Subsarcolemmal Cytoskeleton In Dysferlin‐Mediated Muscle Membrane Repair

Abstract

Mutations in the protein dysferlin have been shown to result in Limb Girdle Muscular Dystrophy 2B and Myoshi Myopathy. In patients and mouse models lacking dysferlin, dysfunctional membrane repair compromises the muscle cell integrity and leads to muscle degeneration and progressive muscle wasting. Dysferlin is a type 2 transmembrane protein with multiple calcium‐dependent, lipid binding C2 domains, that localizes to the t‐tubules and lateral sarcolemma of the muscle fiber. Previous work from our laboratory has shown that following injury, dysferlin is rapidly recruited from the lateral sarcolemma to the wound site and this can be inhibited by pharmacological agents that disrupt the actin cytoskeleton. However, the isoform responsible for dysferlin recruitment remains unknown. The three major actin isoforms expressed in muscle, α‐, β‐, and γ‐actin, are hypothesized to have distinct functions within the myofiber. Cytoplasmic β‐and γ‐actins localize to the costameres of the muscle fiber and are the major actin isoforms that form the subsarcolemmal actin cytoskeleton, while α‐actin is largely associated with the thin filaments of the sarcomere. Interestingly, genetic disruption of γ‐actin in muscle of mice causes a mild degenerative myopathy comparable to that observed in dysferlin deficiency. Therefore, in the current study, we studied mice with genetic loss of γ‐actin in differentiated muscle to investigate whether γ‐actin may be a critical actin isoform in dysferlin recruitment and thus efficient membrane repair.While we hypothesized that γ‐actin may play a role in trafficking sarcolemma‐derived dysferlin to the site of an injury, our data suggest that dysferlin recruitment is unaffected by the genetic loss of γ‐actin. Independent of defects in dysferlin recruitment, muscle fibers isolated from the flexor digitorum brevis of γ‐actin knockout mice show increased calcium uptake following injury, indicative of a defect in membrane repair. We also show that Actg1−/− muscle fibers have an increased uptake of FM1‐43 following injury, a commonly used assay of membrane repair. However, we've previously shown that FM1‐43 uptake following injury may actually be an indicator of wound‐induced endocytosis. Therefore, we hypothesize that calcium overload in membrane repair‐defective muscle fibers from γ‐actin knockout mice may contribute to overactive‐wound induced endocytosis, leading to the observed increase in FM1‐43 uptake. We are currently investigating whether increased wound‐induced endocytosis may result in greater dysferlin endocytosis. Overall, our results suggest that γ‐actin may have an important role in membrane repair in muscle which may contribute to the myopathy observed in the γ‐actin deficient mouse model.Support or Funding InformationMTN has been supported by the NIH Cellular and Molecular Biology Training Grant T‐32‐GM007315 and the NIH Cardiovascular Research and Entrepreneurship Training Grant T32‐HL125242 and research support was from NIH NIAMS AR066213 to DM.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.