Severe vitamin E deficiency results in lethal myopathy in animal models. Membrane repair is an important myocyte response to plasma membrane disruption injury as when repair fails, myocytes die and muscular dystrophy ensues. Here we show that supplementation of cultured cells with α-tocopherol, the most common form of vitamin E, promotes plasma membrane repair. Conversely, in the absence of α-tocopherol supplementation, exposure of cultured cells to an oxidant challenge strikingly inhibits repair. Comparative measurements reveal that, to promote repair, an anti-oxidant must associate with membranes, as α-tocopherol does, or be capable of α-tocopherol regeneration. Finally, we show that myocytes in intact muscle cannot repair membranes when exposed to an oxidant challenge, but show enhanced repair when supplemented with vitamin E. Our work suggests a novel biological function for vitamin E in promoting myocyte plasma membrane repair. We propose that this function is essential for maintenance of skeletal muscle homeostasis.

Ca2؉ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca 2؉ , binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca 2؉ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca 2؉ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.Plasma membrane disruption, a common form of cell injury, is provoked in many mammalian tissues by physiological levels of mechanical stress (1). Disease can result from a failure either to prevent or repair disruptions. In Duchenne muscular dystrophy, for example, normal muscle contractions result in a pathological level of disruption injury because of increased fiber fragility (2). In limb-girdle muscular dystrophy, on the other hand, the failure is in the repair of normally occurring disruptions (3). Indeed, membrane repair, or resealing, is the universal survival response that eukaryotic cells mount when confronted with a life-threatening disruption in the integrity of their plasma membrane.The mechanism of resealing is now well characterized at the cellular level. A large plasma membrane disruption (Ͼ1 m diameter) locally and rapidly (subsecond to second time scale) elicits Ca 2ϩ -activated, homotypic vesicle fusion (4, 5). The "patch" vesicle thus formed then fuses exocytotically with the plasma membrane surrounding the defect site, restoring barrier continuity (6, 7). Identification of the protein components of this process is under way, and members of the SNARE 2 family (membrane proteins thought to be required for many fusion events) are implicated in resealing (8). However, as an emergency response it must be rapid and Ca 2ϩ -responsive and yet capable of being activated with a great level of temporal and spatial flexibility. Therefore, it has been hypothesized that resealing-based fusion may utilize, in addition to canonical elements such as the SNAREs, a specialized subset of fusion components (9). Dysferlin, a Ca 2ϩ -activated membrane-binding protein that mediates exocytotic fusion events in nematodes (10), is one candidate emergency fusion component. Thus, it was recently shown that skeletal muscle cells from dysferlin null mice fail to reseal disruptions (3).One protein shown by immunolocalization and immunoprecipitation to associate with dysferlin in normal, undisturbed skeletal muscle is annexin A1 (11), a member of the annexin family of Ca 2ϩ -regulated membrane-binding proteins (12). This observation, as well as the following additional po...

OBJECTIVESkeletal muscle myopathy is a common diabetes complication. One possible cause of myopathy is myocyte failure to repair contraction-generated plasma membrane injuries. Here, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes.RESEARCH DESIGN AND METHODSMyocytes in intact muscle from type 1 (INS2Akita+/−) and type 2 (db/db) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency. Membrane repair defects were also assessed in diabetic mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo. A cell culture model was used to investigate the role of advanced glycation end products (AGEs) and the receptor for AGE (RAGE) in development of this repair defect.RESULTSDiabetic myocytes displayed significantly more dye influx after laser injury than controls, indicating a repair deficiency. Downhill running also resulted in a higher level of repair failure in diabetic mice. This repair defect was mimicked in cultured cells by prolonged exposure to high glucose. Inhibition of the formation of AGE eliminated this glucose-induced repair defect. However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE.CONCLUSIONSBecause one consequence of repair failure is rapid cell death (via necrosis), our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes.

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.

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo.