RESUMEN
Polymerizing laminins are multi-domain basement membrane (BM) glycoproteins that self-assemble into cell-anchored planar lattices to establish the initial BM scaffold. Nidogens, collagen-IV and proteoglycans then bind to the scaffold at different domain loci to create a mature BM. The LN domains of adjacent laminins bind to each other to form a polymer node, while the LG domains attach to cytoskeletal-anchoring integrins and dystroglycan, as well as to sulfatides and heparan sulfates. The polymer node, the repeating unit of the polymer scaffold, is organized into a near-symmetrical triskelion. The structure, recently solved by cryo-electron microscopy in combination with AlphaFold2 modeling and biochemical studies, reveals how the LN surface residues interact with each other and how mutations cause failures of self-assembly in an emerging group of diseases, the LN-lamininopathies, that include LAMA2-related dystrophy and Pierson syndrome.
Asunto(s)
Membrana Basal , Laminina , Humanos , Laminina/metabolismo , Laminina/química , Laminina/genética , Animales , Membrana Basal/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/genética , Deformidades Congénitas de las Extremidades/metabolismo , Deformidades Congénitas de las Extremidades/genética , Mutación , Síndrome Nefrótico , Trastornos de la Pupila , Síndromes Miasténicos CongénitosRESUMEN
Muscular dystrophy is a group of genetic disorders that lead to muscle wasting and loss of muscle function. Identifying genetic modifiers that alleviate symptoms or enhance the severity of a primary disease helps to understand mechanisms behind disease pathology and facilitates discovery of molecular targets for therapy. Several muscular dystrophies are caused by genetic defects in the components of the dystrophin-glycoprotein adhesion complex (DGC). Thrombospondin-4 overexpression has been shown to mitigate dystrophic disease in mouse models for Duchenne muscular dystrophy (dystrophin deficiency) and limb-girdle muscular dystrophy type 2F (LGMD2F, δ-sarcoglycan deficiency), while deletion of the thrombospondin-4 gene exacerbated the diseases. Hence, thrombospondin-4 has been considered a candidate molecule for therapy of muscular dystrophies involving the DGC. We have investigated whether thrombospondin-4 could act as a genetic modifier for other DGC-associated diseases: limb-girdle muscular dystrophy type 2E (LGMD2E, ß-sarcoglycan deficiency) and laminin α2 chain-deficient muscular dystrophy (LAMA2-RD). Deletion of the thrombospondin-4 gene in mouse models for LGMD2E and LAMA2-RD, respectively, did not result in worsening of the dystrophic phenotype. Loss of thrombospondin-4 did not enhance sarcolemma damage and did not impair trafficking of transmembrane receptors integrin α7ß1 and dystroglycan in double knockout muscles. Our results suggest that thrombospondin-4 might not be a relevant therapeutic target for all muscular dystrophies involving the DGC. This data also demonstrates that molecular pathology between very similar diseases like LGMD2E and 2F can differ significantly.
Asunto(s)
Laminina , Ratones Noqueados , Sarcoglicanos , Trombospondinas , Animales , Laminina/metabolismo , Laminina/genética , Laminina/deficiencia , Sarcoglicanos/genética , Sarcoglicanos/deficiencia , Sarcoglicanos/metabolismo , Ratones , Trombospondinas/genética , Trombospondinas/metabolismo , Trombospondinas/deficiencia , Modelos Animales de Enfermedad , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Eliminación de Gen , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologíaRESUMEN
Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in CHKB, encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in Chkb -/- mice there is a failure of the α7ß1 integrin complex that is specific to affected muscle. We observed that in Chkb -/- hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P2 binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P2 reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of CHKB.
Asunto(s)
Colina Quinasa , Integrinas , Ratones Noqueados , Distrofias Musculares , Sarcolema , Vinculina , Animales , Ratones , Vinculina/metabolismo , Vinculina/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/genética , Integrinas/metabolismo , Colina Quinasa/metabolismo , Colina Quinasa/genética , Sarcolema/metabolismo , Humanos , Adhesiones Focales/metabolismo , Membrana Celular/metabolismo , Actinina/metabolismo , Actinina/genética , Músculo Esquelético/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Actinas/metabolismo , Modelos Animales de EnfermedadRESUMEN
Desminopathy R350P is a human myopathy that is characterized by the progressive loss of muscle fiber organization. This results in the loss of muscle size, mobility, and strength. In desminopathy, inflammation affects muscle homeostasis and repair, and contributes to progressive muscle deterioration. Mitochondria morphology was also suggested to affect desminopathy progression. Epicatechin (Epi)-a natural compound found in cacao-has been proposed to regulate inflammatory signaling and mitochondria morphology in human and animal models. Hence, we hypothesize chronic Epi consumption to improve inflammatory pathway and mitochondria morphology in the peripheral blood mononuclear cells (PBMCs) of a desminopathy R350P patient. We found that 12 weeks of Epi consumption partially restored TRL4 signaling, indicative of inflammatory signaling and mitochondria morphology in the desminopathy patient. Moreover, Epi consumption improved blood health parameters, including reduced HOMA-IR and IL-6 levels in the desminopathy patient. This indicates that Epi consumption could be a useful tool to slow disease progression in desminopathy patients.
Asunto(s)
Catequina , Leucocitos Mononucleares , Mitocondrias , Humanos , Catequina/farmacología , Catequina/administración & dosificación , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Masculino , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofias Musculares/tratamiento farmacológico , Distrofias Musculares/genética , Adulto , Femenino , Inflamación/metabolismo , Inflamación/patología , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/tratamiento farmacológico , Desmina/metabolismo , Desmina/genéticaRESUMEN
Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.
Asunto(s)
Diferenciación Celular , Movimiento Celular , Disferlina , Matriz Extracelular , Mioblastos , Sarcoglicanos , Animales , Mioblastos/metabolismo , Mioblastos/citología , Matriz Extracelular/metabolismo , Ratones , Sarcoglicanos/genética , Sarcoglicanos/metabolismo , Disferlina/genética , Disferlina/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofina/genética , Distrofina/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Decorina/genética , Decorina/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Músculo Esquelético/metabolismoRESUMEN
Cell therapies derived from induced pluripotent stem cells (iPSCs) offer a promising avenue in the field of regenerative medicine due to iPSCs' expandability, immune compatibility, and pluripotent potential. An increasing number of preclinical and clinical trials have been carried out, exploring the application of iPSC-based therapies for challenging diseases, such as muscular dystrophies. The unique syncytial nature of skeletal muscle allows stem/progenitor cells to integrate, forming new myonuclei and restoring the expression of genes affected by myopathies. This characteristic makes genome-editing techniques especially attractive in these therapies. With genetic modification and iPSC lineage specification methodologies, immune-compatible healthy iPSC-derived muscle cells can be manufactured to reverse the progression of muscle diseases or facilitate tissue regeneration. Despite this exciting advancement, much of the development of iPSC-based therapies for muscle diseases and tissue regeneration is limited to academic settings, with no successful clinical translation reported. The unknown differentiation process in vivo, potential tumorigenicity, and epigenetic abnormality of transplanted cells are preventing their clinical application. In this review, we give an overview on preclinical development of iPSC-derived myogenic cell transplantation therapies including processes related to iPSC-derived myogenic cells such as differentiation, scaling-up, delivery, and cGMP compliance. And we discuss the potential challenges of each step of clinical translation. Additionally, preclinical model systems for testing myogenic cells intended for clinical applications are described.
Asunto(s)
Células Madre Pluripotentes Inducidas , Distrofias Musculares , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Músculo Esquelético/fisiología , Distrofias Musculares/metabolismo , Tratamiento Basado en Trasplante de Células y Tejidos , Diferenciación CelularRESUMEN
LMNA-related congenital muscular dystrophy (L-CMD) is caused by mutations in the LMNA gene, encoding lamin A/C. To further understand the molecular mechanisms of L-CMD, proteomic profiling using DIA mass spectrometry was conducted on immortalized myoblasts and myotubes from controls and L-CMD donors each harbouring a different LMNA mutation (R249W, del.32 K and L380S). Compared to controls, 124 and 228 differentially abundant proteins were detected in L-CMD myoblasts and myotubes, respectively, and were associated with enriched canonical pathways including synaptogenesis and necroptosis in myoblasts, and Huntington's disease and insulin secretion in myotubes. Abnormal nuclear morphology and reduced lamin A/C and emerin abundance was evident in all L-CMD cell lines compared to controls, while nucleoplasmic aggregation of lamin A/C was restricted to del.32 K cells, and mislocalization of emerin was restricted to R249W cells. Abnormal nuclear morphology indicates loss of nuclear lamina integrity as a common feature of L-CMD, likely rendering muscle cells vulnerable to mechanically induced stress, while differences between L-CMD cell lines in emerin and lamin A localization suggests that some molecular alterations in L-CMD are mutation specific. Nonetheless, identifying common proteomic alterations and molecular pathways across all three L-CMD lines has highlighted potential targets for the development of non-mutation specific therapies.
Asunto(s)
Lamina Tipo A , Distrofias Musculares , Proteómica , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Mutación , Mioblastos/metabolismo , Masculino , Línea Celular , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
SNURPORTIN-1, encoded by SNUPN, plays a central role in the nuclear import of spliceosomal small nuclear ribonucleoproteins. However, its physiological function remains unexplored. In this study, we investigate 18 children from 15 unrelated families who present with atypical muscular dystrophy and neurological defects. Nine hypomorphic SNUPN biallelic variants, predominantly clustered in the last coding exon, are ascertained to segregate with the disease. We demonstrate that mutant SPN1 failed to oligomerize leading to cytoplasmic aggregation in patients' primary fibroblasts and CRISPR/Cas9-mediated mutant cell lines. Additionally, mutant nuclei exhibit defective spliceosomal maturation and breakdown of Cajal bodies. Transcriptome analyses reveal splicing and mRNA expression dysregulation, particularly in sarcolemmal components, causing disruption of cytoskeletal organization in mutant cells and patient muscle tissues. Our findings establish SNUPN deficiency as the genetic etiology of a previously unrecognized subtype of muscular dystrophy and provide robust evidence of the role of SPN1 for muscle homeostasis.
Asunto(s)
Distrofias Musculares , Niño , Humanos , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , ARN/metabolismo , Empalme del ARN/genética , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
Dystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, α-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane ß-DG whose, cytodomain binds to the actin cytoskeleton. Glycosylation of α-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of α-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. ß-DG acts as a scaffold for several cytoskeletal and nuclear proteins and very little is known about the fine regulation of some of these intracellular interactions and how they are perturbed in diseases. To start filling this gap by identifying uncharacterized intracellular networks preferentially associated with ß-DG, HEK-293 cells were transiently transfected with a plasmid carrying the ß-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing ß-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with α-DG. Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis led to the identification of an interactome formed by 313 exclusive protein matches for ß-DG binding. A series of already known ß-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel ß-DG interactors and their related networks, were identified in diverse subcellular compartments, such as cytoskeleton, endoplasmic reticulum/Golgi, mitochondria, nuclear membrane and the nucleus itself. Of particular interest amongst the novel identified matches, Lamina-Associated Polypeptide-1B (LAP1B), an inner nuclear membrane protein, whose mutations are known to cause nuclear envelopathies characterized by muscular dystrophy, was found to interact with ß-DG in HEK-293 cells. This evidence was confirmed by immunoprecipitation, Western blotting and immunofluorescence experiments. We also found by immunofluorescence experiments that LAP1B looses its nuclear envelope localization in C2C12 DG-knock-out cells, suggesting that LAP1B requires ß-DG for a proper nuclear localization. These results expand the role of ß-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy.
Asunto(s)
Distroglicanos , Distrofias Musculares , Humanos , Distroglicanos/química , Células HEK293 , Proteómica , Distrofias Musculares/metabolismo , Membrana Nuclear/metabolismoRESUMEN
Plectin, a highly versatile and multifunctional cytolinker, has been implicated in several multisystemic disorders. Most sequence variations in the human plectin gene (PLEC) cause epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), an autosomal recessive skin-blistering disorder associated with progressive muscle weakness. In this study, we performed a comprehensive cell biological analysis of dermal fibroblasts from three different patients with EBS-MD, where PLEC expression analyses revealed preserved mRNA levels in all cases, whereas full-length plectin protein content was significantly reduced or completely absent. Downstream effects of pathogenic PLEC sequence alterations included massive bundling of vimentin intermediate filament networks, including the occurrence of ring-like nuclei-encasing filament bundles, elongated mitochondrial networks, and abnormal nuclear morphologies. We found that essential fibroblast functions such as wound healing, migration, or orientation upon cyclic stretch were significantly impaired in the cells of patients with EBS-MD. Finally, EBS-MD fibroblasts displayed reduced adhesion capacities, which could be attributed to smaller focal adhesion contacts. Our study not only emphasizes plectin's functional role in human skin fibroblasts, it also provides further insights into the understanding of EBS-MD-associated disease mechanisms.
Asunto(s)
Epidermólisis Ampollosa Simple , Distrofia Muscular de Cinturas , Distrofias Musculares , Humanos , Filamentos Intermedios/metabolismo , Plectina/genética , Epidermólisis Ampollosa Simple/patología , Distrofias Musculares/complicaciones , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Mitocondrias/metabolismo , Fibroblastos/metabolismo , Proteínas de Filamentos Intermediarios/metabolismoRESUMEN
Pathogenetic mechanism recognition and proof-of-concept clinical trials were performed in our patients affected by collagen VI-related myopathies. This study, which included 69 patients, aimed to identify innovative clinical data to better design future trials. Among the patients, 33 had Bethlem myopathy (BM), 24 had Ullrich congenital muscular dystrophy (UCMD), 7 had an intermediate phenotype (INTM), and five had myosclerosis myopathy (MM). We obtained data on muscle strength, the degree of contracture, immunofluorescence, and genetics. In our BM group, only one third had a knee extension strength greater than 50% of the predicted value, while only one in ten showed similar retention of elbow flexion. These findings should be considered when recruiting BM patients for future trials. All the MM patients had axial and limb contractures that limited both the flexion and extension ranges of motion, and a limitation in mouth opening. The immunofluorescence analysis of collagen VI in 55 biopsies from 37 patients confirmed the correlation between collagen VI defects and the severity of the clinical phenotype. However, biopsies from the same patient or from patients with the same mutation taken at different times showed a progressive increase in protein expression with age. The new finding of the time-dependent modulation of collagen VI expression should be considered in genetic correction trials.
Asunto(s)
Contractura , Distrofias Musculares , Miopatías Estructurales Congénitas , Humanos , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Distrofias Musculares/metabolismo , Contractura/genética , Contractura/patología , MutaciónRESUMEN
Infancy- and childhood-onset muscular dystrophies are associated with a characteristic distribution and progression of motor dysfunction. The underlying causes of progressive childhood muscular dystrophies are heterogeneous involving diverse genetic pathways and genes that encode proteins of the plasma membrane, extracellular matrix, sarcomere, and nuclear membrane components. The prototypical clinicopathological features in an affected child may be adequate to fully distinguish it from other likely diagnoses based on four common features: (1) weakness and wasting of pelvic-femoral and scapular muscles with involvement of heart muscle; (2) elevation of serum muscle enzymes in particular serum creatine kinase; (3) necrosis and regeneration of myofibers; and (4) molecular neurogenetic assessment particularly utilizing next-generation sequencing of the genome of the likeliest candidates genes in an index case or family proband. A number of different animal models of therapeutic strategies have been developed for gene transfer therapy, but so far these techniques have not yet entered clinical practice. Treatment remains for the most part symptomatic with the goal of ameliorating locomotor and cardiorespiratory manifestations of the disease.
Asunto(s)
Distrofias Musculares , Animales , Niño , Humanos , Distrofias Musculares/genética , Distrofias Musculares/terapia , Distrofias Musculares/metabolismo , ProteínasRESUMEN
Tetraspanins organize protein complexes at the cell membrane and are responsible for assembling diverse binding partners in changing cellular states. Tetraspanin CD82 is a useful cell surface marker for prospective isolation of human myogenic progenitors and its expression is decreased in Duchenne muscular dystrophy (DMD) cell lines. The function of CD82 in skeletal muscle remains elusive, partly because the binding partners of this tetraspanin in muscle cells have not been identified. CD82-associated proteins are sought to be identified in human myotubes via mass spectrometry proteomics, which identifies dysferlin and myoferlin as CD82-binding partners. In human dysferlinopathy (Limb girdle muscular dystrophy R2, LGMDR2) myogenic cell lines, expression of CD82 protein is near absent in two of four patient samples. In the cell lines where CD82 protein levels are unaffected, increased expression of the ≈72 kDa mini-dysferlin product is identified using an antibody recognizing the dysferlin C-terminus. These data demonstrate that CD82 binds dysferlin/myoferlin in differentiating muscle cells and its expression can be affected by loss of dysferlin in human myogenic cells.
Asunto(s)
Proteínas Musculares , Distrofias Musculares , Humanos , Disferlina/genética , Proteína Kangai-1 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofias Musculares/metabolismo , TetraspaninasRESUMEN
Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2 F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of ß-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.
Asunto(s)
Distrofia Muscular de Cinturas , Distrofias Musculares , Ratones , Animales , Sarcoglicanos/genética , Sarcoglicanos/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Músculo Esquelético/fisiología , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Ratones NoqueadosRESUMEN
BACKGROUND: Congenital muscular dystrophies (CMDs) result from genetically inherited defects in the biosynthesis and/or the posttranslational modification (glycosylation) of laminin-α2 and α-dystroglycan (α-DG), respectively. The interaction between both proteins is responsible for the stability and integrity of the muscle cell. We aimed to study the expression profiles of both proteins in two classes of CMDs. SUBJECTS AND METHODS: Whole-exome sequencing (WES) was done for four patients with neuromuscular manifestations. The expression of core α-DG and laminin-α2 subunit in skin fibroblasts and MCF-7 cells was assessed by western blot. RESULTS: WES revealed two cases with nonsense mutations; c.2938G > T and c.4348 C > T, in LAMA2 encodes laminin-α2. It revealed also two cases with mutations in POMGNT1 encode protein O-mannose beta-1,2-N-acetylglucosaminyltransferase mutations. One patient had a missense mutation c.1325G > A, and the other had a synonymous variant c.636 C > T. Immunodetection of core α-DG in skin fibroblasts revealed the expression of truncated forms of core α-DG accompanied by reduced expression of laminin-α2 in POMGNT1-CMD patients and one patient with LAMA2-CMD. One patient with LAMA2-CMD had overexpression of laminin-α2 and expression of a low level of an abnormal form of increased molecular weight core α-DG. MCF-7 cells showed truncated forms of core α-CDG with an absent laminin-α2. CONCLUSION: A correlation between the expression pattern/level of core α-DG and laminin-α2 could be found in patients with different types of CMD.
Asunto(s)
Laminina , Distrofias Musculares , Humanos , Distroglicanos/genética , Distroglicanos/metabolismo , Fibroblastos/metabolismo , Laminina/genética , Distrofias Musculares/genética , Distrofias Musculares/complicaciones , Distrofias Musculares/metabolismo , Mutación/genéticaRESUMEN
Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by a CTG expansion resulting in significant transcriptomic dysregulation that leads to muscle weakness and wasting. While strength training is clinically beneficial in DM1, molecular effects had not been studied. To determine whether training rescued transcriptomic defects, RNA-Seq was performed on vastus lateralis samples from 9 male patients with DM1 before and after a 12-week strength-training program and 6 male controls who did not undergo training. Differential gene expression and alternative splicing analysis were correlated with the one-repetition maximum strength evaluation method (leg extension, leg press, hip abduction, and squat). While training program-induced improvements in splicing were similar among most individuals, rescued splicing events varied considerably between individuals. Gene expression improvements were highly varied between individuals, and the percentage of differentially expressed genes rescued after training were strongly correlated with strength improvements. Evaluating transcriptome changes individually revealed responses to the training not evident from grouped analysis, likely due to disease heterogeneity and individual exercise response differences. Our analyses indicate that transcriptomic changes are associated with clinical outcomes in patients with DM1 undergoing training and that these changes are often specific to the individual and should be analyzed accordingly.
Asunto(s)
Distrofias Musculares , Distrofia Miotónica , Entrenamiento de Fuerza , Adulto , Humanos , Masculino , Distrofia Miotónica/genética , Distrofia Miotónica/terapia , Músculo Esquelético/metabolismo , Transcriptoma , Distrofias Musculares/metabolismoRESUMEN
Human TOR1AIP1 encodes LAP1, a nuclear envelope protein expressed in most human tissues, which has been linked to various biological processes and human diseases. The clinical spectrum of diseases related to mutations in TOR1AIP1 is broad, including muscular dystrophy, congenital myasthenic syndrome, cardiomyopathy, and multisystemic disease with or without progeroid features. Although rare, these recessively inherited disorders often lead to early death or considerable functional impairment. Developing a better understanding of the roles of LAP1 and mutant TOR1AIP1-associated phenotypes is paramount to allow therapeutic development. To facilitate further studies, this review provides an overview of the known interactions of LAP1 and summarizes the evidence for the function of this protein in human health. We then review the mutations in the TOR1AIP1 gene and the clinical and pathological characteristics of subjects with these mutations. Lastly, we discuss challenges to be addressed in the future.
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Proteínas del Citoesqueleto , Proteínas de la Membrana , Distrofias Musculares , Humanos , Proteínas de la Membrana/metabolismo , Distrofias Musculares/metabolismo , Mutación , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas del Citoesqueleto/metabolismoRESUMEN
Sarcopenia is a geriatric syndrome characterized by an age-related decline in skeletal muscle mass and strength. Here, we show that suppression of mitochondrial calcium uniporter (MCU)-mediated Ca2+ influx into mitochondria in the body wall muscles of the nematode Caenorhabditis elegans improved the sarcopenic phenotypes, blunting movement and mitochondrial structural and functional decline with age. We found that normally aged muscle cells exhibited elevated resting mitochondrial Ca2+ levels and increased mitophagy to eliminate damaged mitochondria. Similar to aging muscle, we found that suppressing MCU function in muscular dystrophy improved movement via reducing elevated resting mitochondrial Ca2+ levels. Taken together, our results reveal that elevated resting mitochondrial Ca2+ levels contribute to muscle decline with age and muscular dystrophy. Further, modulation of MCU activity may act as a potential pharmacological target in various conditions involving muscle loss.
Asunto(s)
Distrofias Musculares , Sarcopenia , Animales , Caenorhabditis elegans , Mitocondrias/patología , Músculo Esquelético/metabolismo , Sarcopenia/patología , Distrofias Musculares/metabolismo , Calcio/metabolismoRESUMEN
The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin α2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.
Asunto(s)
Laminina , Distrofias Musculares , Humanos , Laminina/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Técnicas de Cultivo de Célula , Distrofias Musculares/metabolismo , Feto , Colágeno Tipo IV , Músculo EsqueléticoRESUMEN
Null mutations of the Lama2-gene cause a severe congenital muscular dystrophy and associated neuropathy. In the absence of laminin-α2 (Lmα2) there is a compensatory replacement by Lmα4, a subunit that lacks the polymerization and α-dystroglycan (αDG)-binding properties of Lmα2. The dystrophic phenotype in the dy3K/dy3K Lama2-/- mouse were evaluated with transgenes driving expression of two synthetic laminin-binding linker proteins. Transgenic muscle-specific expression of αLNNd, a chimeric protein that enables α4-laminin polymerization, and miniagrin (mag), a protein that increases laminin binding to the receptor αDG, separately improved median mouse survival two-fold. The double transgenes (DT) improved mean survival three-fold with increases in overall body weight, muscle size, and grip strength, but, given absence of neuronal expression, did not prevent hindlimb paresis. Muscle improvements included increased myofiber size and number and reduced fibrosis. Myofiber hypertrophy with increased mTOR and Akt phosphorylation were characteristics of mag-dy3K/dy3K and DT-dy3K/dy3K muscle. Elevations of matrix-bound α4-, ß1 and γ1 laminin subunits were detected in muscle extracts and immunostained sections in response to DT expression. Collectively, these findings reveal a complimentary polymerization and αDG-binding benefit to Lama2-/- mouse muscle largely mediated through modified laminin-411.