Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.
A common aspect of many diseases and traumatic injuries affecting skeletal muscle is the necrotic death of muscle fibers. Necrotic death of muscle fibers involves the breakdown of the sarcolemmal membrane that can be exacerbated by increased fragility of the membrane or by compromised endogenous sarcolemmal membrane repair processes. Increasing the efficacy of these repair mechanisms could act as a therapeutic approach for several muscular diseases and injuries. Limb‐girdle muscular dystrophy 2B (LGMD2B), a subtype of LGMD, is an autosomal recessive neuromuscular disorder caused by mutations in the DYSF gene that encodes dysferlin, a protein that is vital in membrane repair. The dysferlin protein has been previously shown to facilitate membrane repair in skeletal muscle and knockout mice for dysferlin develop progressive muscular dystrophy. When dysferlin is absent or inactivated through mutation, membrane damage that cannot be repaired accumulates until muscle fibers undergo necrosis that eventually overwhelms the regenerative capacity of the muscle. This causes skeletal muscle deterioration in LGMD2B patients, particularly in the pelvic and shoulder girdle muscles. There is currently no cure for LGMD2B and treatments that stimulate the membrane repair process in muscle fibers have been greatly overlooked and underutilized as a potential therapeutic approach. This project investigates a novel membrane repair signaling cascade in skeletal cells by utilizing SC79, an Akt activator, to increase membrane repair capacity in skeletal muscle. We hypothesize that activation of the phosphoinositide‐3 kinase (PI3K)/Akt1 signaling axis regulates membrane repair in cultured muscle cells and mouse tissue. Based on our data, we find that SC79 injection leads to decreased sarcolemmal membrane injury during treadmill exercises where injection of SC79 decreases the entry of Evans blue dye into muscle fibers. We determined that various doses of SC79 can increase membrane repair in cultured muscle cells and Bla/J dysferlin deficient mice. In these studies, multi‐photon infrared laser microscopy was used to damage the cell membrane of myoblasts, transdifferentiated from skin fibroblasts isolated from LGMD2B patients, in the presence of FM4‐64 dye, a lipophilic dye that fluoresces when it enters the cells and binds to the phospholipids of the cell membrane and intracellular organelles. The extent of localized FM4‐64 dye fluorescence provides a measurement of how well the membrane is repaired. We found SC79 could increase membrane repair in these human patient myoblasts in a dose dependent fashion. This assay was also used on isolated complete muscles from the Bla/J dysferlin deficient mice, where SC79 also increased membrane repair responses. We conclude that activation of the PI3K/Akt1 signaling axis increases membrane repair in dysferlin deficient skeletal muscle. This project explores a novel signaling cascade controlling membrane repair that could be leveraged to develop new therapies for muscle disease and injury.
Duchenne Muscular Dystrophy (DMD) is a fatal X‐linked genetic disease that is hallmarked by progressive muscle weakness and degeneration. DMD is the most common form of all the muscular dystrophies, affecting 1:5,000 live male births. DMD is caused by mutations in the dystrophin gene, which ablate the expression of dystrophin resulting in compromised structural integrity of the muscle cell plasma membrane, also known as the sarcolemma. There is still an unmet need for novel therapies that address the underlying molecular cause of the disease. Previous work has shown that the tripartite motif protein 72/mitsugumin 53 (TRIM72/MG53) is critical for an effective cell membrane repair response after injury. Our previous results demonstrated that overexpressing TRIM72/MG53 or exogenously delivering recombinant human MG53 protein (rhMG53) can increase membrane repair capacity in many different cell types and improve pathology in multiple animal models of muscular dystrophy. TRIM72/MG53 mediates this effect on membrane repair by binding phosphatidylserine (PS) at membrane injury sites. However, there is a poor understanding of the structural basis of PS binding by TRIM72/MG53 and how this contributes to the membrane repair effects of the protein when expressed endogenously or delivered exogenously. Here we aimed to address this knowledge gap through structure/function analysis. In these studies, we examined the mechanistic basis for MG53 binding to PS by conducting a systematic analysis of MG53 canonical protein domains. We generated a catalytic mutant of TRIM72/MG53 (hMG53(ΔE3)) to delineate its native E3 ubiquitin ligase enzymatic activity and its effect on membrane repair. To identify which protein domains are required for the effect on membrane repair, we generated a panel of nine deletion and fusion protein constructs of human TRIM72/MG53. We confirmed protein expression by transient transfection of human embryonic kidney (HEK293) cells and found that specific protein domains mediated the subcellular location of the protein mutant. Moreover, we evaluated ensemble membrane repair capacity by overexpressing our mutant panel in mouse neuroblastoma (N2a) cells and measuring lactate dehydrogenase (LDH) release after damage with ~500 micrometer glass beads. We found that hMG53(ΔE3) displays comparable subcellular localization to wild‐type hMG53 and improves membrane resealing when endogenously expressed in HEK293 cells or made available exogenously as recombinant protein. We observed that hMG53(ΔE3) can also bind phosphatidylserine (PS)‐coated beads. We found that at least two deletion constructs display enhanced membrane repair response comparable to full length TRIM72/MG53. Taken together, these data suggest that the therapeutic effect of TRIM72/MG53 on membrane repair involves specific protein domains but does not require its E3 ligase enzymatic activity.
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