Dysferlin In Muscle Research Articles

Syntaxin 4-enhanced plasma membrane repair is independent of dysferlin in skeletal muscle.

Plasma membrane repair (PMR) restores membrane integrity of cells, preventing cell death in vital organs, and has been studied extensively in skeletal muscle. Dysferlin, a sarcolemmal Ca2+-binding protein, plays a crucial role in PMR in skeletal muscle. Previous studies have suggested that PMR uses membrane trafficking and membrane fusion, similar to neurotransmission. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion in neurotransmission with the help of synaptotagmin, a crucial Ca2+-binding protein. Interestingly, dysferlin shares structural similarity with synaptotagmin and was shown to promote SNARE-mediated membrane fusion in a liposome-based assay. However, whether dysferlin facilitates SNARE-mediated membrane fusion in PMR in muscle cells remains unclear. In this study, we aimed to test if SNARE-mediated PMR requires dysferlin in muscle cells with pharmacological and genetic approaches. TAT-NSF700, which disrupts the disassembly of SNARE complexes, was used to disrupt functions of SNAREs in muscle cells. We found that human-induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) treated with TAT-NSF700 showed a higher loss of membrane integrity after repetitive mechanical strains. Moreover, laser-wounded mouse flexor digitorum brevis (FDB) fibers treated with TAT-NSF700 showed an increased Ca2+ influx, but a decreased FM1-43 uptake, which depends on dynamin-regulated endocytosis as we previously showed in FDB fibers. Importantly, overexpression of STX4-mCitrine or eGFP-SNAP23 decreased Ca2+ influx in laser-wounded FDB fibers. Furthermore, overexpression of STX4-mCitrine also decreased Ca2+ influx in laser-wounded dysferlin-deficient FDB fibers. Overall, these results suggest that disassembly of SNARE complexes is required for efficient PMR and STX4-enhanced PMR does not require dysferlin in skeletal muscle.NEW & NOTEWORTHY Dysferlin, a crucial Ca2+-binding protein in plasma membrane repair (PMR), shares homology with synaptotagmin, which binds Ca2+ and regulates SNARE-mediated vesicle fusion in neurons. Dysferlin was thus hypothesized to function as synaptotagmin in PMR. We demonstrate here that the activity of SNAREs is important for PMR, and overexpression of STX4 enhances PMR in both intact and dysferlin-deficient skeletal muscle. These data suggest that SNARE-mediated PMR may be independent of dysferlin in skeletal muscle.

G.O.9: AAV gene transfer utilizing homologous overlap vectors mediates functional recovery of dysferlin deficiency

There is no cure for dysferlinopathies, a group of related disorders caused by absent or mutant dysferlin (DYSF). Lack of dysferlin leads to progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement and fibrosis leading to deteriorating muscle weakness. Dysferlin has multiple functions including T-tubule formation and membrane repair. Pre-clinical studies show that only delivery of full-length DYSF corrects the histopathological defects in DYSF−/− mice. As DYSF is too large to be accommodated with canonical packaging, we developed a unique dual vector system using AAV to deliver and express DYSF in muscle. This two vector system (AAV.DYSF.DV) packaged in the rh.74 serotype is defined by a 1 kb region of homology between the two vectors. We compared the efficiency and safety of DYSF.DV delivery in dysferlin deficient mice and in non-human primates (NHPs) in preparation for clinical trials. Dysf−/− mice were treated by intramuscular injection, isolated limb perfusion, and systemic injection with AAV.DYSF.DV. IM injections were performed in the flexor digitorum brevis (FDB) muscle to assess expression and restoration of membrane repair capacity using ascending vector doses (6e9–6e10vg). The FDB was extracted after 10 wks and an ex vivo laser injury membrane repair assay was performed. DYSF.DV restored repair capacity in a dose-dependent manner comparable to WT findings. DYSF.DV was also efficacious when delivered through arterial circulation to hind limbs or systemically via the tail vein. A systemic dose of 6e12vg resulted in widespread gene expression exceeding 50% of muscle fibers; restoring functional deficits in the diaphragm. In NHPs, DYSF.DV delivery was analyzed for gene expression and toxicity using FLAG tag. There was no evidence of toxicity in DYSF-null mice or NHPs showing overexpression of dysferlin. Findings showed clear evidence that functional dysferlin was translated without toxicity laying the foundation for clinical trial.

Involvement of oxidative stress, Nuclear Factor kappa B and the Ubiquitin proteasomal pathway in dysferlinopathy

AimsDysferlinopathies are autosomal recessive neuromuscular disorders arising from mutations of the protein dysferlin that preferentially affect the limbs which waste and weaken. The pathomechanisms of the diseases are not known and effective treatment is not available. Although free radicals and upstream signaling by the redox sensitive transcription factor, NF-κB, in activation of the ubiquitin pathway are shown to occur in several muscle wasting disorders, their involvement in dysferlinopathy is not known. This study analyzed the role of oxidative stress, NF-κB and the ubiquitin pathway in dysferlinopathic muscle and in dysferlin knockdown human myoblasts and myotubes. Main methodsFourteen dysferlinopathic muscle biopsies and 8 healthy control muscle biopsies were analyzed for oxidative stress, NF-κB activation and protein ubiquitinylation and human primary myoblasts and myotubes knocked down for dysferlin were studied for their state of oxidative stress. Key findingsDysferlinopathic muscle biopsies showed NF-κB p65 signaling induced protein ubiquitinylation in response to oxidative stress. Dysferlin knock down primary muscle cell cultures confirmed that oxidative stress is induced in the absence of dysferlin in muscle. SignificanceAnti-oxidants that also inhibit nitrosative stress and NF-κB activation, might prove to be of therapeutic benefit in slowing the progression of muscle wasting that occurs with dysferlinopathy.

Miyoshi-like distal myopathy with mutations in anoctamin 5 gene

Miyoshi myopathy is the most common form of recessive distal myopathy. Recessive mutations in the ANO5 gene have been recently identified in Northern Europe as a cause of non dysferlin-linked distal myopathy and limb girdle muscular dystrophy. We report here the first French cases of anoctamin 5 myopathy in 2 brothers presenting with a Miyoshi-like pattern. Comparing these patients with 12 other cases from the literature shows that all cases share a homogeneous clinical pattern, characterized by initial calf muscles involvement. Asymmetric muscle atrophy often precedes weakness. In this setting, high CK level and normal expression of dysferlin in muscle should lead to consider the diagnosis, which will be confirmed by ANO5 gene testing. The c.191dupA mutation, already reported as a founder mutation in Caucasian patients with anoctamin myopathies, was found in our family in a heterozygous state.

Phenotypic Study in 40 Patients With Dysferlin Gene Mutations

To describe the phenotypic spectrum of dysferlin (DYSF) gene mutations (which cause dysferlinopathies, autosomal recessive muscular dystrophies) in patients with a dysferlin protein deficiency. Clinical, biological, and pathological data from 40 patients were reviewed. The diagnosis of dysferlinopathy was based on the absence or strong reduction of dysferlin in muscle, and confirmed by mutational screening of the DYSF gene. Two French neuromuscular diseases centers (in Paris and Marseilles). Two main dysferlinopathy phenotypes are well recognized: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B were found in 20 (50%) patients only. Unusual phenotypes included a mixed phenotype, referred to as "proximodistal," combining distal and proximal onset in 14 (35%) patients, pseudometabolic myopathy in 4 (10%), and asymptomatic hyperCKemia (an increased serum creatine kinase level) in 2 (5%). The disease may worsen rapidly, and 10 (25%) patients were initially misdiagnosed as having polymyositis. We suggest a relationship between proximodistal phenotype, inflammation, and severity. In addition to typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, dysferlinopathies are a clinically heterogeneous group of disorders ranging from asymptomatism to severe functional disability.