Disruption of the plasma membrane often accompanies cellular injury, and in muscle, plasma membrane resealing is essential for efficient recovery from injury. Muscle contraction, especially of lengthened muscle, disrupts the sarcolemma. To define the molecular machinery that directs repair, we applied laser wounding to live mammalian myofibers and assessed translocation of fluorescently tagged proteins using high-resolution microscopy. Within seconds of membrane disruption, annexins A1, A2, A5, and A6 formed a tight repair “cap.” Actin was recruited to the site of damage, and annexin A6 cap formation was both actin dependent and Ca2+ regulated. Repair proteins, including dysferlin, EHD1, EHD2, MG53, and BIN1, localized adjacent to the repair cap in a “shoulder” region enriched with phosphatidlyserine. Dye influx into muscle fibers lacking both dysferlin and the related protein myoferlin was substantially greater than control or individual null muscle fibers, underscoring the importance of shoulder-localized proteins. These data define the cap and shoulder as subdomains within the repair complex accumulating distinct and nonoverlapping components.

Significance: Many forms of muscular dystrophy produce muscle weakness through injury to skeletal muscle myofibers and specifically disruption of the muscle plasma membrane. Using a mouse model of muscular dystrophy in a genetically diverse background, a genome-wide scan for genetic modifiers was undertaken. A modifier locus that altered plasma membrane leak was interrogated, and a splice site variant in Anxa6, encoding annexin A6, was identified. The Anxa6 splice site produces a truncated annexin A6 protein. The truncated annexin A6 protein was found to inhibit membrane repair by disrupting the formation of the normal annexin A6-rich cap and repair zone. These data demonstrate annexin A6's role in muscle membrane leak and repair in muscular dystrophy.

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion.

Recent reports have suggested that among individuals previously infected with SARS-CoV-2, a single mRNA vaccine dose is sufficient to elicit high levels of immunity. We compared anti-SARS-CoV-2 spike receptor binding domain (RBD) IgG antibody concentrations and antibody-mediated neutralization of spike-angiotensin-converting enzyme (ACE2) receptor binding in vitro following vaccination of non-hospitalized participants by sero-status and acute virus diagnosis history. Participants were analysed before and after mRNA vaccination (BNT162b2/Pfizer or mRNA-1273/Moderna) in a community-based, home-collected, longitudinal serosurvey in the Chicago area (USA); none reported hospitalization for COVID-19. Samples were collected in January and February 2021. Before vaccination, some reported prior positive acute viral diagnostic testing and were seropositive (COVID-19+); the others who did not report acute viral diagnostic testing were categorized as seropositive or seronegative based on anti-spike RBD IgG test results. Of 307 unique vaccine recipients, 46 reported a prior COVID-19 diagnosis and were seropositive (COVID-19 +). Of the 261 with no history of acute viral diagnostic testing, 117 were seropositive and 144 seronegative before vaccination. The median age was 38 years (range 21–83) with 67 female and 33% male; 40% were non-White. Responses were evaluated after one (n = 142) or two (n = 191) doses of BNT162b2 or mRNA-1273 vaccine. After one dose, median post-vaccine IgG concentration and percent surrogate neutralization were each significantly higher among the COVID-19+ (median 48·2 µg/ml, IgG; > 99.9% neutralization) compared to the seropositives (3·6 µg /ml IgG; 56.5% neutralization) and seronegatives (2·6 µg /ml IgG; 38·3% neutralization). The latter two groups reached > 95% neutralization after the second vaccine dose. After one dose of mRNA vaccine, individuals previously diagnosed with COVID-19 responded with high levels of anti-RBD IgG and surrogate neutralization of spike-ACE2 interaction. One dose of mRNA vaccine was not sufficient to generate comparably high responses among most persons previously infected with SARS-CoV-2 without a clinical COVID-19 diagnosis, nor among seronegative persons. National Science Foundation 2035114, NIH 3UL1TR001422–06S4, and Northwestern University Office of Research.

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.