Dysferlin Expression Research Articles

Locally increased dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Abstract Introduction Dysferlin, a transmembrane protein with multiple Ca2+-binding C2-domains, is expressed in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca2+ entry, Dysferlin is thought to mediate vesicle fusion and membrane repair events in order to rapidly seal sarcolemmal lesions. Dysferlin mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, the precise role of Dysferlin in protection of membrane nanodomains in ventricular myocytes (VMs) after myocardial infarction (MI) remains elusive. Purpose We hypothesized that Dysferlin is crucial to prevent VMs from loss-of-function and cell death by stabilizing membrane nanodomains like the transverse-axial tubule (TAT) system and the intercalated disc (ICD) cell-cell contact sites post-MI. Methods and Results 1 week post-MI induced by LAD artery ligation, investigator-blinded echocardiography revealed increased infarct sizes and decreased left-ventricular (LV) ejection fraction of Dysferlin-knockout (KO) vs. WT mice (mean±SEM: 21±2 vs. 28±2%, n=20/30 mice, P<0.05). Immunoblotting of WT LV tissue lysates showed an upregulated Dysferlin expression to 148% compared to sham-operated controls. Importantly, immunohistology identified a maximal Dysferlin expression of 230% in the MI border zone that faces a high ischemic and mechanical stress. Hence, we used data-independent mass spectrometry (DIA-MS) to analyse the proteomic profile of the infarction zone, border zone and remote zone from WT vs. KO hearts 1 week post-MI (each n = 5 mice). Overall, we reproducibly quantified 4360 proteins across all LV samples, identifying local genotype-specific proteotypes post-MI. In WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the zone pairwise comparison, highlighting a vital role of Dysferlin for LV remodelling post-MI. In addition, superresolution stimulated emission depletion (STED) microscopy was applied to study the subcellular localization and function of Dysferlin in the MI border zone. Here, we observed VMs with disrupted TAT endomembrane networks and significantly extended ICD regions. Interestingly, STED imaging revealed Dysferlin clusters that highly decorated residual TAT structures and interdigitated ICD membrane folds in the MI border zone. DIA-MS analysis of anti-Dysferlin co-imunoprecipitation experiments from WT vs. KO LV tissues characterized the cardiac interactome of Dysferlin, confirming interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43. Conclusions Our data demonstrate Dysferlin as an endogenous membrane repair protein necessary to stabilize sarcolemmal nanodomains in the MI border zone. Dysferlin not only prevents from loss of TAT structures, but also compensates for increased mechanical stress at the ICDs. Hence, Dysferlin emerges as a novel therapeutic target to control membrane integrity in VMs to prevent from loss-of-function and cell death post-MI.

Plasma membrane repair defect in Alzheimer's disease neurons is driven by the reduced dysferlin expression.

Alzheimer's disease (AD) is the most common neurodegenerative disease, and a defect in neuronal plasma membrane repair could exacerbate neurotoxicity, neuronal death, and disease progression. In this study, application of AD patient cerebrospinal fluid (CSF) and recombinant human Aβ to otherwise healthy neurons induces defective neuronal plasma membrane repair invitro and exvivo. We identified Aβ as the biochemical component in patient CSF leading to compromised repair capacity and depleting Aβ rescued repair capacity. These elevated Aβ levels reduced expression of dysferlin, a protein that facilitates membrane repair, by altering autophagy and reducing dysferlin trafficking to sites of membrane injury. Overexpression of dysferlin and autophagy inhibition rescued membrane repair. Overall, these findings indicate an AD pathogenic mechanism where Aβ impairs neuronal membrane repair capacity and increases susceptibility to cell death. This suggests that membrane repair could be therapeutically targeted in AD to restore membrane integrity and reduce neurotoxicity and neuronal death.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

Compromised Plasma Membrane Repair Contributes to Neuronal Cell Death and Neurodegeneration During Alzheimer’s Disease

Alzheimer’s Disease (AD) is the most common neurodegenerative disease and involves accumulation of intracellular amyloid beta (Aβ) in the early stages of the disease. Aβ has been shown to compromise neuronal cell membrane integrity by increasing membrane permeability, elevating intracellular calcium concentrations, and Aβ directly penetrating and damaging the neuronal cell membrane. Damage to the cell membrane requires a rapid and effcient repair mechanism to restore the barrier function of the membrane and avoid cell death. Here, we aimed to study the effects of intracellular Aβ on neuronal cell membrane repair. To determine if membrane permeability is compromised in vivo, we stained 6-month FVB and APP/PS1 brain sections with anti-IgG. We observed a significant increase in IgG positivity in the APP/PS1 tissue as compared to the FVB brains. We repeated this with post-mortem frontal lobe tissue and observed a significant increase in IgG-positivity in the AD samples as compared to the non-AD. To assess the effect of neuronal membrane repair in AD patients, we treated mouse neuroblastomas (N2A) and primary mouse hippocampal neurons with non-AD and AD patient cerebrospinal fluid (CSF) at a 1:100 dilution or 1μM recombinant human Aβ42. We conducted laser damage assays and observed a membrane repair defect in these neuronal cell models. We explored the biochemical components of the CSF that may be contributing to this induced repair defect, we measured the Aβ42 by ELISA and stratified Aβ42 concentration by the corresponding CSF AUC. We observed a positive correlation between Aβ42 concentrations and reduced membrane repair capacity. We sought to determine the mechanism of the reduced membrane repair capacity by assessing the expression levels of membrane repair proteins and observed decreased dysferlin in cell models, 6-month APP/PS1 mouse models, and human postmortem tissues with high levels of Aβ. Overexpression of dysferlin in the AD cell models can increase dysferlin expression and restore membrane repair capacity in neurons treated with AD CSF samples or recombinant Aβ42. We have determined this reduction in repair capacity is due to reduced dysferlin expression, and future studies will determine the mechanism leading to dysferlin downregulation. This work was supported by The Ohio State University College of Medicine Dean’s Discovery Grants Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2.

Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.

Differential Dysferlin Expression in Human Fast and Slow Muscle: Clinical Correlation with LGMD-D2 Evolution (P6-8.003)

<h3>Objective:</h3> To evaluate human dysferlin level in slow and fast human muscle and correlate its quantity with LGMD-D2 dystrophy evolution. <h3>Background:</h3> Dysferlinopathy is a common form of limb girdle muscular dystrophy (LGMD) presenting either as LGMDR2/2B or distal Miyoshi myopathy, that show different clinical course. They are caused by mutations in the DYSF gene encoding the protein dysferlin, a transmembrane sarcolemmal protein which has a role in the repair of sarcolemma. Dysferlin is also a T-tubule protein. Dysferlin expression in skeletal muscle fibre types has not been studied so far. <h3>Design/Methods:</h3> We investigated by Western Blotting human skeletal muscles, one proximal in the arm (biceps brachii) and the other distal in the leg (tibialis anterior), which are affected differently during the course of LGMDR2: with onset first in tibialis anterior (around the age of 20 years) and only late involvement the biceps brachii, around the age of 45 years. Muscle specimen were collected after a few hours either in control from sudden death or in muscle biopsies (male 60, 63 year/old; female 48, 52 year/old) of biceps brachii and tibialis anterior and in the 4 muscles analysed, Dysferlin/Myosin ratio was calculated. <h3>Results:</h3> Western blotting results from skeletal muscles showed that in arm the biceps brachii and in leg distal tibialis mucle contains a different amont of dysferlin protein. Dysferlin to myosin heavy chain ratio was higher in biceps brachii (2.26–2.36) than in tibialis anterior muscle (1.17–1.56). Our results show a higher “content” of dysferlin in fast skeletal muscle, i.e. biceps brachii, compared to slow, i.e. tibialis anterior, related to increased resistance to eccentric exercise and muscle fiber injury. <h3>Conclusions:</h3> We found a higher level of dysferlin in biceps brachii, which is involved later in LGMD-D2 disease course versus tibialis anterior muscle, that degenerates earlier, this difference might explain the different muscle involvement observed in LGMDR2/2B and Miyoshi myopathy. <b>Disclosure:</b> Dr. MEZNARIC has nothing to disclose. Dr. Angelini has nothing to disclose.

Patch repair protects cells from the small pore-forming toxin aerolysin.

Aerolysin family pore-forming toxins damage the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four proposed membrane repair mechanisms include toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which repair mechanism aerolysin triggers is unknown. Membrane repair requires Ca2+, but it is controversial if Ca2+ flux is triggered by aerolysin. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to what is seen with cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Intracellular Ca2+ chelation increased cell death, indicating that Ca2+-dependent repair pathways were triggered. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to what is seen with CDCs, expression of the patch repair protein dysferlin protected cells from aerolysin. We propose aerolysin triggers a Ca2+-dependent death mechanism that obscures repair, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

Ancestry dependent balancing selection of placental dysferlin at high-altitude.

Introduction: The placenta mediates fetal growth by regulating gas and nutrient exchange between the mother and the fetus. The cell type in the placenta where this nutrient exchange occurs is called the syncytiotrophoblast, which is the barrier between the fetal and maternal blood. Residence at high-altitude is strongly associated with reduced 3rd trimester fetal growth and increased rates of complications such as preeclampsia. We asked whether altitude and/or ancestry-related placental gene expression contributes to differential fetal growth under high-altitude conditions, as native populations have greater fetal growth than migrants to high-altitude. Methods: We have previously shown that methylation differences largely accounted for altitude-associated differences in placental gene expression that favor improved fetal growth among high-altitude natives. We tested for differences in DNA methylation between Andean and European placental samples from Bolivia [La Paz (∼3,600m) and Santa Cruz, Bolivia (∼400m)]. One group of genes showing significant altitude-related differences are those involved in cell fusion and membrane repair in the syncytiotrophoblast. Dysferlin (DYSF) shows greater expression levels in high- vs. low-altitude placentas, regardless of ancestry. DYSF has a single nucleotide variant (rs10166384;G/A) located at a methylation site that can potentially stimulate or repress DYSF expression. Following up with individual DNA genotyping in an expanded sample size, we observed three classes of DNA methylation that corresponded to individual genotypes of rs10166384 (A/A < A/G < G/G). We tested whether these genotypes are under Darwinian selection pressure by sequencing a ∼2.5kb fragment including the DYSF variants from 96 Bolivian samples and compared them to data from the 1000 genomes project. Results: We found that balancing selection (Tajima's D = 2.37) was acting on this fragment among Andeans regardless of altitude, and in Europeans at high-altitude (Tajima's D = 1.85). Discussion: This supports that balancing selection acting on dysferlin is capable of altering DNA methylation patterns based on environmental exposure to high-altitude hypoxia. This finding is analogous to balancing selection seen frequency-dependent selection, implying both alleles are advantageous in different ways depending on environmental circumstances. Preservation of the adenine (A) and guanine (G) alleles may therefore aid both Andeans and Europeans in an altitude dependent fashion.

Dysferlin facilitates the de novo biogenesis of tubular membranes in hypertrophic remodelling

Abstract Introduction Ferlins are transmembrane proteins with multiple C2 domains that may mediate Ca2+ dependent membrane fusion and repair events. In ventricles, Dysferlin expression is dominant, and patient mutations have been associated with cardiomyopathies. Hence, we hypothesized that Dysferlin is essential for the functional integrity of the tubular endomembrane system in ventricular myocytes (VM), and may facilitate the cellular hypertrophic remodelling in pressure-overload. Results In 30 weeks old Dysferlin knockout (KO) mice, echocardiography revealed dilative cardiomyopathy with reduced left-ventricular ejection fractions (difference between means KO vs. wildtype (WT) −13.45±2.17%, p&amp;lt;0.001, n=16/20 mice). Asking for the subcellular relevance of Dysferlin, we investigated VM nanodomains by immunolabelling in STimulated Emission Depletion (STED) nanoscopy and visualized punctual Dysferlin clusters decorating the T-tubule system of mouse and human VM. Precisely, Dysferlin clusters localized to junctions of the tubular system and the sarcoplasmic reticulum (SR) in nanometric proximity to RyR2 Ca2+ release units. Complexome profiling (LC-MS/MS) also suggested interactions of Dysferlin with RyR2 and the SR membrane-tethering protein Junctophilin-2 (JP2) in high molecular weight complexes. In JP2-overexpressing mice, live-cell STED membrane imaging captured polyadic junctional membrane structures in VM, specifically surrounded by dense Dysferlin clusters, which proposes Dysferlin as stabilizing protein of tubule-SR junctions. Further, we studied the ultrastructural remodelling of the tubular system in a mouse model of cardiac hypertrophy and heart failure induced by transaortic constriction (TAC). Interestingly, mortality four weeks post TAC was not increased in KO mice. However, echocardiography uncovered that Dysf-KO prevents left-ventricular hypertrophy in response to pressure-overload, which was confirmed by single cell measurements (VM area: WT 3559±120 μm2 vs. KO 2671±87 μm2, n=163/158 VM, p&amp;lt;0.001). Thus, we assumed Dysferlin to mediate tubular network proliferation during hypertrophic remodelling. Indeed, WT VM showed an augmented Dysferlin expression post TAC (158±0,125%, p=0,005, n=7/7 mice), and immunofluorescence STED nanoscopy detected an increased Dysferlin network complexity. In detail, we visualized highly abundant Dysferlin clusters decorating newly shaped axial tubules, which were quantified by component-specific tubule network analysis. Conclusion Nanoscale imaging proposes Dysferlin as stabilizer of tubule-SR junctions, thereby promoting functional excitation-contraction coupling, and demonstrates the crucial importance of Dysferlin in pressure-overload induced hypertrophic remodelling, facilitating the de novo biogenesis of axial tubule membranes and tubule-SR junctions necessary for VM growth. Thus, Dysferlin may emerge as potential novel therapeutic rationale to control subcellular membrane remodelling in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Multiscale Bioimaging Cluster of Excellence: From Molecular Machines to Networks of Excitable Cells;German Research Foundation (DFG) - Collaborative Research Centre 1002 (SFB 1002): Modulatory Units in Heart Failure

Proteins implicated in muscular dystrophy and cancer are functional constituents of the centrosome.

Aberrant expression of dystrophin, utrophin, dysferlin, or calpain-3 was originally identified in muscular dystrophies (MDs). Increasing evidence now indicates that these proteins might act as tumor suppressors in myogenic and non-myogenic cancers. As DNA damage and somatic aneuploidy, hallmarks of cancer, are early pathological signs in MDs, we hypothesized that a common pathway might involve the centrosome. Here, we show that dystrophin, utrophin, dysferlin, and calpain-3 are functional constituents of the centrosome. In myoblasts, lack of any of these proteins caused excess centrosomes, centrosome misorientation, nuclear abnormalities, and impaired microtubule nucleation. In dystrophin double-mutants, these defects were significantly aggravated. Moreover, we demonstrate that also in non-myogenic cells, all four MD-related proteins localize to the centrosome, including the muscle-specific full-length dystrophin isoform. Therefore, MD-related proteins might share a convergent function at the centrosome in addition to their diverse, well-established muscle-specific functions. Thus, our findings support the notion that cancer-like centrosome-related defects underlie MDs and establish a novel concept linking MDs to cancer.

Structural and ultrastructural changes in the skeletal muscles of dysferlin-deficient mice during postnatal ontogenesis

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

DYSF promotes monocyte activation in atherosclerotic cardiovascular disease as a DNA methylation-driven gene

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

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.

Effect of Dysferlin Deficiency on Atherosclerosis and Plasma Lipoprotein Composition Under Normal and Hyperlipidemic Conditions.

Dysferlinopathies are a group of muscle disorders caused by mutations to dysferlin, a transmembrane protein involved in membrane patching events following physical damage to skeletal myofibers. We documented dysferlin expression in vascular tissues including non-muscle endothelial cells, suggesting that blood vessels may have an endogenous repair system that helps promote vascular homeostasis. To test this hypothesis, we generated dysferlin-null mice lacking apolipoprotein E (ApoE), a common model of atherosclerosis, dyslipidemia and endothelial injury when stressed with a high fat, and cholesterol-rich diet. Despite high dysferlin expression in mouse and human atheromatous plaques, loss of dysferlin did not affect atherosclerotic burden as measured in the aortic root, arch, thoracic, and abdominal aortic regions. Interestingly, we observed that dysferlin-null mice exhibit lower plasma high-density lipoprotein cholesterol (HDL-C) levels than their WT controls at all measured stages of the disease process. Western blotting revealed abundant dysferlin expression in protein extracts from mouse livers, the main regulator of plasma lipoprotein levels. Despite abnormal lipoprotein levels, Dysf/ApoE double knockout mice responded to cholesterol absorption blockade with lower total cholesterol and blunted atherosclerosis. Our study suggests that dysferlin does not protect against atherosclerosis or participate in cholesterol absorption blockade but regulates basal plasma lipoprotein composition. Dysferlinopathic patients may be dyslipidemic without greater atherosclerotic burden while remaining responsive to cholesterol absorption blockade.

Integrated Genome and Transcriptome Sequencing to Solve a Neuromuscular Puzzle: Miyoshi Muscular Dystrophy and Early Onset Primary Dystonia in Siblings of the Same Family.

BackgroundNeuromuscular disorders (NMD), many of which are hereditary, affect muscular function. Due to advances in high-throughput sequencing technologies, the diagnosis of hereditary NMDs has dramatically improved in recent years.Methods and ResultsIn this study, we report an family with two siblings exhibiting two different NMD, Miyoshi muscular dystrophy (MMD) and early onset primary dystonia (EOPD). Whole exome sequencing (WES) identified a novel monoallelic frameshift deletion mutation (dysferlin: c.4404delC/p.I1469Sfs∗17) in the Dysferlin gene in the index patient who suffered from MMD. This deletion was inherited from his unaffected father and was carried by his younger sister with EOPD. However, immunostaining staining revealed an absence of dysferlin expression in the proband’s muscle tissue and thus suggested the presence of the second underlying mutant allele in dysferlin. Using integrated RNA sequencing (RNA-seq) and whole genome sequencing (WGS) of muscle tissue, a novel deep intronic mutation in dysferlin (dysferlin: c.5341-415A > G) was discovered in the index patient. This mutation caused aberrant mRNA splicing and inclusion of an additional pseudoexon (PE) which we termed PE48.1. This PE was inherited from his unaffected mother. PE48.1 inclusion altered the Dysferlin sequence, causing premature termination of translation.ConclusionUsing integrated genome and transcriptome sequencing, we discovered hereditary MMD and EOPD affecting two siblings of same family. Our results added further weight to the combined use of RNA-seq and WGS as an important method for detection of deep intronic gene mutations, and suggest that integrated sequencing assays are an effective strategy for the diagnosis of hereditary NMDs.

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.

Abnormal Expression of Dysferlin in Blood Monocytes Supports Primary Dysferlinopathy in Patients Confirmed by Genetic Analyses.

Objective: Dysferlin deficiency causes dysferlinopathy. This study aimed to expand the mutational spectrum of dysferlinopathies, to further study one case with diagnostic ambiguity, and to identify the diagnostic value of dysferlin expression in total peripheral blood mononuclear cells (PBMC).Methods: The clinical and molecular profiles of dysferlinopathies in eight Chinese patients were evaluated. We also conducted magnetic resonance imaging (6/8) and determined dysferlin protein expression in muscle (7/8) and PBMC (3/8).Results: Nine of the 13 DYSF mutations identified were novel. One patient was homozygous for the Gln111Ter mutation by genomic DNA sequencing but was found to be heterozygous by sequencing of cDNA from total PBMC. A daughter of this patient did not carry any Gln111Ter mutation. Abnormal muscle MRI with predominant involvement of the medial gastrocnemius and soleus muscle was observed in 5/6 patients. Dysferlin levels were significantly reduced (immunohistochemistry/immunoblot) or absent (immunohistochemistry) in muscle and total PBMC (26–39%) for most patients. Sarcoplasmic accumulation of dysferlin was detected in one patient.Conclusion: Genomic DNA sequencing detects frequent homozygous mutations, while fewer heterozygous mutations in cDNA are detected after posttranscription. Total PBMC may serve as an alternative to confirm diagnosis and to guide further testing in dysferlinopathies. Our results contribute to the mutational spectrum of dysferlinopathies.

Proteasome inhibitors reduce thrombospondin-1 release in human dysferlin-deficient myotubes

BackgroundDysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of both human and mouse models of dysferlinopathy suggest dysferlin deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patients with two missense mutations in exon 44.MethodsTo assess proteasome inhibition we treated dysferlin deficient myotubes with EB1089, a vitamin D3 analog, oprozomib and ixazomib. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury and fusion index was also measured with the different treatments. Data were analyzed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p ≤ 0.05 was considered statistically significant.ResultsTreatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on the repair of muscle membrane after injury.ConclusionsOur findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

A Rare Case of Dysferlinopathy Causing Cardiomyopathy

Introduction We report a case of a 30-year-old man who presented with advanced heart failure secondary to dilated cardiomyopathy. The patient was diagnosed with limb girdle muscular dystrophy type 2B (LGMD2B), a rare autosomal recessive disease. Cardiomyopathy is only rarely described in dysferlinopathies. Case This patient is a 30-year old African American male with medical history of left foot crush injury in 2016. The following year after injury he developed progressive exercise intolerance and palpitations. An electrocardiogram (ECG) showed sinus tachycardia. Echocardiogram revealed a severely dilated left ventricle, with increased wall thickness, apical trabeculations and ejection fraction (EF) of 20%. Right ventricle had normal size and function, and there was no significant valvular disease. His functional capacity and cardiac performance on imaging tests did not improve despite guideline directed medical therapy. He also had persistently elevated transaminases. Chronic liver disease workup was negative with liver biopsy showing mild sinusoidal dilatation without significant fibrosis. Persistently elevated transaminases in the absence of liver disease and significant systemic congestion prompted testing for muscle enzymes, which were severely elevated (CK-16,196; Aldolase 84.3 U/l). Neurologic examined revealed mild proximal muscle weakness. Further evaluation included electromyography (EMG), which was consistent with chronic myopathy. Genetic testing revealed a pathogenic splice site variant (c.2643+1GA), and two variants of uncertain significance (p.Asn367Lys, p.Arg553His) in the dysferlin gene. Muscle biopsy was performed which showed active myopathic features with myofiber necrosis and regeneration (Figure 1A, 1B). No chronic remodeling was present. Immunohistochemical staining showed loss of dysferlin expression (figure 1C; insert normal control). Expression of other sarcolemmal proteins like dystrophin was preserved (figure 1D). The biopsy findings support a diagnosis of a dysferlinopathy in the context of the described genetic variants and argue for the combination of the described variants to be disease causing. Conclusion LGMD2B is a rare limb girdle muscular dystrophy with very few previously reported cases of cardiac muscle involvement. Our case highlights the importance of a comprehensive diagnostic evaluation of idiopathic cardiomyopathy in young patients. Chronically elevated transaminases in young patients should prompt evaluation for myopathy, and genetic evaluation in the appropriate clinical context.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.147 A simple and rapid neutrophils immunoassay for dysferlinopathies

Dysferlin is a protein of approximately 237 kDa predominantly expressed in striated muscle. Mutations in the gene encoding for dysferlin (DYSF) are responsible for various types of rare autosomal recessive muscular dystrophies such as limb girdle muscular dystrophy R2 (formerly LGMD2B) and distal Miyoshi Myopathy. Absence of dysferlin expression assessed by immunoblot of muscle biopsies or CD14+ peripheral blood monocytes is a highly specific diagnostic indicator of disease. CD14+ immunoblot is generally preferred over the muscle biopsy as being a less invasive and more cost-effective assay.