultraestructuraRESUMEN
KEY POINTS: The pathogenic mechanism and the neuromuscular reflex-related phenotype (e.g. tremors accompanied by clonus) of Amish nemaline myopathy, as well as of other recessively inherited TNNT1 myopathies, remain to be clarified. The truncated slow skeletal muscle isoform of troponin T (ssTnT) encoded by the mutant TNNT1 gene is unable to incorporate into myofilaments and is degraded in muscle cells. By contrast to extrafusal muscle fibres, spindle intrafusal fibres of normal mice contain a significant level of cardiac TnT and a low molecular weight splice form of ssTnT. Intrafusal fibres of ssTnT-knockout mice have significantly increased cardiac TnT. Rotarod and balance beam tests have revealed abnormal neuromuscular co-ordination in ssTnT-knockout mice and a blunted response to a spindle sensitizer, succinylcholine. The loss of ssTnT and a compensatory increase of cardiac TnT in intrafusal nuclear bag fibres may increase myofilament Ca2+ -sensitivity and tension, impairing spindle function, thus identifying a novel mechanism for the development of targeted treatment. ABSTRACT: A nonsense mutation at codon Glu180 of TNNT1 gene causes Amish nemaline myopathy (ANM), a recessively inherited disease with infantile lethality. TNNT1 encodes the slow skeletal muscle isoform of troponin T (ssTnT). The truncated ssTnT is unable to incorporate into myofilament and is degraded in muscle cells. The symptoms of ANM include muscle weakness, atrophy, contracture and tremors accompanied by clonus. An ssTnT-knockout (KO) mouse model recapitulates key features of ANM such as atrophy of extrafusal slow muscle fibres and increased fatigability. However, the neuromuscular reflex-related symptoms of ANM have not been explained. By isolating muscle spindles from ssTnT-KO and control mice aiming to examine the composition of myofilament proteins, we found that, in contrast to extrafusal fibres, intrafusal fibres contain a significant level of cardiac TnT and the low molecular weight splice form of ssTnT. Intrafusal fibres from ssTnT-KO mice have significantly increased cardiac TnT. Rotarod and balance beam tests revealed impaired neuromuscular co-ordination in ssTnT-KO mice, indicating abnormality in spindle functions. Unlike the wild-type control, the beam running ability of ssTnT-KO mice had a blunted response to a spindle sensitizer, succinylcholine. Immunohistochemistry detected ssTnT and cardiac TnT in nuclear bag fibres, whereas fast skeletal muscle TnT was detected in nuclear chain fibres, and cardiac α-myosin was present in one of the two nuclear bag fibres. The loss of ssTnT and a compensatory increase of cardiac TnT in nuclear bag fibres would increase myofilament Ca2+ -sensitivity and tension, thus affecting spindle activities. This mechanism provides an explanation for the pathophysiology of ANM, as well as a novel target for treatment.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Husos Musculares/metabolismo , Miopatías Nemalínicas/genética , Troponina T/genética , Animales , Células Cultivadas , Locomoción , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/fisiología , Miofibrillas/metabolismo , Miopatías Nemalínicas/metabolismo , Miopatías Nemalínicas/fisiopatologíaRESUMEN
OBJECTIVE: To describe the clinical phenotype, long-term treatment outcome, and overall survival of sporadic late-onset nemaline myopathy (SLONM) with or without a monoclonal protein (MP). METHODS: We conducted a retrospective chart review of patients seen between September 2000 and June 2017 and collected clinical, laboratory, and survival data. Treatment response was classified as mild, moderate, or marked as adjudged by predefined criteria. RESULTS: We identified 28 patients with SLONM; 17 (61%) had an associated MP. Median age at symptom onset was 62 years. Diagnosis was often delayed by a median of 35 months from symptom onset. There was no difference in clinical or laboratory features between patients with or without MP. Although the majority of patients had proximal or axial weakness at onset, about 18% of patients had atypical presentations. A total of 7/9 (78%) patients receiving IV immunoglobulin (IVIg), 6/8 (75%) receiving hematologic therapy as either autologous stem cell transplant (ASCT) or chemotherapy, and 1/8 (13%) receiving immunosuppressive therapies responded to treatment (p = 0.001). All 3 patients with marked response were treated with IVIg; 2 of them had an MP. The 5-year and 10-year overall survival from symptom onset was 92% and 68%, respectively, with no difference between patients with or without MP. CONCLUSION: SLONM has a wide spectrum of clinical presentations. In this contemporary case series, overall survival of patients did not seem to be affected by the presence of an MP. Initial treatment with IVIg is reasonable in all patients, followed by ASCT or chemotherapy as second-line therapy in patients with an associated MP.
Asunto(s)
Inmunoglobulinas Intravenosas/uso terapéutico , Inmunosupresores/uso terapéutico , Miopatías Nemalínicas/fisiopatología , Paraproteinemias/terapia , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Factores Inmunológicos/uso terapéutico , Lenalidomida/uso terapéutico , Masculino , Persona de Mediana Edad , Miopatías Nemalínicas/complicaciones , Miopatías Nemalínicas/metabolismo , Miopatías Nemalínicas/terapia , Paraproteinemias/complicaciones , Paraproteinemias/metabolismo , Estudios Retrospectivos , Trasplante de Células Madre , Tasa de Supervivencia , Talidomida/uso terapéutico , Trasplante AutólogoRESUMEN
Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.
Asunto(s)
Actinina/metabolismo , Proteínas Cullin/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miopatías Nemalínicas/metabolismo , Animales , Proteínas Cullin/genética , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados/embriología , Proteínas Musculares/genética , Debilidad Muscular/embriología , Debilidad Muscular/genética , Debilidad Muscular/metabolismo , Músculo Esquelético/embriología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Mutación , Miopatías Nemalínicas/embriología , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/patología , Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific adaptors for Cullin 3 (CUL3) E3 ubiquitin ligase to regulate protein turnover through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM; however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, nebulin-related anchoring protein (NRAP). KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric proteins contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease-causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miopatías Nemalínicas/metabolismo , Ubiquitinación , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Ratones , Miofibrillas/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/fisiopatología , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
L-tyrosine supplementation may provide benefit to nemaline myopathy (NM) patients, however previous studies are inconclusive, with no elevation of L-tyrosine levels in blood or tissue reported. We evaluated the ability of L-tyrosine treatments to improve skeletal muscle function in all three published animal models of NM caused by dominant skeletal muscle α-actin (ACTA1) mutations. Highest safe L-tyrosine concentrations were determined for dosing water and feed of wildtype zebrafish and mice respectively. NM TgACTA1D286G-eGFP zebrafish treated with 10 µM L-tyrosine from 24 hours to 6 days post fertilization displayed no improvement in swimming distance. NM TgACTA1D286G mice consuming 2% L-tyrosine supplemented feed from preconception had significant elevations in free L-tyrosine levels in sera (57%) and quadriceps muscle (45%) when examined at 6-7 weeks old. However indicators of skeletal muscle integrity (voluntary exercise, bodyweight, rotarod performance) were not improved. Additionally no benefit on the mechanical properties, energy metabolism, or atrophy of skeletal muscles of 6-7 month old TgACTA1D286G and KIActa1H40Y mice eventuated from consuming a 2% L-tyrosine supplemented diet for 4 weeks. Therefore this study yields important information on aspects of the clinical utility of L-tyrosine for ACTA1 NM.
Asunto(s)
Actinas/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miopatías Nemalínicas/tratamiento farmacológico , Miopatías Nemalínicas/metabolismo , Tirosina/administración & dosificación , Pez Cebra/metabolismo , Animales , Suplementos Dietéticos , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/efectos de los fármacosRESUMEN
The E41K mutation in TPM2 gene encoding muscle regulatory protein beta-tropomyosin is associated with nemaline myopathy and cap disease. The mutation results in a reduced Ca2+-sensitivity of the thin filaments and in muscle weakness. To elucidate the structural basis of the reduced Ca2+-sensitivity of the thin filaments, we studied multistep changes in spatial arrangement of tropomyosin (Tpm), actin and myosin heads during the ATPase cycle in reconstituted fibers, using the polarized fluorescence microscopy. The E41K mutation inhibits troponin's ability to shift Tpm to the closed position at high Ca2+, thus restraining the transition of the thin filaments from the "off" to the "on" state. The mutation also inhibits the ability of S1 to shift Tpm to the open position, decreases the amount of the myosin heads bound strongly to actin at high Ca2+, but increases the number of such heads at low Ca2+. These changes may contribute to the low Ca2+-sensitivity and muscle weakness. As the mutation has no effect on troponin's ability to switch actin monomers on at high Ca2+ and inhibits their switching off at low Ca2+, the use of reagents that increase the Ca2+-sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.