Noah Weisleder

MG53 nucleates assembly of cell membrane repair machinery

Dynamic membrane repair and remodelling is an elemental process that maintains cell integrity and mediates efficient cellular function. Here we report that MG53, a muscle-specific tripartite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that traffic to and fuse with sarcolemmal membranes. Mice null for MG53 show progressive myopathy and reduced exercise capability, associated with defective membrane-repair capacity. Injury of the sarcolemmal membrane leads to entry of the extracellular oxidative environment and MG53 oligomerization, resulting in recruitment of MG53-containing vesicles to the injury site. After vesicle translocation, entry of extracellular Ca2+ facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca2+-dependent membrane fusion are distinct steps involved in the repair of membrane damage and that MG53 may initiate the assembly of the membrane repair machinery in an oxidation-dependent manner.

Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.

Defective membrane repair can contribute to the progression of muscular dystrophy. Although mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy in human patients, the molecular mechanism underlying the functional interplay between Cav3 and dysferlin in membrane repair of muscle physiology and disease has not been fully resolved. We recently discovered that mitsugumin 53 (MG53), a muscle-specific TRIM (Tri-partite motif) family protein (TRIM72), contributes to intracellular vesicle trafficking and is an essential component of the membrane repair machinery in striated muscle. Here we show that MG53 interacts with dysferlin and Cav3 to regulate membrane repair in skeletal muscle. MG53 mediates active trafficking of intracellular vesicles to the sarcolemma and is required for movement of dysferlin to sites of cell injury during repair patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair. Our data reveal that a molecular complex formed by MG53, dysferlin, and Cav3 is essential for repair of muscle membrane damage and also provide a therapeutic target for treatment of muscular and cardiovascular diseases that are linked to compromised membrane repair.

Uncontrolled calcium sparks act as a dystrophic signal for mammalian skeletal muscle

Most excitable cells maintain tight control of intracellular Ca2+ through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca2+ release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca2+ sparks in intact mammalian skeletal muscle. Spontaneous Ca2+ sparks can be reversibly induced by osmotic shock, and participate in a normal physiological response to exercise. In dystrophic muscle with fragile membrane integrity, stress-induced Ca2+ sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca2+ spark response. Thus, membrane-deformation-induced Ca2+ sparks have an important role in physiological and pathophysiological regulation of Ca2+ signalling, and uncontrolled Ca2+ spark activity in connection with chronic activation of store-operated Ca2+ entry may function as a dystrophic signal in mammalian skeletal muscle.

MG53 Constitutes a Primary Determinant of Cardiac Ischemic Preconditioning

Background— Ischemic heart disease is the greatest cause of death in Western countries. The deleterious effects of cardiac ischemia are ameliorated by ischemic preconditioning (IPC), in which transient ischemia protects against subsequent severe ischemia/reperfusion injury. IPC activates multiple signaling pathways, including the reperfusion injury salvage kinase pathway (mainly PI3K-Akt-glycogen synthase kinase-3&bgr; [GSK3&bgr;] and ERK1/2) and the survivor activating factor enhancement pathway involving activation of the JAK-STAT3 axis. Nevertheless, the fundamental mechanism underlying IPC is poorly understood. Methods and Results— In the present study, we define MG53, a muscle-specific TRIM-family protein, as a crucial component of cardiac IPC machinery. Ischemia/reperfusion or hypoxia/oxidative stress applied to perfused mouse hearts or neonatal rat cardiomyocytes, respectively, causes downregulation of MG53, and IPC can prevent ischemia/reperfusion-induced decrease in MG53 expression. MG53 deficiency increases myocardial vulnerability to ischemia/reperfusion injury and abolishes IPC protection. Overexpression of MG53 attenuates whereas knockdown of MG53 enhances hypoxia- and H2O2-induced cardiomyocyte death. The cardiac protective effects of MG53 are attributable to MG53-dependent interaction of caveolin-3 with phosphatidylinositol 3 kinase and subsequent activation of the reperfusion injury salvage kinase pathway without altering the survivor activating factor enhancement pathway. Conclusions— These results establish MG53 as a primary component of the cardiac IPC response, thus identifying a potentially important novel therapeutic target for the treatment of ischemic heart disease.

TRIC channels are essential for Ca2+ handling in intracellular stores.

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ ‘spark’ signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation–contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.

Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals

The mitochondrion is essential for energy metabolism and production of reactive oxygen species (ROS). In intact cells, respiratory mitochondria exhibit spontaneous “superoxide flashes”, the quantal ROS-producing events consequential to transient mitochondrial permeability transition (tMPT). Here we perform the first in vivo imaging of mitochondrial superoxide flashes and tMPT activity in living mice expressing the superoxide biosensor mt-cpYFP, and demonstrate their coupling to whole-body glucose metabolism. Robust tMPT/superoxide flash activity occurred in skeletal muscle and sciatic nerve of anesthetized transgenic mice. In skeletal muscle, imaging tMPT/superoxide flashes revealed labyrinthine three-dimensional networks of mitochondria that operate synchronously. The tMPT/superoxide flash activity surged in response to systemic glucose challenge or insulin stimulation, in an apparently frequency-modulated manner and involving also a shift in the gating mode of tMPT. Thus, in vivo imaging of tMPT-dependent mitochondrial ROS signals and the discovery of the metabolism-tMPT-superoxide flash coupling mark important technological and conceptual advances for the study of mitochondrial function and ROS signaling in health and disease.

Cardioprotection of Ischemia/Reperfusion Injury by Cholesterol-Dependent MG53-Mediated Membrane Repair

Rationale: Unrepaired cardiomyocyte membrane injury causes irreplaceable cell loss, leading to myocardial fibrosis and eventually heart failure. However, the cellular and molecular mechanisms of cardiac membrane repair are largely unknown. MG53, a newly identified striated muscle-specific protein, is involved in skeletal muscle membrane repair. But the role of MG53 in the heart has not been determined. Objective: We sought to investigate whether MG53 mediates membrane repair in cardiomyocytes and, if so, the cellular and molecular mechanism underlying MG53-mediated membrane repair in cardiomyocytes. Moreover, we determined possible cardioprotective effect of MG53-mediated membrane repair. Methods and Results: We demonstrated that MG53 is crucial to the emergency membrane repair response in cardiomyocytes and protects the heart from stress-induced loss of cardiomyocytes. Disruption of the sarcolemmal membrane by mechanical, electric, chemical, or metabolic insults caused rapid and robust translocation of MG53 toward the injury sites. Ablation of MG53 prevented sarcolemmal resealing after infrared laser–induced membrane damage in intact heart, and exacerbated mitochondrial dysfunction and loss of cardiomyocytes during ischemia/reperfusion injury. Unexpectedly, the MG53-mediated cardiac membrane repair was mediated by a cholesterol-dependent mechanism: depletion of membrane cholesterol abolished, and its recovery restored injury-induced membrane translocation of MG53. The redox status of MG53 did not affect initiation of MG53 translocation, whereas MG53 oxidation conferred stability to the membrane repair patch. Conclusions: Thus, cholesterol-dependent MG53-mediated membrane repair is a vital, heretofore unappreciated cardioprotective mechanism against a multitude of insults and may bear important therapeutic implications.

Azumolene Inhibits a Component of Store-operated Calcium Entry Coupled to the Skeletal Muscle Ryanodine Receptor

Dantrolene reduces the elevated myoplasmic Ca2+ generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca2+ entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.

Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy

Recombinant human MG53 protein can increase membrane repair after injury in cells and can reduce pathology in animal models of muscle injury and muscular dystrophy. Mending Muscle To repair a torn muscle, one might require a little bit of ice and a lot of rest. For those with Duchenne muscular dystrophy (DMD), however, muscle degeneration is not as easily repaired, and patients ultimately experience difficulty standing, walking, and breathing. DMD results from a lack of the protein dystrophin, which is located at the cell membrane to help muscle fibers repair themselves. There is no cure for DMD, but Weisleder and colleagues have now shown that exogenous delivery of a different repair protein, Mitsugumin 53 (MG53), to cells can prevent muscle damage in cell culture and in mice. The authors first showed that muscle and nonmuscle cells treated with recombinant human MG53 (rhMG53) in vitro were resistant to mechanical, chemical, and photo damage because MG53 localized to the injury site and provided protection. In vivo, Weisleder and colleagues showed that dystrophin-deficient mdx mice treated intramuscularly or intravenously with rhMG53 displayed reduced muscle damage and decreased muscle pathology compared to saline-treated controls, even in the presence of a membrane-damaging toxin. This repair process also worked in muscle fibers isolated from mdx mice that were deficient in either of two natural repair proteins, MG53 or dysferlin, suggesting that exogenous delivery of rhMG53 works by a new mechanism—other than the intracellular machinery—to patch up damaged cell membrane. Soluble MG53 protein therapy could be a viable treatment for DMD that avoids the well-known limitations of dystrophin gene replacement therapy. Toward translation, Weisleder et al. have further demonstrated that exogenous MG53 is nontoxic and safe in animals. The ability of the protein to preserve muscle function and to enhance repair capacity in humans has yet to be shown, but additional studies in larger animals and human muscle fibers will give a clearer indication of its therapeutic potential. Mitsugumin 53 (MG53), a muscle-specific TRIM family protein, is an essential component of the cell membrane repair machinery. Here, we examined the translational value of targeting MG53 function in tissue repair and regenerative medicine. Although native MG53 protein is principally restricted to skeletal and cardiac muscle tissues, beneficial effects that protect against cellular injuries are present in nonmuscle cells with overexpression of MG53. In addition to the intracellular action of MG53, injury to the cell membrane exposes a signal that can be detected by MG53, allowing recombinant MG53 protein to repair membrane damage when provided in the extracellular space. Recombinant human MG53 (rhMG53) protein purified from Escherichia coli fermentation provided dose-dependent protection against chemical, mechanical, or ultraviolet-induced damage to both muscle and nonmuscle cells. Injection of rhMG53 through multiple routes decreased muscle pathology in the mdx dystrophic mouse model. Our data support the concept of targeted cell membrane repair in regenerative medicine, and present MG53 protein as an attractive biological reagent for restoration of membrane repair defects in human diseases.