Limb-girdle muscular dystrophies are a genetically diverse group of diseases characterized by chronic muscle wasting and weakness. Recessive mutations in ANO5 (TMEM16E) have been directly linked to several clinical phenotypes including limb-girdle muscular dystrophy type 2L and Miyoshi myopathy type 3, although the pathogenic mechanism has remained elusive. ANO5 is a member of the Anoctamin/TMEM16 superfamily that encodes both ion channels and regulators of membrane phospholipid scrambling. The phenotypic overlap of ANO5 myopathies with dysferlin-associated muscular dystrophies has inspired the hypothesis that ANO5, like dysferlin, may be involved in the repair of muscle membranes following injury. Here we show that Ano5-deficient mice have reduced capacity to repair the sarcolemma following laser-induced damage, exhibit delayed regeneration after cardiotoxin injury and suffer from defective myoblast fusion necessary for the proper repair and regeneration of multinucleated myotubes. Together, these data suggest that ANO5 plays an important role in sarcolemmal membrane dynamics. Genbank Mouse Genome Informatics accession no. 3576659.

ImportanceICU-acquired muscle atrophy occurs commonly and worsens outcomes in adults. The incidence and severity of muscle atrophy in critically ill children are poorly characterized.ObjectiveTo determine incidence, severity and risk factors for muscle atrophy in critically ill children.Design, setting and participantsA single-center, prospective cohort study of 34 children receiving invasive mechanical ventilation for ≥48 hours. Patients 1 week– 18 years old with respiratory failure and without preexisting neuromuscular disease or skeletal trauma were recruited from a tertiary Pediatric Intensive Care Unit (PICU) between June 2015 and May 2016. We used serial bedside ultrasound to assess thickness of the diaphragm, biceps brachii/brachialis, quadriceps femoris and tibialis anterior. Serial electrical impedance myography (EIM) was assessed in children >1 year old. Medical records were abstracted from an electronic database.ExposuresRespiratory failure requiring endotracheal intubation for ≥48 hours.Main outcome and measuresThe primary outcome was percent change in muscle thickness. Secondary outcomes were changes in EIM-derived fat percentage and “quality”.ResultsOf 34 enrolled patients, 30 completed ≥2 ultrasound assessments with a median interval of 6 (IQR 6–7) days. Mean age was 5.42 years, with 12 infants <1 year (40%) and 18 children >1 year old (60%). In the entire cohort, diaphragm thickness decreased 11.1% (95%CI, -19.7% to -2.52%) between the first two assessments or 2.2%/day. Quadriceps thickness decreased 8.62% (95%CI, -15.7% to -1.54%) or 1.5%/day. Biceps (-1.71%; 95%CI, -8.15% to 4.73%) and tibialis (0.52%; 95%CI, -5.81% to 3.40%) thicknesses did not change. Among the entire cohort, 47% (14/30) experienced diaphragm atrophy (defined a priori as ≥10% decrease in thickness). Eighty three percent of patients (25/30) experienced atrophy in ≥1 muscle group, and 47% (14/30)—in ≥2 muscle groups. On multivariate linear regression, increasing age and traumatic brain injury (TBI) were associated with greater muscle loss. EIM revealed increased fat percentage and decreased muscle “quality”.Conclusions and relevanceIn children receiving invasive mechanical ventilation, diaphragm and other skeletal muscle atrophy is common and rapid. Increasing age and TBI may increase severity of limb muscle atrophy. Prospective studies are required to link muscle atrophy to functional outcomes in critically ill children.

ObjectiveDysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment.MethodsTraditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety.ResultsTreated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns.InterpretationTreated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.

No abstract

؊/؊ mice. These results indicate that PCho promotes bacterial resistance to pulmonary clearance early in infection in a manner that is at least partially independent of the TLR4 pathway.

No abstract

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.