Limb Girdle Muscular Dystrophy 2B Research Articles

LMNA-related muscular dystrophy: Identification of variants in alternative genes and personalized clinical translation.

Background: Laminopathies are caused by rare alterations in LMNA, leading to a wide clinical spectrum. Though muscular dystrophy begins at early ages, disease progression is different in each patient. We investigated variability in laminopathy phenotypes by performing a targeted genetic analysis of patients diagnosed with LMNA-related muscular dystrophy to identify rare variants in alternative genes, thereby explaining phenotypic differences. Methods: We analyzed 105 genes associated with muscular diseases by targeted sequencing in 26 pediatric patients of different countries, diagnosed with any LMNA-related muscular dystrophy. Family members were also clinically assessed and genetically analyzed. Results: All patients carried a pathogenic rare variant in LMNA. Clinical diagnoses included Emery-Dreifuss muscular dystrophy (EDMD, 13 patients), LMNA-related congenital muscular dystrophy (L-CMD, 11 patients), and limb-girdle muscular dystrophy 1B (LGMD1B, 2 patients). In 9 patients, 10 additional rare genetic variants were identified in 8 genes other than LMNA. Genotype-phenotype correlation showed additional deleterious rare variants in five of the nine patients (3 L-CMD and 2 EDMD) with severe phenotypes. Conclusion: Analysis f known genes related to muscular diseases in close correlation with personalized clinical assessments may help identify additional rare variants of LMNA potentially associated with early onset or most severe disease progression.

P2 Receptor Signaling in Motor Units in Muscular Dystrophy.

The purine signaling system is represented by purine and pyrimidine nucleotides and nucleosides that exert their effects through the adenosine, P2X and P2Y receptor families. It is known that, under physiological conditions, P2 receptors play only a minor role in modulating the functions of cells and systems; however, their role significantly increases under some pathophysiological conditions, such as stress, ischemia or hypothermia, when they can play a dominant role as a signaling molecule. The diversity of P2 receptors and their wide distribution in the body make them very attractive as a target for the pharmacological action of drugs with a new mechanism of action. The review is devoted to the involvement of P2 signaling in the development of pathologies associated with a loss of muscle mass. The contribution of adenosine triphosphate (ATP) as a signal molecule in the pathogenesis of a number of muscular dystrophies (Duchenne, Becker and limb girdle muscular dystrophy 2B) is considered. To understand the processes involving the purinergic system, the role of the ATP and P2 receptors in several models associated with skeletal muscle degradation is also discussed.

Evaluation of cardiomyopathy with two-dimensional speckle tracking echocardiography in limb-girdle muscular dystrophy type 2A and 2B.

Cardiac involvement in limb-girdle muscular dystrophy (LGMD)2A and LGMD2B, the most common subgroups of LGMD, is controversial. Our study aims to determine whether myocardial dysfunction develops in LGMD2A and LGMD2B patients. The study included 16 LGMD2A, 12 LGMD2B patients, and 48 healthy individuals. Comparisons included demographic, clinical, and laboratory parameters of LGMD2A and LGMD2B subgroups and traditional echocardiography and two-dimensional speckle tracking echocardiography (2D-STE) parameters with the normal population. The median age was 33 (22-39 interquartile range [IQR]) in the LGMD2A group, 33 (27-38 IQR) in the LGMD2B group, and 28 (25-35 IQR) in the control group. The left ventricular (LV) ejection fraction of both LGMD2A and LGMD2B groups was similar to the control group (p=0.296 and p=0.918). Apical 4-chamber longitudinal strain (LS), Apical 2-chamber LS, Apical 3-chamber LS, left ventricular global longitudinal strain (LVGLS)-mid-myocardial, LVGLS-endocardium, and LVGLS-epicardium were lower (less negative) in the LGMD2B group compared to the control group (p=0.006, p=0.001, p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively). LV 2D-STE parameters of LGMD2A patients were similar to the control group, while they decreased significantly (less negative) in LGMD2B patients, indicating that LV subclinical myocardial dysfunction may develop in LGMD2B patients.

Clinical and pathological features of immune-mediated necrotising myopathies in a single-centre muscle biopsy cohort.

ObjectiveImmune-mediated necrotising myopathy (IMNM) is a subset of idiopathic inflammatory myopathies (IIM) characterized by significantly elevated creatine kinase level, muscle weakness and predominant muscle fibre necrosis in muscle biopsy. This study aimed to investigate the clinical and pathological characteristics of patients with IMNM in a single-centre muscle biopsy cohort.MethodsA total of 860 patients who had muscle biopsy reports in our centre from May 2008 to December 2017 were enrolled in this study. IMNM was diagnosed according to the 2018 European Neuromuscular Centre (ENMC) clinicopathological diagnostic criteria for IMNM.ResultsThe muscle biopsy cohort consisted of 531 patients with IIM (61.7%), 253 patients with non-IIM (29.4%), and 76 undiagnosed patients (8.8%). IIM cases were classified as IMNM (68[7.9%]), dermatomyositis (346[40.2%]), anti-synthetase syndrome (82[9.5%]), polymyositis (32[3.7%]), and sporadic inclusion body myositis (3[0.3%]). Limb girdle muscular dystrophy (LGMD) 2B and lipid storage myopathy (LSM) are the two most common non-IIM disorders in our muscle biopsy cohort. IMNM patients had a higher onset age (41.57 ± 14.45 vs 21.66 ± 7.86 and 24.56 ± 10.78, p < .0001), shorter duration (21.79 ± 26.01 vs 66.69 ± 67.67 and 24.56 ± 10.78, p < .0001), and more frequent dysphagia (35.3% vs. 3.4 and 6.3%, p = .001) than LGMD 2B and LSM patients. Muscle biopsy from IMNM showed more frequent muscle fibre necrosis (95.6% vs 72.4 and 56.3%, p < .0001), overexpression of major histocompatibility complex-I on sarcolemma (83.8% vs 37.9 and 12.9%, p < .0001), and CD4+ T cell endomysia infiltration (89.7% vs 53.6 and 50%, p < .0001) compared with those from LGMD 2B and LSM patients.ConclusionsIt is easy to distinguish IMNM from other IIM subtypes according to clinical symptoms and myositis specific antibodies profiles. However, distinguishing IMNM from disorders clinically similar to non-IIM needs combined clinical, serological and pathological features.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12891-022-05372-z.

Key Facilitator Proteins that Mediate Sarcolemma Membrane Repair have Potential as Therapeutics for Muscle Disease and Injury

A common aspect of many diseases and traumatic injuries affecting skeletal muscle is the necrotic death of muscle fibers. Necrotic death of muscle fibers involves the breakdown of the sarcolemmal membrane that can be exacerbated by increased fragility of the membrane or by compromised endogenous sarcolemmal membrane repair processes. Increasing the efficacy of these repair mechanisms could act as a therapeutic approach for several muscular diseases and injuries. Limb‐girdle muscular dystrophy 2B (LGMD2B), a subtype of LGMD, is an autosomal recessive neuromuscular disorder caused by mutations in the DYSF gene that encodes dysferlin, a protein that is vital in membrane repair. The dysferlin protein has been previously shown to facilitate membrane repair in skeletal muscle and knockout mice for dysferlin develop progressive muscular dystrophy. When dysferlin is absent or inactivated through mutation, membrane damage that cannot be repaired accumulates until muscle fibers undergo necrosis that eventually overwhelms the regenerative capacity of the muscle. This causes skeletal muscle deterioration in LGMD2B patients, particularly in the pelvic and shoulder girdle muscles. There is currently no cure for LGMD2B and treatments that stimulate the membrane repair process in muscle fibers have been greatly overlooked and underutilized as a potential therapeutic approach. This project investigates a novel membrane repair signaling cascade in skeletal cells by utilizing SC79, an Akt activator, to increase membrane repair capacity in skeletal muscle. We hypothesize that activation of the phosphoinositide‐3 kinase (PI3K)/Akt1 signaling axis regulates membrane repair in cultured muscle cells and mouse tissue. Based on our data, we find that SC79 injection leads to decreased sarcolemmal membrane injury during treadmill exercises where injection of SC79 decreases the entry of Evans blue dye into muscle fibers. We determined that various doses of SC79 can increase membrane repair in cultured muscle cells and Bla/J dysferlin deficient mice. In these studies, multi‐photon infrared laser microscopy was used to damage the cell membrane of myoblasts, transdifferentiated from skin fibroblasts isolated from LGMD2B patients, in the presence of FM4‐64 dye, a lipophilic dye that fluoresces when it enters the cells and binds to the phospholipids of the cell membrane and intracellular organelles. The extent of localized FM4‐64 dye fluorescence provides a measurement of how well the membrane is repaired. We found SC79 could increase membrane repair in these human patient myoblasts in a dose dependent fashion. This assay was also used on isolated complete muscles from the Bla/J dysferlin deficient mice, where SC79 also increased membrane repair responses. We conclude that activation of the PI3K/Akt1 signaling axis increases membrane repair in dysferlin deficient skeletal muscle. This project explores a novel signaling cascade controlling membrane repair that could be leveraged to develop new therapies for muscle disease and injury.

The C2 domains of dysferlin: roles in membrane localization, Ca2+ signalling and sarcolemmal repair.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.

Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B.

Efficient sarcolemmal repair is required for muscle cell survival, with deficits in this process leading to muscle degeneration. Lack of the sarcolemmal protein dysferlin impairs sarcolemmal repair by reducing secretion of the enzyme acid sphingomyelinase (ASM), and causes limb girdle muscular dystrophy 2B (LGMD2B). The large size of the dysferlin gene poses a challenge for LGMD2B gene therapy efforts aimed at restoring dysferlin expression in skeletal muscle fibers. Here, we present an alternative gene therapy approach targeting reduced ASM secretion, the consequence of dysferlin deficit. We showed that the bulk endocytic ability is compromised in LGMD2B patient cells, which was addressed by extracellularly treating cells with ASM. Expression of secreted human ASM (hASM) using a liver-specific adeno-associated virus (AAV) vector restored membrane repair capacity of patient cells to healthy levels. A single in vivo dose of hASM-AAV in the LGMD2B mouse model restored myofiber repair capacity, enabling efficient recovery of myofibers from focal or lengthening contraction–induced injury. hASM-AAV treatment was safe, attenuated fibro-fatty muscle degeneration, increased myofiber size, and restored muscle strength, similar to dysferlin gene therapy. These findings elucidate the role of ASM in dysferlin-mediated plasma membrane repair and to our knowledge offer the first non–muscle-targeted gene therapy for LGMD2B.

Functional recovery of a novel knockin mouse model of dysferlinopathy by readthrough of nonsense mutation

Biallelic mutations in the dysferlin gene cause limb-girdle muscular dystrophy 2B or Miyoshi distal myopathy. We found that nonsense mutations are the most common mutation type among Korean patients with dysferlinopathy; more than half of the patients have at least one nonsense allele, which may be amenable to readthrough therapy. We generated a knockin mouse, dqx, harboring DYSF p.Q832∗ mutation. Homozygous dqx mice lacked dysferlin in skeletal muscle, while 2 weeks of oral ataluren restored dysferlin expression and ameliorated skeletal muscle pathology. Their physical performance improved, and protection against eccentric contractions was noted. The improvement was most evident in mice treated with oral ataluren of 0.9 mg/mL. These improvements were sustained for 8 weeks in ataluren-treated dqx mice, while the parameters of A/J mice treated with ataluren over the same period did not improve. These results support that readthrough therapy by oral ataluren may also be applicable to dysferlinopathy patients with nonsense mutation.

A Patient With Late-onset Limb-girdle Muscular Dystrophy Type 2B Mimicking Dermatomyositis: A Case Report and Review.

Limb-Girdle Muscular Dystrophy 2B (LGMD2B) presents with proximal and/or distal muscle weakness and markedly high creatine kinase level. It is caused by the loss of dysferlin due to mutations in the DYSF gene. Due to its similar clinical features as inflammatory myopathy, it is often difficult to distinguish between the two. We present a case of a 48-year-old male who developed progressive proximal muscle weakness, papulosquamous lesions on the knuckles, elevated levels of muscle enzymes, and electromyogram abnormalities. Based on the clinical presentation, the initial impression was dermatomyositis, yet it was refractory to immunosuppressive therapy. Subsequently, dysferlin immunostaining and genetic analysis led to the final diagnosis of LGMD2B. This case shows that LGMD2B can present with extramuscular symptoms mimicking inflammatory myopathy in later stages of life. Dysferlin immunostaining and/or genetic analysis of the DYSF gene are essential for its diagnosis.

Over three decades of natural history of limb girdle muscular dystrophy type R1/2A and R2/2B: Mathematical modelling of a multifactorial study

We aimed to describe the natural history of Limb Girdle Muscular Dystrophy type 2A and 2B over more than three decades by considering muscular strength, motor, cardiac and respiratory function. 428 visits of nineteen 2A and twenty 2B patients were retrospectively analysed through a regression model to create the curves of evolution with disease duration of muscle strength (through Medical Research Council grading), motor function measure scale (D1, D2 and D3 domains) and cardio-pulmonary function tests. Clinically relevant muscular and motor function alterations occurred after the first decade of disease, while mild respiratory function alterations started after the second, with preserved cardiac function. Although type 2A showed relatively stronger distal lower limb muscles, while type 2B started with relatively stronger upper limb muscles, the corresponding motor functions were similar, becoming severely compromised after 25 years of disease. This was the longest retrospective study in types 2A and 2B. It defined curves of disease evolution not only from a neuromuscular, but also from functional, cardiac, and respiratory points of view, to be used to evaluate how the natural progression is changed by therapies. Due to slow disease progression, it was not possible to identify time sensitive endpoints.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.148 Semiquantitative CT assessment of muscle involvement in limb girdle muscular dystrophy 2A and 2B with the wide range of duration

Limb girdle muscular dystrophy (LGMD) 2A and 2B represent the most frequent forms of LGMDs in the Japanese population. We investigated muscle CT images of both LGMDs with the wide range of disease duration to characterize muscle involvement of this disease and to determine the rate of progression. We applied the Mercuri grading system to CT scans cross-sectional images of six cross section (shoulder, upper and lower trunk, pelvis, thigh and calf). This study included 15 patients with LGMD2A (8 male, 7 female, 41.2±21.6) and 27 patients with LGMD2B (13 male, 14 female, 50.5±17.4 years) registered in IBIC (Integrative Brain Imaging Center:the database of skeletal muscle images in Japan). The patients were classified into 3 groups according to disease duration: stage1 (<10years), stage 2 (11∼30years) and stage 3 (31<years). In stage1, both types showed early predominant degeneration of subscapularis, paraspinal, gluteus minimus and the medial head of the gastrocnemius. There was mild muscle involvement in 2A, but moderate involvement in 2B at shoulder level. The involvement of the pelvic muscles in 2A was equal to, or slightly more than those of 2B. The degeneration of the posterior segment of the thigh with sparing of the gracilis muscle was observed in both. In stage2, biceps brachii muscle was much more severely involved than triceps brachii muscle in 2A, but this finding was not true for 2B. Patchy involvement of the quadriceps was observed in 2B. In stage3, almost all muscles were replaced by fat in 2B whereas there were some residual muscles in 2A at the thigh and calf level. The rate of disease progression was much slower in LGMD2A than LGMD2B. This finding is concordant with our previous study using %MVI.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.146 A multi-center LGMD2B-related DYSF mutation analysis in China

Limb-girdle muscular dystrophy 2B (LGMD2B) is the most prevalent LGMD subtype in Asian population. It is characterized by progressive limb muscle weakness and caused by recessive DYSF gene mutations. To date, there has not been a systemic evaluation in the prevalence of mutations in LGMD2B patients. We analyzed DYSF gene mutations in 243 LGMD2B patients recruited from four diagnostic centers (2010 January to 2019 July) in China. A total of 230 mutational alleles were extracted (NM_003494.4). MutationAssessor was used for assessing the pathogenicity of single nucleotide variants.

Insights into lipid accumulation in skeletal muscle in dysferlin-deficient mice

Loss of dysferlin (DYSF) protein in humans results in limb-girdle muscular dystrophy 2B, characterized by progressive loss of muscles in the distal limbs with impaired locomotion. The DYSF-null (Bla/J) mouse develops severe steatotic muscles upon aging. Here, we report a marked increase in adipocytes, especially in the psoas and gluteus muscles but not in the soleus and tibialis anterior muscles in aged Bla/J mice compared with WT mice. There was a robust upregulation in the mRNA expression of enzymes involved in lipogenesis and triacylglycerol (TAG) synthesis pathways in the steatotic skeletal muscles. Lipidomic analysis of the steatotic skeletal muscles revealed an increase in several molecular species of TAG, although it is unclear whether it was at the expense of phosphatidylcholine and phosphatidylserine. The adipocytes in steatotic muscles were extramyocellular, as determined by the increased expression of caveolin 1 (a cellular marker for adipocytes) and lipid-droplet protein, perilipin 1. This increase in adipocytes occured as a consequence of the loss of myocytes.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.

Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers. When plasmids encoding the human calpain3 or dysferlin cDNA sequences were injected into quadriceps muscles of LGMD2A and LGMD2B mouse models, respectively, in 3-month studies, robust levels of calpain3 and dysferlin proteins were detected. We observed a statistically significant decrease in Evans blue dye penetration in LGMD2B mouse muscles after delivery of the dysferlin gene, consistent with repair of the muscle membrane defect in these mice. The therapeutic value of delivery of the genes for alpha-sarcoglycan and follistatin was documented by significant drops in Evans blue dye penetration in gastrocnemius muscles of LGMD2D mice. These results indicated for the first time that a combined gene therapy involving both alpha-sarcoglycan and follistatin would be valuable for LGMD2D patients. We suggest that this non-viral gene delivery method should be explored for its translational potential in patients.

Anesthetic management of a patient with limb-girdle muscular dystrophy 2B:CARE-compliant case report and literature review

BackgroundLimb-girdle muscular dystrophies (LGMDs) belong to few neuromuscular disorders mainly involving pelvic and shoulder girdle muscles. Also, cardiac or pulmonary complications, increased rhabdomyolysis risk when exposed to volatile anesthetics and succinylcholine may increase anesthesia related risks. However, current reports about the anesthesia management of these patients are limited.Case presentationWe described our anesthetic management of a 36 years old woman with LGMD 2B receiving arthroscopic knee surgery. In consideration of the high risk of rhabdomyolysis, total intravenous anesthesia (TIVA) was selected for her surgery. Considering the unpredictable respiratory depression, opioid based patient-controlled intravenous analgesia was replaced with an intra-articular cocktail therapy consisting of 20 ml of 0.2% ropivacaine. Also, we reviewed the literatures on anesthetic management of LGMD through searching PubMed, in order to provide a comprehensive and safe guidance for the surgery.ConclusionsCarefully conducted general anesthesia with TIVA technique is a good choice for LGMD patients. Neuraxial anesthesia may be used if general anesthesia needs to be avoided. To warrant safe anesthesia for surgery, any decision must be well thought out during perioperative period.

Fibroadipogenic progenitors are responsible for muscle loss in limb girdle muscular dystrophy 2B

Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) – a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.

Subsarcolemmal Cytoskeleton In Dysferlin‐Mediated Muscle Membrane Repair

Mutations in the protein dysferlin have been shown to result in Limb Girdle Muscular Dystrophy 2B and Myoshi Myopathy. In patients and mouse models lacking dysferlin, dysfunctional membrane repair compromises the muscle cell integrity and leads to muscle degeneration and progressive muscle wasting. Dysferlin is a type 2 transmembrane protein with multiple calcium‐dependent, lipid binding C2 domains, that localizes to the t‐tubules and lateral sarcolemma of the muscle fiber. Previous work from our laboratory has shown that following injury, dysferlin is rapidly recruited from the lateral sarcolemma to the wound site and this can be inhibited by pharmacological agents that disrupt the actin cytoskeleton. However, the isoform responsible for dysferlin recruitment remains unknown. The three major actin isoforms expressed in muscle, α‐, β‐, and γ‐actin, are hypothesized to have distinct functions within the myofiber. Cytoplasmic β‐and γ‐actins localize to the costameres of the muscle fiber and are the major actin isoforms that form the subsarcolemmal actin cytoskeleton, while α‐actin is largely associated with the thin filaments of the sarcomere. Interestingly, genetic disruption of γ‐actin in muscle of mice causes a mild degenerative myopathy comparable to that observed in dysferlin deficiency. Therefore, in the current study, we studied mice with genetic loss of γ‐actin in differentiated muscle to investigate whether γ‐actin may be a critical actin isoform in dysferlin recruitment and thus efficient membrane repair.While we hypothesized that γ‐actin may play a role in trafficking sarcolemma‐derived dysferlin to the site of an injury, our data suggest that dysferlin recruitment is unaffected by the genetic loss of γ‐actin. Independent of defects in dysferlin recruitment, muscle fibers isolated from the flexor digitorum brevis of γ‐actin knockout mice show increased calcium uptake following injury, indicative of a defect in membrane repair. We also show that Actg1−/− muscle fibers have an increased uptake of FM1‐43 following injury, a commonly used assay of membrane repair. However, we've previously shown that FM1‐43 uptake following injury may actually be an indicator of wound‐induced endocytosis. Therefore, we hypothesize that calcium overload in membrane repair‐defective muscle fibers from γ‐actin knockout mice may contribute to overactive‐wound induced endocytosis, leading to the observed increase in FM1‐43 uptake. We are currently investigating whether increased wound‐induced endocytosis may result in greater dysferlin endocytosis. Overall, our results suggest that γ‐actin may have an important role in membrane repair in muscle which may contribute to the myopathy observed in the γ‐actin deficient mouse model.Support or Funding InformationMTN has been supported by the NIH Cellular and Molecular Biology Training Grant T‐32‐GM007315 and the NIH Cardiovascular Research and Entrepreneurship Training Grant T32‐HL125242 and research support was from NIH NIAMS AR066213 to DM.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modeling Skeletal Muscle Laminopathies Using Human Induced Pluripotent Stem Cells Carrying Pathogenic LMNA Mutations.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in LMNA. The main proteins encoded by LMNA are Lamin A and C, which together with Lamin B1 and B2, form the nuclear lamina: a mesh-like structure located underneath the inner nuclear membrane. Laminopathies show striking tissue specificity, with subtypes affecting striated muscle, peripheral nerve, and adipose tissue, while others cause multisystem disease with accelerated aging. Although several pathogenic mechanisms have been proposed, the exact pathophysiology of laminopathies remains unclear, compounded by the rarity of these disorders and lack of easily accessible cell types to study. To overcome this limitation, we used induced pluripotent stem cells (iPSCs) from patients with skeletal muscle laminopathies such as LMNA-related congenital muscular dystrophy and limb-girdle muscular dystrophy 1B, to model disease phenotypes in vitro. iPSCs can be derived from readily accessible cell types, have unlimited proliferation potential and can be differentiated into cell types that would otherwise be difficult and invasive to obtain. iPSC lines from three skeletal muscle laminopathy patients were differentiated into inducible myogenic cells and myotubes. Disease-associated phenotypes were observed in these cells, including abnormal nuclear shape and mislocalization of nuclear lamina proteins. Nuclear abnormalities were less pronounced in monolayer cultures of terminally differentiated skeletal myotubes than in proliferating myogenic cells. Notably, skeletal myogenic differentiation of LMNA-mutant iPSCs in artificial muscle constructs improved detection of myonuclear abnormalities compared to conventional monolayer cultures across multiple pathogenic genotypes, providing a high-fidelity modeling platform for skeletal muscle laminopathies. Our results lay the foundation for future iPSC-based therapy development and screening platforms for skeletal muscle laminopathies.

LIMB-GIRDLE MUSCULAR DYSTROPHY I: P.6Functional recovery by readthrough therapy in a knock-in mouse model with nonsense dysferlin mutation

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.