Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B.

Limb girdle muscular dystrophy type 2B (LGMD2B) and other dysferlinopathies are degenerative muscle diseases that result from mutations in the dysferlin gene and have limited treatment options. The dysferlin protein has been linked to multiple cellular functions including a Ca2+-dependent membrane repair process that reseals disruptions in the sarcolemmal membrane. Recombinant human MG53 protein (rhMG53) can increase the membrane repair process in multiple cell types both in vitro and in vivo. Here, we tested whether rhMG53 protein can improve membrane repair in a dysferlin-deficient mouse model of LGMD2B (B6.129-Dysftm1Kcam/J). We found that rhMG53 can increase the integrity of the sarcolemmal membrane of isolated muscle fibers and whole muscles in a Ca2+-independent fashion when assayed by a multi-photon laser wounding assay. Intraperitoneal injection of rhMG53 into mice before acute eccentric treadmill exercise can decrease the release of intracellular enzymes from skeletal muscle and decrease the entry of immunoglobulin G and Evans blue dye into muscle fibers in vivo. These results indicate that short-term rhMG53 treatment can ameliorate one of the underlying defects in dysferlin-deficient muscle by increasing sarcolemmal membrane integrity. We also provide evidence that rhMG53 protein increases membrane integrity independently of the canonical dysferlin-mediated, Ca2+-dependent pathway known to be important for sarcolemmal membrane repair.

Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone.

Osteocytes sense loading in bone, but their mechanosensation mechanisms remain poorly understood. Plasma membrane disruptions (PMD) develop with loading under physiological conditions in many cell types (e.g., myocytes, endothelial cells). These PMD foster molecular flux across cell membranes that promotes tissue adaptation, but this mechanosensation mechanism had not been explored in osteocytes. Our goal was to investigate whether PMD occur and initiate consequent mechanotransduction in osteocytes during physiological loading. We found that osteocytes experience PMD during in vitro (fluid flow) and in vivo (treadmill exercise) mechanical loading, in proportion to the level of stress experienced. In fluid flow studies, osteocyte PMD preferentially formed with rapid as compared to gradual application of loading. In treadmill studies, osteocyte PMD increased with loading in weight bearing locations (tibia), but this trend was not seen in non-weight bearing locations (skull). PMD initiated osteocyte mechanotransduction including calcium signaling and expression of c-fos, and repair rates of these PMD could be enhanced or inhibited pharmacologically to alter downstream mechanotransduction and osteocyte survival. PMD may represent a novel mechanosensation pathway in bone and a target for modifying skeletal adaptation signaling in osteocytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:653-662, 2018.

Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses.

Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak-Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann-Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane.