RARE DISEASE
Miyoshi myopathy
Miyoshi myopathy
Miyoshi myopathy
Drug discovery
0
drugs
With orphan designations
Overview
Miyoshi myopathy is an autosomal recessive dysferlinopathy caused by DYSF mutations, leading to dysferlin deficiency and impaired muscle membrane repair. It typically presents in adolescence/early adulthood (median onset: 19 years) with distal calf weakness, difficulty standing on toes, and marked CK elevation (20–150× normal). Progressive atrophy spreads proximally, with possible wheelchair dependence within 10–30 years. Diagnosis combines genetic testing, muscle biopsy (dysferlin staining), and MRI showing fatty calf infiltration [1][6][15].
Categories: rare genetic diseases, rare neurological diseases
Research Papers
92 drug discovery papers about Miyoshi myopathy, with 1 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
92 drug discovery papers about Miyoshi myopathy, with 1 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
small molecules
2024-08-20 | Brain of miyoshi myopathy/dysferlinopathy patients presents with structural and metabolic anomalies.
Miyoshi myopathy/dysferlinopathy (MMD) is a rare muscle disease caused by DYSF gene mutations. Apart from skeletal muscles, DYSF is also expressed in the brain. However, the impact of MMD-causing DYSF variants on brain structure and function remains unexplored. To investigate this, we utilized magnetic resonance (MR) modalities (MR volumetry and 31P MR spectroscopy) in a family with seven children, four of whom have the illness. The MMD siblings showed distinct differences from healthy controls: (1) a significant (p < 0.001) right-sided volume asymmetry (+ 232 mm3) of the inferior lateral ventricles; and (2) a significant (p < 0.001) decrease in [Mg2+], along with a modified energy metabolism profile and altered membrane turnover in the hippocampus and motor and premotor cortices. The patients' [Mg2+], energy metabolism, and membrane turnover measures returned to those of healthy relatives after a month of 400 mg/day magnesium supplementation. This work is the first to describe anatomical and functional abnormalities characteristic of neurodegeneration in the MMD brain. Therefore, we call for further examination of brain functions in larger cohorts of MMD patients and testing of magnesium supplementation, which has proven to be an effective corrective approach in our study.
2022-11-19 | Inhibition of the immunoproteasome modulates innate immunity to ameliorate muscle pathology of dysferlin-deficient BlAJ mice
Abstract Muscle repair in dysferlinopathies is defective. Although macrophage (Mø)-rich infiltrates are prominent in damaged skeletal muscles of patients with dysferlinopathy, the contribution of the immune system to the disease pathology remains to be fully explored. Numbers of both pro-inflammatory M1 Mø and effector T cells are increased in muscle of dysferlin-deficient BlAJ mice. In addition, symptomatic BlAJ mice have increased muscle production of immunoproteasome. In vitro analyses using bone marrow-derived Mø of BlAJ mice show that immunoproteasome inhibition results in C3aR1 and C5aR1 downregulation and upregulation of M2-associated signaling. Administration of immunoproteasome inhibitor ONX-0914 to BlAJ mice rescues muscle function by reducing muscle infiltrates and fibro-adipogenesis. These findings reveal an important role of immunoproteasome in the progression of muscular dystrophy in BlAJ mouse and suggest that inhibition of immunoproteasome may produce therapeutic benefit in dysferlinopathy.
2019-07-18 | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells
Abstract Autosomal recessive mutations in Anoctamin 5 ( ANO5/TMEM16E ), a member of the transmembrane 16 (TMEM16) family of Ca 2+ -activated ion channels and phospholipid scramblases, cause adult-onset muscular dystrophies (limb girdle muscular dystrophy 2L (LGMD2L) and Miyoshi Muscular Dystrophy (MMD3). However, the molecular role of ANO5 is unclear and ANO5 knockout mouse models show conflicting requirements of ANO5 in muscle. To study the role of ANO5 in human muscle cells we generated a myoblast line from a MMD3-patient carrying the c.2272C>T mutation, which we find causes the mutant protein to be degraded. The patient myoblasts exhibit normal myogenesis, but are compromised in their plasma membrane repair (PMR) ability. The repair deficit is linked to the poor ability of the endoplasmic reticulum (ER) to clear cytosolic Ca 2+ increase caused by focal plasma membrane injury. Expression of wild-type ANO5 or pharmacological prevention of injury-triggered cytosolic Ca 2+ overload enable injured patient muscle cells to repair. A homology model of ANO5 shows that several of the known LGMD2L/MMD3 patient mutations line the transmembrane region of the protein implicated in its channel activity. These results point to a role of cytosolic Ca 2+ homeostasis in PMR, indicate a role for ANO5 in ER-mediated cytosolic Ca 2+ uptake and identify normalization of cytosolic Ca 2+ homeostasis as a potential therapeutic approach to treat muscular dystrophies caused by ANO5 deficit.
2019-06-28 | Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells
Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.
2018-09-05 | LIMB-GIRDLE MUSCULAR DYSTROPHY I
Defects in dysferlin gene cause limb-girdle muscular dystrophy 2B or Miyoshi distal myopathy. Nonsense mutations have been reported to be most common among Korean patients with dysferlinopathy. More than half of the patients have at least one nonsense allele in dysferlin. Readthrough by ataluren is a promising strategy to overcome nonsense mutation, in use for the nonsense-mediated Duchenne muscular dystrophy. We generated a knock-in mouse with p.Q832* mutation, most frequently found among our dysferlinopathy cases. Mice with homozygous p.Q832* mutation lost dysferlin expression at sarcolemma. It performed worse on the Rotarod and forelimb grip test than wild type C57BL6 mice and moved less on activity monitoring. On eccentric contraction ex vivo, extensor digitorum longus muscle from these mice tended to break easily. These abnormalities could successfully be alleviated when the mice were orally administered with ataluren in liquid. Furthermore, ataluren decreased the number of damaged muscle, fiber size variation and serum creatine kinase level. This results support that readthrough strategy with ataluren will also be applicable to patients with nonsense dysferlin mutation.
gene therapies
2024-03-29 | The Dysferlinopathies Conundrum: Clinical Spectra, Disease Mechanism and Genetic Approaches for Treatments.
Dysferlinopathies refer to a spectrum of muscular dystrophies that cause progressive muscle weakness and degeneration. They are caused by mutations in the DYSF gene, which encodes the dysferlin protein that is crucial for repairing muscle membranes. This review delves into the clinical spectra of dysferlinopathies, their molecular mechanisms, and the spectrum of emerging therapeutic strategies. We examine the phenotypic heterogeneity of dysferlinopathies, highlighting the incomplete understanding of genotype-phenotype correlations and discussing the implications of various DYSF mutations. In addition, we explore the potential of symptomatic, pharmacological, molecular, and genetic therapies in mitigating the disease's progression. We also consider the roles of diet and metabolism in managing dysferlinopathies, as well as the impact of clinical trials on treatment paradigms. Furthermore, we examine the utility of animal models in elucidating disease mechanisms. By culminating the complexities inherent in dysferlinopathies, this write up emphasizes the need for multidisciplinary approaches, precision medicine, and extensive collaboration in research and clinical trial design to advance our understanding and treatment of these challenging disorders.
2023-09-16 | Portrait of Dysferlinopathy: Diagnosis and Development of Therapy
Dysferlinopathy is a disease caused by a dysferlin deficiency due to mutations in the DYSF gene. Dysferlin is a membrane protein in the sarcolemma and is involved in different functions, such as membrane repair and vesicle fusion, T-tubule development and maintenance, Ca2+ signalling, and the regulation of various molecules. Miyoshi Myopathy type 1 (MMD1) and Limb–Girdle Muscular Dystrophy 2B/R2 (LGMD2B/LGMDR2) are two possible clinical presentations, yet the same mutations can cause both presentations in the same family. They are therefore grouped under the name dysferlinopathy. Onset is typically during the teenage years or young adulthood and is characterized by a loss of Achilles tendon reflexes and difficulty in standing on tiptoes or climbing stairs, followed by a slow progressive loss of strength in limb muscles. The MRI pattern of patient muscles and their biopsies show various fibre sizes, necrotic and regenerative fibres, and fat and connective tissue accumulation. Recent tools were developed for diagnosis and research, especially to evaluate the evolution of the patient condition and to prevent misdiagnosis caused by similarities with polymyositis and Charcot–Marie–Tooth disease. The specific characteristic of dysferlinopathy is dysferlin deficiency. Recently, mouse models with patient mutations were developed to study genetic approaches to treat dysferlinopathy. The research fields for dysferlinopathy therapy include symptomatic treatments, as well as antisense-mediated exon skipping, myoblast transplantation, and gene editing.
2017-06-01 | Coupling of excitation to Ca2+ release is modulated by dysferlin.
Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca2+ channels protect voltage-induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+ -free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca2+ leak.Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca2+ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca2+ leak from the SR into the cytoplasm.
2015-03-24 | A comparison of AAV strategies distinguishes overlapping vectors for efficient systemic delivery of the 6.2 kb Dysferlin coding sequence
Recombinant adeno-associated virus (rAAV) is currently the best vector for gene delivery into the skeletal muscle. However, the 5-kb packaging size of this virus is a major obstacle for large gene transfer. This past decade, many different strategies were developed to circumvent this issue (concatemerization-splicing, overlapping vectors, hybrid dual or fragmented AAV). Loss of function mutations in the DYSF gene whose coding sequence is 6.2kb lead to progressive muscular dystrophies (LGMD2B: OMIM_253601; MM: OMIM_254130; DMAT: OMIM_606768). In this study, we compared large gene transfer techniques to deliver the DYSF gene into the skeletal muscle. After rAAV8s intramuscular injection into dysferlin deficient mice, we showed that the overlap strategy is the most effective approach to reconstitute a full-length messenger. After systemic administration, the level of dysferlin obtained on different muscles corresponded to 0.5- to 2-fold compared to the normal level. We further demonstrated that the overlapping vector set was efficient to correct the histopathology, resistance to eccentric contractions and whole body force in the dysferlin deficient mice. Altogether, these data indicate that using overlapping vectors could be a promising approach for a potential clinical treatment of dysferlinopathies.
2013-09-13 | Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.
The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb‐girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides ( AON s), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON ‐treated blood‐derived CD 133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/bl AJ dysferlin null mice. We thus extended these experiments producing the full‐length dysferlin mediated by a lentiviral vector in blood‐derived CD 133+ stem cells isolated from the same patients. Transplantation of engineered blood‐derived CD 133+ stem cells into scid/bl AJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus‐mediated delivery of full‐length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies.
oligonucleotides
2026-07-14 | PPMO therapy for dysferlinopathy induces pseudoexon skipping and restoration of functional protein.
The dysferlinopathies are a spectrum of autosomal recessive muscle diseases caused by mutations in the dysferlin gene (DYSF) gene. Clinical manifestations vary from asymptomatic hyperCKemia to severe muscle pathology and loss of muscle function. These are designated limb-girdle muscular dystrophy type 2R or LGMDR2 (formerly LGMD2B or Miyoshi myopathy). Among other functions, dysferlin is crucial for plasma membrane repair and maintenance of intracellular calcium homeostasis. In previous studies, we identified in two independent point mutations deep within introns that cause aberrant DYSF mRNA splicing and the inclusion of pseudoexons within transcripts that disrupt protein expression. In this study, we generated and characterized a novel mouse model for one of these mutations (within DYSF intron 44). In these mice, a segment of human DYSF DNA containing the mutant intronic sequence flanked by surrounding human exon sequences replaces the normal homologous mouse DNA. These mice exhibit aberrant Dysf pre-mRNA splicing, pseudoexon inclusion, loss of DYSF protein expression, and muscle pathology similar to that observed in patients. Using this new model, we identified antisense oligonucleotides and then a PPMO that blocks the mouse Dysf pre-mRNA splicing complexes from binding the mutant pre-mRNA, thereby restoring nearly normal muscle histology and function.
2025-07-28 | An Overview of Recent Advances and Clinical Applications of Exon Skipping and Splice Modulation for Muscular Dystrophy and Various Genetic Diseases.
Exon skipping and splice modulation using synthetic antisense oligonucleotides (AONs) are therapeutic approaches to delay or arrest the progression of genetic diseases. AON-mediated exon skipping modifies pre-mRNA splicing to correct the reading frame of genes responsible for disease and modulate a protein structure, providing therapeutic benefits. Studied for over two decades, exon skipping AONs have received conditional marketing authorization for Duchenne muscular dystrophy (DMD). The US Food and Drug Administration (FDA) has granted accelerated approval for four AONs to skip a single DMD exon for treating patients with DMD: eteplirsen for exon 51 skipping, golodirsen and viltolarsen for exon 53 skipping, and casimersen for exon 45 skipping. Various preclinical and clinical trials are underway with AONs targeting other exons and ones loading bioconjugates, such as cell-penetrating peptides and antibodies to enhance the exon skipping ability. Besides AONs targeting pre-mRNA, genomic DNA target exon skipping using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, which holds promise in current preclinical trials for DMD. In hopes of achieving clinical success parallel to DMD, exon skipping and splice modulation are studied in other muscular dystrophies, such as Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy including limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy (DMAT), myotonic dystrophy, and merosin-deficient congenital muscular dystrophy type 1A (MDC1A). Splice-modulating AONs have also been extensively tested in other inherited diseases, such as Usher syndrome, dystrophic epidermolysis bullosa (DEB), fibrodysplasia ossificans progressiva (FOP), and allergic diseases. This chapter will introduce the developmental status of exon skipping and splice-modulating therapies for genetic diseases.
2024-06-19 | Limb Girdle Muscular Dystrophy Type 2B (LGMD2B): Diagnosis and Therapeutic Possibilities.
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.
2022-11-22 | Morpholino-Mediated Exons 28-29 Skipping of Dysferlin and Characterization of Multiexon-skipped Dysferlin using RT-PCR, Immunoblotting, and Membrane Wounding Assay.
Dysferlinopathies are a group of disabling muscular dystrophies that includes limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT) as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for many patients with dysferlinopathies. It was reported that exons 28-29 of DYSF are dispensable for dysferlin functions. Here, we present a method for multiexon skipping of DYSF exons 28-29 using a cocktail of two phosphorodiamidate morpholino oligomers (PMOs) on cells derived from a dystrophinopathy patient. Also, we describe assays to characterize the multiexon skipped dysferlin at several levels by using one-step RT-PCR, immunoblotting, and a membrane wounding assay.
2020-04-01 | Antisense-Mediated Skipping of Dysferlin Exons in Control and Dysferlinopathy Patient-Derived Cells
Dysferlinopathies encompass a spectrum of progressive muscular dystrophies caused by the lack of dysferlin due to missense mutations in the dysferlin gene or mutations causing premature truncation of protein translation. Dysferlin is a modular protein, and dysferlins lacking one or more repetitive domains have been shown to retain functionality. As such, antisense-mediated exon skipping has been proposed as a therapy for dysferlinopathy. By skipping the mutated exon, the reading frame would be maintained, while the mutation would be bypassed, thus allowing production of an internally deleted, but partially functional, dysferlin. We previously showed that dysferlin exon skipping is feasible in control cell lines. We here evaluated exon skipping and dysferlin protein restoration in patient-derived cells requiring the skipping of exon 9, 29, 30, or 34. Exon 30 skipping was possible at high efficiency, but did not result in increased dysferlin. We discovered that the alleged exon 30 mutation was in fact a polymorphism and identified a splicing mutation in intron 28 as the disease-causing mutation. While exon skipping was feasible for each of the other cell lines, no increases in dysferlin protein could be detected by western blotting.
proteins
2026-04-09 | Dysferlinopathies: phenotypic study of a Moroccan series of 28 cases.
Dysferlinopathies are a spectrum of autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene (DYSF), leading to heterogeneous phenotypes, mainly limb-girdle muscular dystrophy type 2R (LGMDR2) and Miyoshi distal myopathy (MMD1). In Morocco, dysferlinopathies accounted for 18% of all limb-girdle muscular dystrophies, ranking second after sarcoglycanopathies. We retrospectively analyzed 28 confirmed cases, diagnosed either by dysferlin deficiency or DYSF mutation. The median age at examination was 25,5 years (21-33). Twelve patients (43%) were female, and 19 (68%) had consanguineous parents. The proximodistal phenotype was predominant (50%), followed by LGMDR2 (39%) and MMD1 (11%). Selective biceps brachii involvement was observed in 68% of patients. Seven patients (25%) showed hypertrophy of the extensor digitorum brevis muscle, and two (7%) presented with severe genu recurvatum. In addition to dystrophic lesions, muscle biopsy revealed inflammatory infiltrates in 15 cases (58%), leading to an initial misdiagnosis of inflammatory myopathy in two cases. Dysferlin deficiency confirmed the diagnosis in 26 cases, while two others had homozygous DYSF variants. Four patients received corticosteroids, with clear improvement in only one. Overall, disease progression was slow, although two patients lost ambulation at the ages of 26 and 32. Regular physical activity appeared to have a beneficial effect in most patients.
2023-09-05 | Translational implications of targeting annexin A2: From membrane repair to muscular dystrophy, cardiovascular disease and cancer
Abstract Background Annexin A2 contributes to several key cellular functions and processes, including membrane repair. Effective repair prevents cell death and degeneration, especially in skeletal or cardiac muscle, epithelia, and endothelial cells. To maintain cell integrity after damage, mammalian cells activate multiple membrane repair mechanisms. One protein family that facilitates membrane repair processes are the Ca 2+ ‐regulated phospholipid‐binding annexins. Body Annexin A2 facilitates repair in association with S100A10 and related S100 proteins by forming a plug and linking repair to other physiologic functions. Deficiency of annexin A2 enhances cellular degeneration, exacerbating muscular dystrophy and degeneration. Downstream of repair, annexin A2 links the membrane with the cytoskeleton, calcium‐dependent endocytosis, exocytosis, cell proliferation, transcription, and apoptosis to extracellular roles, including vascular fibrinolysis, and angiogenesis. These roles regulate cardiovascular disease progression. Finally, annexin A2 protects cancer cells from membrane damage due to immune cells or chemotherapy. Since these functions are regulated by post‐translational modifications, they represent a therapeutic target for reducing the negative consequences of annexin A2 expression. Conclusion Thus, connecting the roles of annexin A2 in repair to its other physiologic functions represents a new translational approach to treating muscular dystrophy and cardiovascular diseases without enhancing its pro‐tumorigenic activities.
2023-08-21 | The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.
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.
2019-07-19 | Functional muscle hypertrophy by increased insulin‐like growth factor 1 does not require dysferlin
Abstract Introduction Dysferlin loss‐of‐function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin‐like growth factor 1 (IGF‐1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF‐1 promotes functional muscle hypertrophy in dysferlin‐null ( Dysf −/− ) mice. Methods Muscle‐specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf −/− and control mice. Increased IGF‐1 levels were confirmed by enzyme‐linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice. Results Muscle hypertrophy occurred in response to increased IGF‐1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF‐1 did not cause loss of force per cross‐sectional area in Dysf −/− muscles. Discussion We conclude that increased local IGF‐1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF‐1 as a potential therapeutic for dysferlinopathies.
2016-06-07 | Membrane repair of human skeletal muscle cells requires Annexin-A5
Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin.
small molecules
2024-08-20 | Brain of miyoshi myopathy/dysferlinopathy patients presents with structural and metabolic anomalies.
Miyoshi myopathy/dysferlinopathy (MMD) is a rare muscle disease caused by DYSF gene mutations. Apart from skeletal muscles, DYSF is also expressed in the brain. However, the impact of MMD-causing DYSF variants on brain structure and function remains unexplored. To investigate this, we utilized magnetic resonance (MR) modalities (MR volumetry and 31P MR spectroscopy) in a family with seven children, four of whom have the illness. The MMD siblings showed distinct differences from healthy controls: (1) a significant (p < 0.001) right-sided volume asymmetry (+ 232 mm3) of the inferior lateral ventricles; and (2) a significant (p < 0.001) decrease in [Mg2+], along with a modified energy metabolism profile and altered membrane turnover in the hippocampus and motor and premotor cortices. The patients' [Mg2+], energy metabolism, and membrane turnover measures returned to those of healthy relatives after a month of 400 mg/day magnesium supplementation. This work is the first to describe anatomical and functional abnormalities characteristic of neurodegeneration in the MMD brain. Therefore, we call for further examination of brain functions in larger cohorts of MMD patients and testing of magnesium supplementation, which has proven to be an effective corrective approach in our study.
2022-11-19 | Inhibition of the immunoproteasome modulates innate immunity to ameliorate muscle pathology of dysferlin-deficient BlAJ mice
Abstract Muscle repair in dysferlinopathies is defective. Although macrophage (Mø)-rich infiltrates are prominent in damaged skeletal muscles of patients with dysferlinopathy, the contribution of the immune system to the disease pathology remains to be fully explored. Numbers of both pro-inflammatory M1 Mø and effector T cells are increased in muscle of dysferlin-deficient BlAJ mice. In addition, symptomatic BlAJ mice have increased muscle production of immunoproteasome. In vitro analyses using bone marrow-derived Mø of BlAJ mice show that immunoproteasome inhibition results in C3aR1 and C5aR1 downregulation and upregulation of M2-associated signaling. Administration of immunoproteasome inhibitor ONX-0914 to BlAJ mice rescues muscle function by reducing muscle infiltrates and fibro-adipogenesis. These findings reveal an important role of immunoproteasome in the progression of muscular dystrophy in BlAJ mouse and suggest that inhibition of immunoproteasome may produce therapeutic benefit in dysferlinopathy.
2019-07-18 | Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells
Abstract Autosomal recessive mutations in Anoctamin 5 ( ANO5/TMEM16E ), a member of the transmembrane 16 (TMEM16) family of Ca 2+ -activated ion channels and phospholipid scramblases, cause adult-onset muscular dystrophies (limb girdle muscular dystrophy 2L (LGMD2L) and Miyoshi Muscular Dystrophy (MMD3). However, the molecular role of ANO5 is unclear and ANO5 knockout mouse models show conflicting requirements of ANO5 in muscle. To study the role of ANO5 in human muscle cells we generated a myoblast line from a MMD3-patient carrying the c.2272C>T mutation, which we find causes the mutant protein to be degraded. The patient myoblasts exhibit normal myogenesis, but are compromised in their plasma membrane repair (PMR) ability. The repair deficit is linked to the poor ability of the endoplasmic reticulum (ER) to clear cytosolic Ca 2+ increase caused by focal plasma membrane injury. Expression of wild-type ANO5 or pharmacological prevention of injury-triggered cytosolic Ca 2+ overload enable injured patient muscle cells to repair. A homology model of ANO5 shows that several of the known LGMD2L/MMD3 patient mutations line the transmembrane region of the protein implicated in its channel activity. These results point to a role of cytosolic Ca 2+ homeostasis in PMR, indicate a role for ANO5 in ER-mediated cytosolic Ca 2+ uptake and identify normalization of cytosolic Ca 2+ homeostasis as a potential therapeutic approach to treat muscular dystrophies caused by ANO5 deficit.
2019-06-28 | Phenotypic Drug Screening for Dysferlinopathy Using Patient-Derived Induced Pluripotent Stem Cells
Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.
2018-09-05 | LIMB-GIRDLE MUSCULAR DYSTROPHY I
Defects in dysferlin gene cause limb-girdle muscular dystrophy 2B or Miyoshi distal myopathy. Nonsense mutations have been reported to be most common among Korean patients with dysferlinopathy. More than half of the patients have at least one nonsense allele in dysferlin. Readthrough by ataluren is a promising strategy to overcome nonsense mutation, in use for the nonsense-mediated Duchenne muscular dystrophy. We generated a knock-in mouse with p.Q832* mutation, most frequently found among our dysferlinopathy cases. Mice with homozygous p.Q832* mutation lost dysferlin expression at sarcolemma. It performed worse on the Rotarod and forelimb grip test than wild type C57BL6 mice and moved less on activity monitoring. On eccentric contraction ex vivo, extensor digitorum longus muscle from these mice tended to break easily. These abnormalities could successfully be alleviated when the mice were orally administered with ataluren in liquid. Furthermore, ataluren decreased the number of damaged muscle, fiber size variation and serum creatine kinase level. This results support that readthrough strategy with ataluren will also be applicable to patients with nonsense dysferlin mutation.
gene therapies
2024-03-29 | The Dysferlinopathies Conundrum: Clinical Spectra, Disease Mechanism and Genetic Approaches for Treatments.
Dysferlinopathies refer to a spectrum of muscular dystrophies that cause progressive muscle weakness and degeneration. They are caused by mutations in the DYSF gene, which encodes the dysferlin protein that is crucial for repairing muscle membranes. This review delves into the clinical spectra of dysferlinopathies, their molecular mechanisms, and the spectrum of emerging therapeutic strategies. We examine the phenotypic heterogeneity of dysferlinopathies, highlighting the incomplete understanding of genotype-phenotype correlations and discussing the implications of various DYSF mutations. In addition, we explore the potential of symptomatic, pharmacological, molecular, and genetic therapies in mitigating the disease's progression. We also consider the roles of diet and metabolism in managing dysferlinopathies, as well as the impact of clinical trials on treatment paradigms. Furthermore, we examine the utility of animal models in elucidating disease mechanisms. By culminating the complexities inherent in dysferlinopathies, this write up emphasizes the need for multidisciplinary approaches, precision medicine, and extensive collaboration in research and clinical trial design to advance our understanding and treatment of these challenging disorders.
2023-09-16 | Portrait of Dysferlinopathy: Diagnosis and Development of Therapy
Dysferlinopathy is a disease caused by a dysferlin deficiency due to mutations in the DYSF gene. Dysferlin is a membrane protein in the sarcolemma and is involved in different functions, such as membrane repair and vesicle fusion, T-tubule development and maintenance, Ca2+ signalling, and the regulation of various molecules. Miyoshi Myopathy type 1 (MMD1) and Limb–Girdle Muscular Dystrophy 2B/R2 (LGMD2B/LGMDR2) are two possible clinical presentations, yet the same mutations can cause both presentations in the same family. They are therefore grouped under the name dysferlinopathy. Onset is typically during the teenage years or young adulthood and is characterized by a loss of Achilles tendon reflexes and difficulty in standing on tiptoes or climbing stairs, followed by a slow progressive loss of strength in limb muscles. The MRI pattern of patient muscles and their biopsies show various fibre sizes, necrotic and regenerative fibres, and fat and connective tissue accumulation. Recent tools were developed for diagnosis and research, especially to evaluate the evolution of the patient condition and to prevent misdiagnosis caused by similarities with polymyositis and Charcot–Marie–Tooth disease. The specific characteristic of dysferlinopathy is dysferlin deficiency. Recently, mouse models with patient mutations were developed to study genetic approaches to treat dysferlinopathy. The research fields for dysferlinopathy therapy include symptomatic treatments, as well as antisense-mediated exon skipping, myoblast transplantation, and gene editing.
2017-06-01 | Coupling of excitation to Ca2+ release is modulated by dysferlin.
Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca2+ channels protect voltage-induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+ -free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca2+ leak.Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca2+ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca2+ leak from the SR into the cytoplasm.
2015-03-24 | A comparison of AAV strategies distinguishes overlapping vectors for efficient systemic delivery of the 6.2 kb Dysferlin coding sequence
Recombinant adeno-associated virus (rAAV) is currently the best vector for gene delivery into the skeletal muscle. However, the 5-kb packaging size of this virus is a major obstacle for large gene transfer. This past decade, many different strategies were developed to circumvent this issue (concatemerization-splicing, overlapping vectors, hybrid dual or fragmented AAV). Loss of function mutations in the DYSF gene whose coding sequence is 6.2kb lead to progressive muscular dystrophies (LGMD2B: OMIM_253601; MM: OMIM_254130; DMAT: OMIM_606768). In this study, we compared large gene transfer techniques to deliver the DYSF gene into the skeletal muscle. After rAAV8s intramuscular injection into dysferlin deficient mice, we showed that the overlap strategy is the most effective approach to reconstitute a full-length messenger. After systemic administration, the level of dysferlin obtained on different muscles corresponded to 0.5- to 2-fold compared to the normal level. We further demonstrated that the overlapping vector set was efficient to correct the histopathology, resistance to eccentric contractions and whole body force in the dysferlin deficient mice. Altogether, these data indicate that using overlapping vectors could be a promising approach for a potential clinical treatment of dysferlinopathies.
2013-09-13 | Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.
The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb‐girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides ( AON s), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON ‐treated blood‐derived CD 133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/bl AJ dysferlin null mice. We thus extended these experiments producing the full‐length dysferlin mediated by a lentiviral vector in blood‐derived CD 133+ stem cells isolated from the same patients. Transplantation of engineered blood‐derived CD 133+ stem cells into scid/bl AJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus‐mediated delivery of full‐length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies.
oligonucleotides
2026-07-14 | PPMO therapy for dysferlinopathy induces pseudoexon skipping and restoration of functional protein.
The dysferlinopathies are a spectrum of autosomal recessive muscle diseases caused by mutations in the dysferlin gene (DYSF) gene. Clinical manifestations vary from asymptomatic hyperCKemia to severe muscle pathology and loss of muscle function. These are designated limb-girdle muscular dystrophy type 2R or LGMDR2 (formerly LGMD2B or Miyoshi myopathy). Among other functions, dysferlin is crucial for plasma membrane repair and maintenance of intracellular calcium homeostasis. In previous studies, we identified in two independent point mutations deep within introns that cause aberrant DYSF mRNA splicing and the inclusion of pseudoexons within transcripts that disrupt protein expression. In this study, we generated and characterized a novel mouse model for one of these mutations (within DYSF intron 44). In these mice, a segment of human DYSF DNA containing the mutant intronic sequence flanked by surrounding human exon sequences replaces the normal homologous mouse DNA. These mice exhibit aberrant Dysf pre-mRNA splicing, pseudoexon inclusion, loss of DYSF protein expression, and muscle pathology similar to that observed in patients. Using this new model, we identified antisense oligonucleotides and then a PPMO that blocks the mouse Dysf pre-mRNA splicing complexes from binding the mutant pre-mRNA, thereby restoring nearly normal muscle histology and function.
2025-07-28 | An Overview of Recent Advances and Clinical Applications of Exon Skipping and Splice Modulation for Muscular Dystrophy and Various Genetic Diseases.
Exon skipping and splice modulation using synthetic antisense oligonucleotides (AONs) are therapeutic approaches to delay or arrest the progression of genetic diseases. AON-mediated exon skipping modifies pre-mRNA splicing to correct the reading frame of genes responsible for disease and modulate a protein structure, providing therapeutic benefits. Studied for over two decades, exon skipping AONs have received conditional marketing authorization for Duchenne muscular dystrophy (DMD). The US Food and Drug Administration (FDA) has granted accelerated approval for four AONs to skip a single DMD exon for treating patients with DMD: eteplirsen for exon 51 skipping, golodirsen and viltolarsen for exon 53 skipping, and casimersen for exon 45 skipping. Various preclinical and clinical trials are underway with AONs targeting other exons and ones loading bioconjugates, such as cell-penetrating peptides and antibodies to enhance the exon skipping ability. Besides AONs targeting pre-mRNA, genomic DNA target exon skipping using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, which holds promise in current preclinical trials for DMD. In hopes of achieving clinical success parallel to DMD, exon skipping and splice modulation are studied in other muscular dystrophies, such as Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy including limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy (DMAT), myotonic dystrophy, and merosin-deficient congenital muscular dystrophy type 1A (MDC1A). Splice-modulating AONs have also been extensively tested in other inherited diseases, such as Usher syndrome, dystrophic epidermolysis bullosa (DEB), fibrodysplasia ossificans progressiva (FOP), and allergic diseases. This chapter will introduce the developmental status of exon skipping and splice-modulating therapies for genetic diseases.
2024-06-19 | Limb Girdle Muscular Dystrophy Type 2B (LGMD2B): Diagnosis and Therapeutic Possibilities.
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.
2022-11-22 | Morpholino-Mediated Exons 28-29 Skipping of Dysferlin and Characterization of Multiexon-skipped Dysferlin using RT-PCR, Immunoblotting, and Membrane Wounding Assay.
Dysferlinopathies are a group of disabling muscular dystrophies that includes limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT) as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for many patients with dysferlinopathies. It was reported that exons 28-29 of DYSF are dispensable for dysferlin functions. Here, we present a method for multiexon skipping of DYSF exons 28-29 using a cocktail of two phosphorodiamidate morpholino oligomers (PMOs) on cells derived from a dystrophinopathy patient. Also, we describe assays to characterize the multiexon skipped dysferlin at several levels by using one-step RT-PCR, immunoblotting, and a membrane wounding assay.
2020-04-01 | Antisense-Mediated Skipping of Dysferlin Exons in Control and Dysferlinopathy Patient-Derived Cells
Dysferlinopathies encompass a spectrum of progressive muscular dystrophies caused by the lack of dysferlin due to missense mutations in the dysferlin gene or mutations causing premature truncation of protein translation. Dysferlin is a modular protein, and dysferlins lacking one or more repetitive domains have been shown to retain functionality. As such, antisense-mediated exon skipping has been proposed as a therapy for dysferlinopathy. By skipping the mutated exon, the reading frame would be maintained, while the mutation would be bypassed, thus allowing production of an internally deleted, but partially functional, dysferlin. We previously showed that dysferlin exon skipping is feasible in control cell lines. We here evaluated exon skipping and dysferlin protein restoration in patient-derived cells requiring the skipping of exon 9, 29, 30, or 34. Exon 30 skipping was possible at high efficiency, but did not result in increased dysferlin. We discovered that the alleged exon 30 mutation was in fact a polymorphism and identified a splicing mutation in intron 28 as the disease-causing mutation. While exon skipping was feasible for each of the other cell lines, no increases in dysferlin protein could be detected by western blotting.
proteins
2026-04-09 | Dysferlinopathies: phenotypic study of a Moroccan series of 28 cases.
Dysferlinopathies are a spectrum of autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene (DYSF), leading to heterogeneous phenotypes, mainly limb-girdle muscular dystrophy type 2R (LGMDR2) and Miyoshi distal myopathy (MMD1). In Morocco, dysferlinopathies accounted for 18% of all limb-girdle muscular dystrophies, ranking second after sarcoglycanopathies. We retrospectively analyzed 28 confirmed cases, diagnosed either by dysferlin deficiency or DYSF mutation. The median age at examination was 25,5 years (21-33). Twelve patients (43%) were female, and 19 (68%) had consanguineous parents. The proximodistal phenotype was predominant (50%), followed by LGMDR2 (39%) and MMD1 (11%). Selective biceps brachii involvement was observed in 68% of patients. Seven patients (25%) showed hypertrophy of the extensor digitorum brevis muscle, and two (7%) presented with severe genu recurvatum. In addition to dystrophic lesions, muscle biopsy revealed inflammatory infiltrates in 15 cases (58%), leading to an initial misdiagnosis of inflammatory myopathy in two cases. Dysferlin deficiency confirmed the diagnosis in 26 cases, while two others had homozygous DYSF variants. Four patients received corticosteroids, with clear improvement in only one. Overall, disease progression was slow, although two patients lost ambulation at the ages of 26 and 32. Regular physical activity appeared to have a beneficial effect in most patients.
2023-09-05 | Translational implications of targeting annexin A2: From membrane repair to muscular dystrophy, cardiovascular disease and cancer
Abstract Background Annexin A2 contributes to several key cellular functions and processes, including membrane repair. Effective repair prevents cell death and degeneration, especially in skeletal or cardiac muscle, epithelia, and endothelial cells. To maintain cell integrity after damage, mammalian cells activate multiple membrane repair mechanisms. One protein family that facilitates membrane repair processes are the Ca 2+ ‐regulated phospholipid‐binding annexins. Body Annexin A2 facilitates repair in association with S100A10 and related S100 proteins by forming a plug and linking repair to other physiologic functions. Deficiency of annexin A2 enhances cellular degeneration, exacerbating muscular dystrophy and degeneration. Downstream of repair, annexin A2 links the membrane with the cytoskeleton, calcium‐dependent endocytosis, exocytosis, cell proliferation, transcription, and apoptosis to extracellular roles, including vascular fibrinolysis, and angiogenesis. These roles regulate cardiovascular disease progression. Finally, annexin A2 protects cancer cells from membrane damage due to immune cells or chemotherapy. Since these functions are regulated by post‐translational modifications, they represent a therapeutic target for reducing the negative consequences of annexin A2 expression. Conclusion Thus, connecting the roles of annexin A2 in repair to its other physiologic functions represents a new translational approach to treating muscular dystrophy and cardiovascular diseases without enhancing its pro‐tumorigenic activities.
2023-08-21 | The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.
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.
2019-07-19 | Functional muscle hypertrophy by increased insulin‐like growth factor 1 does not require dysferlin
Abstract Introduction Dysferlin loss‐of‐function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin‐like growth factor 1 (IGF‐1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF‐1 promotes functional muscle hypertrophy in dysferlin‐null ( Dysf −/− ) mice. Methods Muscle‐specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf −/− and control mice. Increased IGF‐1 levels were confirmed by enzyme‐linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice. Results Muscle hypertrophy occurred in response to increased IGF‐1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF‐1 did not cause loss of force per cross‐sectional area in Dysf −/− muscles. Discussion We conclude that increased local IGF‐1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF‐1 as a potential therapeutic for dysferlinopathies.
2016-06-07 | Membrane repair of human skeletal muscle cells requires Annexin-A5
Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin.
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