Dysferlin and Myoferlin Regulate Transverse Tubule Formation and Glycerol Sensitivity

Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β (TGFβ) pathway, osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways. We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy

High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay

OBJECTIVE: Serological testing is needed to investigate the extent of transmission of SARS-CoV-2 from front-line essential workers to their household members. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, DBS). We describe assay performance and demonstrate its utility for remote sampling with results from a community-based study. METHODS: An ELISA to the receptor binding domain of the SARS-CoV-2 spike protein was optimized to quantify IgG antibodies in DBS. Samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known COVID-19 cases and their household members. RESULTS: Among 30 individuals sharing a household with a virus-confirmed case of COVID-19, 80% were seropositive. Of 202 community individuals without prior confirmed acute COVID-19 diagnoses, 36% were seropositive. Of documented convalescent COVID-19 cases from the community, 29 of 30 (97%) were seropositive for IgG antibodies to the receptor binding domain. CONCLUSION: DBS ELISA provides a minimally-invasive alternative to venous blood collection. Early analysis suggests a high rate of transmission among household members. High rates of seroconversion were also noted following recovery from infection. Serological testing for SARS-CoV-2 IgG antibodies in DBS samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β (TGFβ) pathway, osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways. We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy

GRAF1 deficiency blunts sarcolemmal injury repair and exacerbates cardiac and skeletal muscle pathology in dystrophin-deficient mice

Background The plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin–amphiphysin–Rvs (BAR)–pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair. Methods We used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice. Results Herein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1 double deficiency phenocopied the severe muscle pathologies observed in dystrophin/dysferlin-double null mice. Consistent with a model that GRAF1 facilitates dysferlin-dependent membrane patching, we found that GRAF1 associates with and regulates plasma membrane deposition of dysferlin. Conclusions Overall, our work indicates that GRAF1 facilitates dysferlin-dependent membrane repair following acute muscle injury. These findings indicate that GRAF1 might play a role in the phenotypic variation and pathological progression of cardiac and skeletal muscle degeneration in muscular dystrophy patients