Comprehensive Proteomic Analysis of Dysferlinopathy Unveiling Molecular Mechanisms and Biomarkers Linked to Pathological Progression.

AIMS: Previous proteomics studies in dysferlinopathy muscle have been limited in scope, often utilizing 2D-electrophoresis and yielding only a small number of differential expression calls. To address this gap, this study aimed to employ high-resolution proteomics to explore the proteomic landscapes of dysferlinopathy and analyze the correlation between muscle pathological changes and alterations in protein expression in muscle biopsies. METHODS: We conducted a comprehensive approach to investigate the proteomic profile and disease-associated changes in the muscle tissue proteome from 15 patients with dysferlinopathy, exhibiting varying degrees of dystrophic pathology, alongside age-matched controls. Our methodology encompasses tandem mass tag (TMT)-labeled liquid chromatography-mass spectrometry (LC-MS/MS)-based proteomics, protein-protein interaction (PPI) network analysis, weighted gene co-expression network analysis, and differential expression analysis. Subsequently, we examined the correlation between the expression of key proteins and the clinical characteristics of the patients to identify pathogenic targets associated with DYSF mutations in dysferlinopathy. RESULTS: A total of 1600 differentially expressed proteins were identified, with 1321 showing high expression levels and 279 expressed at lower levels. Our investigation yields a molecular profile delineating the altered protein networks in dysferlinopathy-afflicted skeletal muscle, uncovering dysregulation across numerous cellular pathways and molecular processes, including mRNA metabolic processes, regulated exocytosis, immune response, muscle system processes, energy metabolic processes, and calcium transmembrane transport. Moreover, we observe significant associations between the protein expression of ANXA1, ANXA2, ANXA4, ANXA5, LMNA, PYGM, and the extent of histopathologic changes in muscle biopsies from patients with dysferlinopathy, validated through immunoblotting and immunofluorescence assays. CONCLUSIONS: Through the aggregation of expression data from dysferlinopathy-impacted muscles exhibiting a range of pathological alterations, we identified multiple key proteins associated with the dystrophic pathology of patients with dysferlinopathy. These findings provide novel insights into the pathogenesis of dysferlinopathy and propose promising targets for future therapeutic endeavors.

Pilot investigations into the mechanistic basis for adverse effects of glucocorticoids in dysferlinopathy.

BACKGROUND: Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by gene mutations resulting in deficiency of the membrane-associated protein dysferlin. They manifest post-growth and are characterised by muscle wasting (primarily in the limb and limb-gridle muscles), inflammation, and replacement of myofibres with adipose tissue. The precise pathomechanism for dysferlinopathy is currently unclear; as such there are no treatments currently available. Glucocorticoids (GCs) are widely used to reduce inflammation and treat muscular dystrophies, but when administered to patients with dysferlinopathy, they have unexpected adverse effects, with accelerated loss of muscle strength. METHODS: To investigate the mechanistic basis for the adverse effects of GCs in dysferlinopathy, the potent GC dexamethasone (Dex) was administered for 4-5 weeks (0.5-0.75 µg/mL in drinking water) to dysferlin-deficient BLA/J and normal wild-type (WT) male mice, sampled at 5 (Study 1) or 10 months (Study 2) of age. A wide range of analyses were conducted. Metabolism- and immune-related gene expression was assessed in psoas muscles at both ages and in quadriceps at 10 months of age. For the 10-month-old mice, quadriceps and psoas muscle histology was assessed. Additionally, we investigated the impact of Dex on the predominantly slow and fast-twitch soleus and extensor digitorum longus (EDL) muscles (respectively) in terms of contractile function, myofibre-type composition, and levels of proteins related to contractile function and metabolism, plus glycogen. RESULTS: At both ages, many complement-related genes were highly expressed in BLA/J muscles, and WT mice were generally more responsive to Dex than BLA/J. The effects of Dex on BLA/J mice included (i) increased expression of inflammasome-related genes in muscles (at 5 months) and (ii) exacerbated histopathology of quadriceps and psoas muscles at 10 months. A novel observation was pronounced staining for glycogen in many myofibres of the damaged quadriceps muscles, with large pale vacuolated myofibres, suggesting possible myofibre death by oncosis. CONCLUSION: These pilot studies provide a new focus for further investigation into the adverse effects of GCs on dysferlinopathic muscles.

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.

Loss of Calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy R1/2A (LGMD R1/2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wild-type (WT) mice, we determined whether loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD R1/2A pathology.

Bioengineered Model of Human LGMD2B Skeletal Muscle Reveals Roles of Intracellular Calcium Overload in Contractile and Metabolic Dysfunction in Dysferlinopathy.

Dysferlin is a multi-functional protein that regulates membrane resealing, calcium homeostasis, and lipid metabolism in skeletal muscle. Genetic loss of dysferlin results in limb girdle muscular dystrophy 2B/2R (LGMD2B/2R) and other dysferlinopathies - rare untreatable muscle diseases that lead to permanent loss of ambulation in humans. The mild disease severity in dysferlin-deficient mice and diverse genotype-phenotype relationships in LGMD2B patients have prompted the development of new in vitro models for personalized studies of dysferlinopathy. Here the first 3-D tissue-engineered hiPSC-derived skeletal muscle ("myobundle") model of LGMD2B is described that exhibits compromised contractile function, calcium-handling, and membrane repair, and transcriptomic changes indicative of impaired oxidative metabolism and mitochondrial dysfunction. In response to the fatty acid (FA) challenge, LGMD2B myobundles display mitochondrial deficits and intracellular lipid droplet (LD) accumulation. Treatment with the ryanodine receptor (RyR) inhibitor dantrolene or the dissociative glucocorticoid vamorolone restores LGMD2B contractility, improves membrane repair, and reduces LD accumulation. Lastly, it is demonstrated that chemically induced chronic RyR leak in healthy myobundles phenocopies LGMD2B contractile and metabolic deficit, but not the loss of membrane repair capacity. Together, these results implicate intramyocellular Ca2+ leak as a critical driver of dysferlinopathic phenotype and validate the myobundle system as a platform to study LGMD2B pathogenesis.

Mitochondrial abnormalities contribute to muscle weakness in a Dnajb6 deficient zebrafish model.

Mutations in DNAJB6 are a well-established cause of limb girdle muscular dystrophy type D1 (LGMD D1). Patients with LGMD D1 develop progressive muscle weakness with histology showing fibre damage, autophagic vacuoles, and aggregates. Whilst there are many reports of LGMD D1 patients, the role of DNAJB6 in the muscle is still unclear. In this study, we developed a loss of function zebrafish model in order to investigate the role of Dnajb6. Using a double dnajb6a and dnajb6b mutant model, we show that loss of Dnajb6 leads to a late onset muscle weakness. Interestingly, we find that adult fish lacking Dnajb6 do not have autophagy or myofibril defects, however, they do show mitochondrial changes and damage. This study demonstrates that loss of Dnajb6 causes mitochondrial defects and suggests that this contributes to muscle weakness in LGMD D1. These findings expand our knowledge of the role of Dnajb6 in the muscle and provides a model to screen novel therapies for LGMD D1.

Validation of a novel western blot assay to monitor patterns and levels of alpha dystroglycan in skeletal muscle of patients with limb girdle muscular dystrophies.

The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.

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.

Gene therapy with bidridistrogene xeboparvovec for limb-girdle muscular dystrophy type 2E/R4: phase 1/2 trial results.

Limb-girdle muscular dystrophy 2E/R4 is caused by mutations in the ß-sarcoglycan (SGCB) gene, leading to SGCB deficiency and consequent muscle loss. We developed a gene therapy approach based on functional replacement of the deficient SCB protein. Here we report interim results from a first-in-human, open-label, nonrandomized, phase 1/2 trial evaluating the safety and efficacy of bidridistrogene xeboparvovec, an adeno-associated virus-based gene therapy containing a codon-optimized, full-length human SGCB transgene. Patients aged 4-15 years with confirmed SGCB mutations at both alleles received one intravenous infusion of either 1.85 × 1013 vector genome copies kg-1 (Cohort 1, n = 3) or 7.41 × 1013 vector gene copies kg-1 (Cohort 2, n = 3). Primary endpoint was safety, and secondary endpoint was change in SGCB expression in skeletal muscle from baseline to Day 60. We report interim Year 2 results (trial ongoing). The most frequent treatment-related adverse events were vomiting (four of six patients) and gamma-glutamyl transferase increase (three of six patients). Serious adverse events resolved with standard therapies. Robust SGCB expression was observed: Day 60 mean (s.d.) percentage of normal expression 36.2% (2.7%) in Cohort 1 and 62.1% (8.7%) in Cohort 2. Post hoc exploratory analysis showed preliminary motor improvements using the North Star Assessment for Limb-girdle Type Muscular Dystrophies maintained through Year 2. The 2-year safety and efficacy of bidridistrogene xeboparvovec support clinical development advancement. Further studies are necessary to confirm the long-term safety and efficacy of this gene therapy. ClinicalTrials.gov registration: NCT03652259 .

Altered expression of proteins involved in metabolism in LGMDR1 muscle is lost in cell culture conditions.

BACKGROUND: Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy due to mutations in the CAPN3 gene. While the pathophysiology of this disease has not been clearly established yet, Wnt and mTOR signaling pathways impairment in LGMDR1 muscles has been reported. RESULTS: A reduction in Akt phosphorylation ratio and upregulated expression of proteins implicated in glycolysis (HK-II) and in fructose and lactate transport (GLUT5 and MCT1) in LGMDR1 muscle was observed. In vitro analysis to establish mitochondrial and glycolytic functions of primary cultures were performed, however, no differences between control and patients were observed. Additionally, gene expression analysis showed a lack of correlation between primary myoblasts/myotubes and LGMDR1 muscle while skin fibroblasts and CD56- cells showed a slightly better correlation with muscle. FRZB gene was upregulated in all the analyzed cell types (except in myoblasts). CONCLUSIONS: Proteins implicated in metabolism are deregulated in LGMDR1 patients' muscle. Obtained results evidence the limited usefulness of primary myoblasts/myotubes for LGMDR1 gene expression and metabolic studies. However, since FRZB is the only gene that showed upregulation in all the analyzed cell types it is suggested its role as a key regulator of the pathophysiology of the LGMDR1 muscle fiber. The Wnt signaling pathway inactivation, secondary to FRZB upregulation, and GLUT5 overexpression may participate in the impaired adipogenesis in LGMD1R patients.

CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy.

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC50) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products.

Dysferlinopathy in Tunisia: clinical spectrum, genetic background and prognostic profile.

Dysferlinopathy is a rare group of hereditary muscular dystrophy with an autosomal recessive mode of inheritance caused by a mutation in the DYSF gene. It encodes for the dysferlin protein, which has a crucial role in multiple cellular processes, including muscle fiber membrane repair. This deficit has heterogeneous clinical presentations. In this study, we collected 20 Tunisian patients with a sex ratio of 1 and a median age of 50.5 years old (Interquartile range (IQR) = [36,5-54,75]). They were followed for periods ranging from 5 to 48 years. The median age at onset was 17 years old (IQR = [16,8-28,4]). Five major phenotypes were identified: Limb-girdle muscular dystrophy (LGMDR2) (35%), a proximodistal phenotype (35%), Miyoshi myopathy (10%),  Distal myopathy with anterior tibial onset (DMAT) (10%), and asymptomatic HyperCKemia (10%). At the last evaluation, more than half of patients (55%) were on wheelchair. Loss of ambulation occurred generally during the fourth decade. After 20 years of disease progression, two patients with a proximodistal phenotype (10%) developed dilated cardiomyopathy and mitral valve regurgitation. Restrictive respiratory syndrome was observed in three patients (DMAT: 1 patient, proximodistal phenotype: 1 patient, LGMDR2: 1 patient). Genetic study disclosed five mutations. We observed clinical heterogeneity between families and even within the same family. Disease progression was mainly slow to intermediate regardless of the phenotype.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2 F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of ß-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.

In Vivo DYSF Gene Viral Delivery Provides a Histoprotective Effect in Skeletal Muscle Tissue in Dysferlin-Deficient Mice.

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

Phenotype Genotype Characterization of FKRP-related Muscular Dystrophy among Indian Patients.

BACKGROUND: The phenotypic spectrum of Fukutin-related protein (FKRP) mutations is highly variable and comprises of limb girdle muscular dystrophy (LGMD) R9 (previously LGMD 2I) and FKRP related congenital muscular dystrophies. OBJECTIVE: To identify the distinct genotype phenotype pattern in Indian patients with FKRP gene mutations. METHODS: We retrospectively reviewed the case files of patients with muscular dystrophy having a genetically confirmed FKRP mutation. All patients had undergone genetic testing using next-generation sequencing. RESULTS: Our patients included five males and four females presenting between 1.5 years and seven years of age (median age - 3 years). The initial symptom was a delayed acquisition of gross motor developmental milestones in seven patients and recurrent falls and poor sucking in one patient each. Two patients had a language delay, with both having abnormalities on the brain MRI. Macroglossia, scapular winging, and facial weakness were noted in one, three and four patients respectively. Calf muscle hypertrophy was seen in eight patients and ankle contractures in six. At the last follow-up, three patients had lost ambulation (median age - 7 years; range 6.5-9 years) and three patients had not attained independent ambulation. Creatine kinase levels ranged between 2793 and 32,396 U/L (mean 12,120 U/L). A common mutation - c.1343C>T was noted in 5 patients in our cohort. Additionally, four novel mutations were identified. Overall, six patients had an LGMD R9 phenotype, and three had a congenital muscular dystrophy phenotype. CONCLUSION: Patients with FKRP mutations can have varied presentations. A Duchenne-like phenotype was the most commonly encountered pattern in our cohort, with c.1343C>T being the most common mutation.

TRIM32 biallelic defects cause limb-girdle muscular dystrophy R8: identification of two novel mutations and investigation of genotype-phenotype correlation.

BACKGROUND: Limb-girdle muscular dystrophy R8 (LGMD R8) is a rare autosomal recessive muscle disease caused by TRIM32 gene biallelic defects. The genotype-phenotype correlation of this disease has been reported poorly. Here, we report a Chinese family with two female LGMD R8 patients. METHODS: We performed whole-genome sequencing (WGS) and Sanger sequencing on the proband. Meanwhile, the function of mutant TRIM32 protein was analyzed by bioinformatics and experimental analysis. In addition, a summary of the reported TRIM32 deletions and point mutations and an investigation of genotype-phenotype correlation were performed through a combined analysis of the two patients and other cases reported in previous literature. RESULTS: The two patients displayed typical symptoms of LGMD R8, which worsened during pregnancy. Genetic analysis by whole-genome sequencing (WGS) and Sanger sequencing showed that the patients were compound heterozygotes of a novel deletion (chr9.hg19:g.119431290_119474250del) and a novel missense mutation (TRIM32:c.1700A > G, p.H567R). The deletion encompassed 43 kb and resulted in the removal of the entire TRIM32 gene. The missense mutation altered the structure and further affected function by interfering with the self-association of the TRIM32 protein. Females with LGMD R8 showed less severe symptoms than males, and patients carrying two mutations in NHL repeats of the TRIM32 protein had earlier disease onset and more severe symptoms than other patients. CONCLUSIONS: This research extended the spectrum of TRIM32 mutations and firstly provided useful data on the genotype-phenotype correlation, which is valuable for the accurate diagnosis and genetic counseling of LGMD R8.

Myostatin and follistatin as monitoring and prognostic biomarkers in dysferlinopathy.

Myostatin is a myokine which acts upon skeletal muscle to inhibit growth and regeneration. Myostatin is endogenously antagonised by follistatin. This study assessed serum myostatin and follistatin concentrations as monitoring or prognostic biomarkers in dysferlinopathy, an autosomal recessively inherited muscular dystrophy. Myostatin was quantified twice with a three-year interval in 76 patients with dysferlinopathy and 38 controls. Follistatin was quantified in 62 of these patients at the same timepoints, and in 31 controls. Correlations with motor function, muscle fat fraction and contractile cross-sectional area were performed. A regression model was used to account for confounding variables. Baseline myostatin, but not follistatin, correlated with baseline function and MRI measures. However, in individual patients, three-year change in myostatin did not correlate with functional or MRI changes. Linear modelling demonstrated that function, serum creatine kinase and C-reactive protein, but not age, were independently related to myostatin concentration. Baseline myostatin concentration predicted loss of ambulation but not rate of change of functional or MRI measures, even when relative inhibition with follistatin was considered. With adjustment for extra-muscular causes of variation, myostatin could form a surrogate measure of functional ability or muscle mass, however myostatin inhibition does not form a promising treatment target in dysferlinopathy.

Assessing the Role of Aquaporin 4 in Skeletal Muscle Function.

Water transport across the biological membranes is mediated by aquaporins (AQPs). AQP4 and AQP1 are the predominantly expressed AQPs in the skeletal muscle. Since the discovery of AQP4, several studies have highlighted reduced AQP4 levels in Duchenne muscular dystrophy (DMD) patients and mouse models, and other neuromuscular disorders (NMDs) such as sarcoglycanopathies and dysferlinopathies. AQP4 loss is attributed to the destabilizing dystrophin-associated protein complex (DAPC) in DMD leading to compromised water permeability in the skeletal muscle fibers. However, AQP4 knockout (KO) mice appear phenotypically normal. AQP4 ablation does not impair physical activity in mice but limits them from achieving the performance demonstrated by wild-type mice. AQP1 levels were found to be upregulated in DMD models and are thought to compensate for AQP4 loss. Several groups investigated the expression of other AQPs in the skeletal muscle; however, these findings remain controversial. In this review, we summarize the role of AQP4 with respect to skeletal muscle function and findings in NMDs as well as the implications from a clinical perspective.

Clinical exome sequencing identifies novel compound heterozygous mutations of the POMT2 gene in patients with limb-girdle muscular dystrophy.

OBJECTIVE: Mutations in protein O-mannosyltransferase 2 (POMT2) (MIM#607439) have been identified in severe congenital muscular dystrophy such as Walker-Warburg syndrome (WWS) and milder limb-girdle muscular dystrophy type 2N (LGMD2N). The aim of this study is to investigate the genetic causes in patients with LGMD2N. METHODS: Three patients diagnosed with mild limb-girdle muscular dystrophy were recruited. The genetically pathogenic variant was identified by clinical exome sequencing, and healthy controls were verified by Sanger sequencing. RESULTS: Novel compound heterozygous mutations c.800A > G and c.1074_1075delinsAT of POMT2 were revealed in one affected individual by clinical exome sequencing. There was no report of these two variants and predicted to be highly damaging to the function of the POMT2. CONCLUSION: The novel variants extend the spectrum of POMT2 mutations, which promotes the prognostic value of testing for POMT2 mutations in patients with LGMD2N.

Golgi Complex form and Function: A Potential Hub Role Also in Skeletal Muscle Pathologies?

A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC's form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC's form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary (e.g., mutation of a glycosylation enzyme), or secondary mechanisms (e.g., GC mis-localization in Duchenne muscles), which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies.