Alexis R. Demonbreun

The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion

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.

Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair

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 A6s role in muscle membrane leak and repair in muscular dystrophy. Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy and its associated cardiomyopathy, we used quantitative trait locus mapping and whole genome sequencing in a mouse model. This approach uncovered a modifier locus on chromosome 11 associated with sarcolemmal membrane damage and heart mass. Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene. A synonymous variant in exon 11 creates a cryptic splice donor, resulting in a truncated annexin A6 protein called ANXA6N32. Live cell imaging showed that annexin A6 orchestrates a repair zone and cap at the site of membrane disruption. In contrast, ANXA6N32 dramatically disrupted the annexin A6-rich cap and the associated repair zone, permitting membrane leak. Anxa6 is a modifier of muscular dystrophy and membrane repair after injury.

Genetic background influences muscular dystrophy

Mutations in the genes encoding dystrophin and its associated proteins, the sarcoglycans, lead to muscular dystrophy in humans and in mouse models. In the presence of identical gene mutations, the muscular dystrophy phenotype can be highly variable. Using a mouse model of limb girdle muscular dystrophy engineered with a null allele of gamma-sarcoglycan, we bred the identical gamma-sarcoglycan mutation into four different genetic backgrounds. We found that the gamma-sarcoglycan mutation is least severe in the129SV/J (129) strain and most severe on the DBA 2J JAX (DBA) strain using quantitative measures of Evans blue dye uptake, as a marker of membrane permeability defects, and hydroxyproline content, as a marker of fibrosis. In addition we show that the DBA mice are most severely affected regardless of gender and age. The enhanced phenotype observed in the DBA strain was not caused by exercise as the DBA mice scored the lowest in a voluntary activity test. The milder phenotype seen in the 129SV/J and C57B6/J strains suggests that these backgrounds contain modifier loci that partially suppress the muscular dystrophy phenotype. Identification of these modifier genes and the associated pathways may lead to novel therapeutic strategies.

Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy

Loss-of-function mutations in dysferlin cause muscular dystrophy, and dysferlin has been implicated in resealing membrane disruption in myofibers. Given the importance of membrane fusion in many aspects of muscle function, we studied the role of dysferlin in muscle growth. We found that dysferlin null myoblasts have a defect in myoblast-myotube fusion, resulting in smaller myotubes in culture. In vivo, dysferlin null muscle was found to have mislocalized nuclei and vacuolation. We found that myoblasts isolated from dysferlin null mice accumulate enlarged, lysosomal-associated membrane protein 2 (LAMP2)-positive lysosomes. Dysferlin null myoblasts accumulate transferrin-488, reflecting abnormal vesicular trafficking. Additionally, dysferlin null myoblasts display abnormal trafficking of the insulin-like growth factor (IGF) receptor, where the receptor is shuttled to LAMP2-positive lysosomes. We studied growth, in vivo, by infusing mice with the growth stimulant IGF1. Control IGF1-treated mice increased myofiber diameter by 30% as expected, whereas dysferlin null muscles had no response to IGF1, indicating a defect in myofiber growth. We also noted that dysferlin null fibroblasts also accumulate acidic vesicles, IGF receptor and transferrin, indicating that dysferlin is important for nonmuscle vesicular trafficking. These data implicate dysferlin in multiple membrane fusion events within the cell and suggest multiple pathways by which loss of dysferlin contributes to muscle disease.

Myoferlin is required for insulin-like growth factor response and muscle growth.

Insulin‐like growth factor (IGF) is a potent stimulus of muscle growth. Myoferlin is a membraneassociated protein important for muscle development and regeneration. Myoferlin‐null mice have smaller muscles and defective myoblast fusion. To understand the mechanism by which myoferlin loss retards muscle growth, we found that myoferlin‐null muscle does not respond to IGF1. In vivo after IGF1 infusion, control muscle increased myofiber diameter by 25%, but myoferlin‐null muscle was unresponsive. Myoblasts cultured from myoferlin‐null muscle and treated with IGF1 αlso failed to show the expected increase in fusion to multinucleate myotubes. The IGF1 receptor colocalized with myoferlin at sites of myoblast fusion. The lack of IGF1 responsiveness in myoferlin‐null myoblasts was linked directly to IGF1 receptor mistrafficking as well as decreased IGF1 signaling. In myoferlin‐null myoblasts, the IGF1 receptor accumulated into large vesicular structures. These vesicles colocalized with a marker of late endosomes/lysosomes, LAMP2, specifying redirection from a recycling to a degradative pathway. Furthermore, ultrastructural analysis showed a marked increase in vacuoles in myoferlin‐null muscle. These data demonstrate that IGF1 receptor recycling is required for normal myogenesis and that myoferlin is a critical mediator of postnatal muscle growth mediated by IGF1.—Demonbreun, A. R., Posey, A. D., Heretis, K., Swaggart, K. A., Earley, J. U., Pytel, P., McNally, E. M. Myoferlin is required for insulin‐like growth factor response and muscle growth. FASEB J. 24, 1284–1295 (2010). www.fasebj.org

Endocytic Recycling Proteins EHD1 and EHD2 Interact with Fer-1-like-5 (Fer1L5) and Mediate Myoblast Fusion

The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.

An actin-dependent annexin complex mediates plasma membrane repair in muscle

Demonbreun et al. visualized muscle membrane repair in real time after laser-induced microdamage. Annexin proteins were observed to form a repair cap at the site of injury, supporting a shoulder-like structure containing EHD1, EHD2, dysferlin, and MG53.

Ferlin Proteins in Myoblast Fusion and Muscle Growth

Myoblast fusion contributes to muscle growth in development and during regeneration of mature muscle. Myoblasts fuse to each other as well as to multinucleate myotubes to enlarge the myofiber. The molecular mechanisms of myoblast fusion are incompletely understood. Adhesion, apposition, and membrane fusion are accompanied by cytoskeletal rearrangements. The ferlin family of proteins is implicated in human muscle disease and has been implicated in fusion events in muscle, including myoblast fusion, vesicle trafficking and membrane repair. Dysferlin was the first mammalian ferlin identified and it is now known that there are six different ferlins. Loss-of-function mutations in the dysferlin gene lead to limb girdle muscular dystrophy and the milder disorder Miyoshi Myopathy. Dysferlin is a membrane-associated protein that has been implicated in resealing disruptions in the muscle plasma membrane. Newer data supports a broader role for dysferlin in intracellular vesicular movement, a process also important for resealing. Myoferlin is highly expressed in myoblasts that undergoing fusion, and the absence of myoferlin leads to impaired myoblast fusion. Myoferlin also regulates intracellular trafficking events, including endocytic recycling, a process where internalized vesicles are returned to the plasma membrane. The trafficking role of ferlin proteins is reviewed herein with a specific focus as to how this machinery alters myogenesis and muscle growth.

Myoferlin regulation by NFAT in muscle injury, regeneration and repair

Ferlin proteins mediate membrane-fusion events in response to Ca2+. Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.

EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.

EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.