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1.
Int J Mol Sci ; 25(11)2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38891760

RESUMO

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.


Assuntos
Disferlina , Terapia Genética , Distrofia Muscular do Cíngulo dos Membros , Mutação , Humanos , Distrofia Muscular do Cíngulo dos Membros/terapia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Disferlina/genética , Disferlina/metabolismo , Terapia Genética/métodos , Oligonucleotídeos Antissenso/uso terapêutico , Animais
2.
J Physiol ; 602(9): 1893-1910, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38615232

RESUMO

Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.


Assuntos
Cálcio , Disferlina , Humanos , Cálcio/metabolismo , Disferlina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Músculo Estriado/metabolismo , Músculo Estriado/fisiologia
3.
Matrix Biol ; 129: 44-58, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38582404

RESUMO

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.


Assuntos
Diferenciação Celular , Movimento Celular , Disferlina , Matriz Extracelular , Mioblastos , Sarcoglicanas , Animais , Mioblastos/metabolismo , Mioblastos/citologia , Matriz Extracelular/metabolismo , Camundongos , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Disferlina/genética , Disferlina/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Distrofina/genética , Distrofina/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Decorina/genética , Decorina/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Músculo Esquelético/metabolismo
4.
J Mol Biol ; 435(17): 168193, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37406927

RESUMO

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.


Assuntos
Proteínas de Ligação ao Cálcio , Cálcio , Disferlina , Proteínas de Membrana , Fosfatidilinositol 4,5-Difosfato , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Disferlina/química , Disferlina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Domínios C2 , Ligação Proteica , Fosfatidilinositol 4,5-Difosfato/química
5.
Bull Exp Biol Med ; 174(6): 768-773, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37160600

RESUMO

We studied the effects of a dual-vector DYSF gene delivery system based on adeno-associated virus serotype 9 capsids on pathological manifestations of dysferlinopathy in skeletal muscles of Bla/J mice lacking DYSF expression. The mice received intravenous injection of 3×1013 genomic copies of the virus containing the dual-vector system. M. gastrocnemius, m. psoas major, m. vastus lateralis, and m. gluteus superficialis were isolated for histological examination in 3, 6, and 12 weeks after treatment. Healthy wild-type (C57BL/6) mice served as positive control and were sacrificed 3 weeks after injection of 150 µl of 0.9% NaCl into the caudal vein. To detect dysferlin in muscle cryosections, immunohistochemical analysis with diagnostic antibodies was performed; paraffin sections were stained with hematoxylin and eosin for morphometric analysis. After administration of gene-therapeutic constructs, muscle fibers with membrane or cytoplasmic dysferlin location were detected in all examined muscles. The proportion of necrotic muscle fibers decreased, the number of muscle fibers with central location of the nucleus increased, and the mean cross-section area of the muscle fibers decreased.


Assuntos
Músculo Esquelético , Distrofia Muscular do Cíngulo dos Membros , Camundongos , Animais , Disferlina/genética , Disferlina/metabolismo , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/terapia , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Técnicas de Transferência de Genes
6.
Int J Mol Sci ; 24(5)2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-36902136

RESUMO

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca2+ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair.


Assuntos
Proteínas Reguladoras de Apoptose , Proteínas de Ligação ao Cálcio , Cálcio , Disferlina , Músculo Esquelético , Regeneração , Proteínas de Ligação a Tacrolimo , Anexina A1/metabolismo , Cálcio/metabolismo , Calpaína/metabolismo , Caveolina 3/metabolismo , Disferlina/metabolismo , Músculo Esquelético/lesões , Músculo Esquelético/fisiologia , Sarcolema/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Ressonância de Plasmônio de Superfície , Proteínas de Ligação a Tacrolimo/metabolismo , Apoptose , Transdução de Sinais , Animais , Ratos
7.
J Phys Chem B ; 127(2): 577-589, 2023 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-36608331

RESUMO

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its ß-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its ß-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.


Assuntos
Membrana Celular , Disferlina , Humanos , Membrana Celular/química , Disferlina/química , Disferlina/metabolismo , Lipídeos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Ligação Proteica , Sarcolema/química
8.
Acta Neuropathol Commun ; 11(1): 15, 2023 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653852

RESUMO

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.


Assuntos
Proteínas de Membrana , Distrofia Muscular do Cíngulo dos Membros , Camundongos , Animais , Disferlina/genética , Disferlina/metabolismo , Camundongos Knockout , Proteínas de Membrana/metabolismo , Calpaína/genética , Proteômica , Distrofia Muscular do Cíngulo dos Membros/patologia , Músculo Esquelético/patologia , Éxons/genética
9.
Cells ; 11(20)2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36291129

RESUMO

The plasma membrane (sarcolemma) of skeletal muscle myofibers is susceptible to injury caused by physical and chemical stresses during normal daily movement and/or under disease conditions. These acute plasma membrane disruptions are normally compensated by an intrinsic membrane resealing process involving interactions of multiple intracellular proteins including dysferlin, annexin, caveolin, and Mitsugumin 53 (MG53)/TRIM72. There is new evidence for compromised muscle sarcolemma repair mechanisms in Amyotrophic Lateral Sclerosis (ALS). Mitochondrial dysfunction in proximity to neuromuscular junctions (NMJs) increases oxidative stress, triggering MG53 aggregation and loss of its function. Compromised membrane repair further worsens sarcolemma fragility and amplifies oxidative stress in a vicious cycle. This article is to review existing literature supporting the concept that ALS is a disease of oxidative-stress induced disruption of muscle membrane repair that compromise the integrity of the NMJs and hence augmenting muscle membrane repair mechanisms could represent a viable therapeutic strategy for ALS.


Assuntos
Esclerose Lateral Amiotrófica , Regeneração , Sarcolema , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Anexinas/metabolismo , Proteínas de Transporte/metabolismo , Caveolinas/metabolismo , Disferlina/metabolismo , Proteínas de Membrana/metabolismo , Sarcolema/metabolismo , Sarcolema/patologia
10.
Ultrastruct Pathol ; 46(4): 359-367, 2022 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-35880824

RESUMO

A number of sarcolemma proteins are responsible for muscle fiber repair. Dysferlin encoded by the DYSF gene is one of these proteins. Dysferlin promotes membrane repair in striated muscle fibers (MFs). Mutations in DYSF lead to loss of or decreased dysferlin expression, impaired membrane repair in MF, and its destruction, clinically manifesting as dysferlinopathy. Preclinical studies of cell and gene therapies aimed at restoring impaired muscle regeneration require well-characterized small animal models. Our investigation aimed to distinguish the histopathological features of a mouse strain lacking dysferlin expression (Bla/J strain). Ultrastructural changes in the sarcolemma, mitochondria and contractile apparatus were observed. It was shown that postnatal histogenesis of skeletal muscles in genetically determined dysferlin deficiency is characterized by a higher proportion of necrotic muscle fibers, compensatory hypertrophy of muscle fibers with their subsequent atrophy, and decreases in proliferative activity and the level of myogenic differentiation of myogenic progenitor cells compared to wild-type mice (C57Bl/6).


Assuntos
Disferlina , Músculo Esquelético , Distrofia Muscular do Cíngulo dos Membros , Animais , Disferlina/genética , Disferlina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia
11.
Muscle Nerve ; 66(4): 513-522, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35859452

RESUMO

INTRODUCTION/AIMS: Most mouse models of muscular dystrophy (MD) show mild phenotypes, which limits the translatability of experimental therapies to patients. A growing body of evidence suggests that MD is accompanied by metabolic abnormalities that could potentially exacerbate the primary muscle wasting process. Since thermoneutral (TN) housing of mice (~30°C) has been shown to affect many metabolic parameters, particularly when combined with a Western diet (WD), our aim was to determine whether the combination of TN and WD exacerbates muscle wasting in dysferlin-deficient BLAJ mice, a common model of limb-girdle MD type 2b (LGMD2b). METHODS: The 2-mo-old wild-type (WT) and BLAJ mice were housed at TN or room temperature (RT) and fed a WD or regular chow for 9 mo. Ambulatory function, muscle histology, and protein immunoblots of skeletal muscle were assessed. RESULTS: BLAJ mice at RT and fed a chow diet showed normal ambulation function similar to WT mice, whereas 90% of BLAJ mice under WD and TN combination showed ambulatory dysfunction (p < 0.001), and an up to 4.1-fold increase in quadriceps and gastrocnemius fat infiltration. Western blotting revealed decreased autophagy marker microtubules-associated protein 1 light chain 3-B (LC3BII/LC3BI) ratio and up-regulation of protein kinase B/AKT and ribosomal protein S6 phosphorylation, suggesting inefficient cellular debris and protein clearance in TN BLAJ mice fed a WD. Male and female BLAJ mice under TN and WD combination showed heterogenous fibro-fatty infiltrate composition. DISCUSSION: TN and WD combination exacerbates rodent LGMD2b without affecting WT mice. This improves rodent modeling of human MD and helps elucidate how metabolic abnormalities may play a causal role in muscle wasting.


Assuntos
Distrofia Muscular do Cíngulo dos Membros , Distrofias Musculares , Animais , Dieta Ocidental/efeitos adversos , Disferlina/genética , Disferlina/metabolismo , Feminino , Habitação , Humanos , Masculino , Camundongos , Músculo Esquelético , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Distrofias Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo
12.
Transl Res ; 247: 19-38, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35460889

RESUMO

Dysferlin (DYSF) has drawn much attention due to its involvement in dysferlinopathy and was reported to affect monocyte functions in recent studies. However, the role of DYSF in the pathogenesis of atherosclerotic cardiovascular diseases (ASCVD) and the regulation mechanism of DYSF expression have not been fully studied. In this study, Gene Expression Omnibus (GEO) database and epigenome-wide association study (EWAS) literatures were searched to find the DNA methylation-driven genes (including DYSF) of ASCVD. The hub genes related to DYSF were also identified through weighted correlation network analysis (WGCNA). Regulation of DYSF expression through its promoter methylation status was verified using peripheral blood leucocytes (PBLs) from ASCVD patients and normal controls, and experiments on THP1 cells and Apoe-/- mice. Similarly, the expressions of DYSF related hub genes, mainly contained SELL, STAT3 and TMX1, were also validated. DYSF functions were then evaluated by phagocytosis, transwell and adhesion assays in DYSF knock-down and overexpressed THP1 cells. The results showed that DYSF promoter hypermethylation up-regulated its expression in clinical samples, THP1 cells and Apoe-/- mice, confirming DYSF as a DNA methylation-driven gene. The combination of DYSF expression and methylation status in PBLs had a considerable prediction value for ASCVD. Besides, DYSF could enhance the phagocytosis, migration and adhesion ability of THP1 cells. Among DYSF related hub genes, SELL was proven to be the downstream target of DYSF by wet experiments. In conclusion, DYSF promoter hypermethylation upregulated its expression and promoted monocytes activation, which further participated in the pathogenesis of ASCVD.


Assuntos
Aterosclerose , Doenças Cardiovasculares , Metilação de DNA , Disferlina , Animais , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Doenças Cardiovasculares/metabolismo , Disferlina/genética , Disferlina/metabolismo , Humanos , Camundongos , Monócitos/metabolismo
13.
Cells ; 10(11)2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34831431

RESUMO

Two of the main pathologies characterizing dysferlinopathies are disrupted muscle membrane repair and chronic inflammation, which lead to symptoms of muscle weakness and wasting. Here, we used recombinant human Galectin-1 (rHsGal-1) as a therapeutic for LGMD2B mouse and human models. Various redox and multimerization states of Gal-1 show that rHsGal-1 is the most effective form in both increasing muscle repair and decreasing inflammation, due to its monomer-dimer equilibrium. Dose-response testing shows an effective 25-fold safety profile between 0.54 and 13.5 mg/kg rHsGal-1 in Bla/J mice. Mice treated weekly with rHsGal-1 showed downregulation of canonical NF-κB inflammation markers, decreased muscle fat deposition, upregulated anti-inflammatory cytokines, increased membrane repair, and increased functional movement compared to non-treated mice. Gal-1 treatment also resulted in a positive self-upregulation loop of increased endogenous Gal-1 expression independent of NF-κB activation. A similar reduction in disease pathologies in patient-derived human cells demonstrates the therapeutic potential of Gal-1 in LGMD2B patients.


Assuntos
Galectina 1/uso terapêutico , Distrofia Muscular do Cíngulo dos Membros/patologia , Animais , Biomarcadores/metabolismo , Citocinas/metabolismo , Disferlina/deficiência , Disferlina/metabolismo , Humanos , Inflamação/patologia , Masculino , Membranas , Camundongos , Fibras Musculares Esqueléticas/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais
14.
Sci Rep ; 11(1): 15865, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354129

RESUMO

Muscular dystrophies are disorders characterized by progressive muscle loss and weakness that are both genotypically and phenotypically heterogenous. Progression of muscle disease arises from impaired regeneration, plasma membrane instability, defective membrane repair, and calcium mishandling. The ferlin protein family, including dysferlin and myoferlin, are calcium-binding, membrane-associated proteins that regulate membrane fusion, trafficking, and tubule formation. Mice lacking dysferlin (Dysf), myoferlin (Myof), and both dysferlin and myoferlin (Fer) on an isogenic inbred 129 background were previously demonstrated that loss of both dysferlin and myoferlin resulted in more severe muscle disease than loss of either gene alone. Furthermore, Fer mice had disordered triad organization with visibly malformed transverse tubules and sarcoplasmic reticulum, suggesting distinct roles of dysferlin and myoferlin. To assess the physiological role of disorganized triads, we now assessed excitation contraction (EC) coupling in these models. We identified differential abnormalities in EC coupling and ryanodine receptor disruption in flexor digitorum brevis myofibers isolated from ferlin mutant mice. We found that loss of dysferlin alone preserved sensitivity for EC coupling and was associated with larger ryanodine receptor clusters compared to wildtype myofibers. Loss of myoferlin alone or together with a loss of dysferlin reduced sensitivity for EC coupling, and produced disorganized and smaller ryanodine receptor cluster size compared to wildtype myofibers. These data reveal impaired EC coupling in Myof and Fer myofibers and slightly potentiated EC coupling in Dysf myofibers. Despite high homology, dysferlin and myoferlin have differential roles in regulating sarcotubular formation and maintenance resulting in unique impairments in calcium handling properties.


Assuntos
Disferlina/metabolismo , Acoplamento Excitação-Contração/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Disferlina/genética , Feminino , Masculino , Fusão de Membrana/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos da Linhagem 129 , Contração Muscular/fisiologia , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Distrofias Musculares/fisiopatologia
15.
Stem Cell Reports ; 16(4): 985-996, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33711268

RESUMO

Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Oligodesoxirribonucleotídeos/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Alelos , Antibacterianos/farmacologia , Sequência de Bases , Miopatias Distais/genética , Miopatias Distais/terapia , Disferlina/genética , Disferlina/metabolismo , Éxons/genética , Edição de Genes , Células HEK293 , Haplótipos/genética , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/terapia , Distrofia Muscular de Duchenne/genética , Mutagênese Insercional/genética , Mutação/genética , Splicing de RNA/genética , RNA Guia de Cinetoplastídeos/metabolismo , Ribonucleases/metabolismo
16.
Biochem J ; 478(1): 197-215, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33449082

RESUMO

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.


Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Disferlina/química , Proteínas Musculares/química , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cristalografia por Raios X , Disferlina/genética , Disferlina/metabolismo , Expressão Gênica , Modelos Moleculares , Proteínas Musculares/metabolismo , Mutagênese , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Eletricidade Estática
17.
J Phys Chem B ; 125(1): 148-157, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33355462

RESUMO

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain ß-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.


Assuntos
Cálcio , Proteínas de Membrana , Cálcio/metabolismo , Análise por Conglomerados , Disferlina/genética , Disferlina/metabolismo , Lipídeos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Ligação Proteica
18.
FEBS J ; 288(1): 160-174, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32893434

RESUMO

Plasma membrane repair is an evolutionarily conserved mechanism by which cells can seal breaches in the plasma membrane. Mutations in several proteins with putative roles in sarcolemma integrity, membrane repair, and membrane transport result in several forms of muscle disease; however, the mechanisms that are activated and responsible for sarcolemma resealing are not well understood. Using the standard assays for membrane repair, which track the uptake of FM 1-43 dye into adult skeletal muscle fibers following laser-induced sarcolemma disruption, we show that labeling of resting fibers by FM1-43 prior to membrane wounding and the induced FM1-43 dye uptake after sarcolemma wounding occurs via dynamin-dependent endocytosis. Dysferlin-deficient muscle fibers show elevated dye uptake following wounding, which is the basis for the assertion that membrane repair is defective in this model. Our data show that dynamin inhibition mitigates the differences in FM1-43 dye uptake between dysferlin-null and wild-type muscle fibers, suggesting that elevated wound-induced FM1-43 uptake in dysferlin-deficient muscle may actually be due to enhanced dynamin-dependent endocytosis following wounding, though dynamin inhibition had no effect on dysferlin trafficking after wounding. By monitoring calcium flux after membrane wounding, we show that reversal of calcium precedes the sustained, slower increase of dynamin-dependent FM1-43 uptake in WT fibers, and that dysferlin-deficient muscle fibers have persistently increased calcium after wounding, consistent with its proposed role in resealing. These data highlight a previously unappreciated role for dynamin-dependent endocytosis in wounded skeletal muscle fibers and identify overactive dynamin-dependent endocytosis following sarcolemma wounding as a potential mechanism or consequence of dysferlin deficiency.


Assuntos
Cálcio/metabolismo , Dinaminas/genética , Disferlina/genética , Endocitose/genética , Sarcolema/genética , Animais , Animais Geneticamente Modificados , Dimetil Sulfóxido/farmacologia , Dinaminas/metabolismo , Disferlina/metabolismo , Corantes Fluorescentes/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrazonas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Sarcolema/patologia , Coloração e Rotulagem/métodos
19.
Muscle Nerve ; 63(2): 239-249, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33125736

RESUMO

INTRODUCTION: We conducted an open-label study to examine the effects of the flavonoid (-)-epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD). METHODS: Seven participants received (-)-epicatechin 50 mg twice per day for 8 weeks. Pre- and postprocedures included biceps brachii biopsy to assess muscle structure and growth-relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests. RESULTS: Western blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5'-adenosine monophosphate-activated protein kinase). Peroxisome proliferator-activated receptor gamma coactivator-1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae-associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor-5, myoblast determination protein 1, myocyte enhancer factor-2, and structure-associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states. DISCUSSION: (-)-Epicatechin, an exercise mimetic, appears to have short-term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.


Assuntos
Catequina/uso terapêutico , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , Adulto , Biópsia , Western Blotting , Creatina Quinase/metabolismo , Disferlina/metabolismo , Teste de Esforço , Folistatina/metabolismo , Frequência Cardíaca , Humanos , Ácido Láctico/sangue , Fatores de Transcrição MEF2/metabolismo , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Tamanho Mitocondrial , Proteínas Musculares/metabolismo , Força Muscular , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/metabolismo , Miogenina/metabolismo , Miostatina/metabolismo , Biogênese de Organelas , Consumo de Oxigênio , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regeneração , Utrofina/metabolismo
20.
PLoS One ; 15(9): e0238441, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881965

RESUMO

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity and myogenic potential of dysferlin-deficient muscle cells and muscle fibers. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, myoblast migration, and membrane repair capacity in dysferlin-deficient myotubes and myofibers. Furthermore, increased membrane repair capacity of dysferlin-deficient myotubes, independent of increased myogenic maturation is apparent and co-localizes on the membrane of myotubes after a brief 10min treatment with labeled rHsGal-1. We show the carbohydrate recognition domain of Gal-1 is necessary for observed membrane repair. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggest mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-1 shows calcium-independent membrane repair in dysferlin-deficient and wild-type myotubes and myofibers. Together our novel results reveal Gal-1 mediates disease pathologies through both changes in integral myogenic protein expression and mechanical membrane stabilization.


Assuntos
Disferlina/genética , Galectina 1/farmacologia , Distrofia Muscular do Cíngulo dos Membros/terapia , Animais , Linhagem Celular , Modelos Animais de Doenças , Disferlina/metabolismo , Galectina 1/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Miofibrilas/metabolismo
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