Marco Brotto

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.

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.

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.

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.

Deficiency of MIP/MTMR14 phosphatase induces a muscle disorder by disrupting Ca 2+ homeostasis

The intracellular Ca2+ concentration ([Ca2+]i) in skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process. However, the signalling components involved in such rapid Ca2+ movement are not fully understood. Here we report that mice deficient in the newly identified PtdInsP (phosphatidylinositol phosphate) phosphatase MIP/MTMR14 (muscle-specific inositol phosphatase) show muscle weakness and fatigue. Muscles isolated from MIP/MTMR14−/− mice produced less contractile force, had markedly prolonged relaxation and showed exacerbated fatigue relative to normal muscles. Further analyses revealed that MIP/MTMR14 deficiency resulted in spontaneous Ca2+ leakage from the internal store — the sarcoplasmic reticulum. This was attributed to decreased metabolism (dephosphorylation) and the subsequent accumulation of MIP/MTMR14 substrates, especially PtdIns(3,5)P2 and PtdIns (3,4)P2. Furthermore, we found that PtdIns(3,5)P2 and PtdIns(3,4)P2 bound to, and directly activated, the Ca2+ release channel (ryanodine receptor 1, RyR1) of the sarcoplasmic reticulum. These studies provide the first evidence that finely controlled PtdInsP levels in muscle cells are essential for maintaining Ca2+ homeostasis and muscle performance.

Prostaglandin E2: From Clinical Applications to Its Potential Role in Bone- Muscle Crosstalk and Myogenic Differentiation

Prostaglandin E(2) (PGE(2)), a prostanoid synthesized from arachidonic acid via the cyclooxygenase pathway, is a modulator of physiological responses including inflammation, fever, and muscle regeneration. Several patents have been filed that are related to PGE(2), one of them being directly related to skeletal muscles. In this report, we first summarize the key patents describing inventions for the utilization of PGE(2) for either diagnostic or therapeutic purposes, including skeletal muscle. In the second part of our work we present new and exciting data that demonstrates that PGE(2) accelerates skeletal muscle myogenic differentiation. Our discovery resulted from our recent and novel concept of bone-muscle crosstalk. Bone and muscle are anatomically intimate endocrine organs and we aimed to determine whether this anatomical intimacy also translates into a biochemical communication from bone cells to muscle cells at the in vitro level. The effects of MLOY4 osteocyte-like cell conditioned medium (CM) and three osteocyte-secreted factors, PGE(2), sclerostin and monocyte chemotactic protein (MCP-3), on C2C12 myogenic differentiation were evaluated using morphological analyses, a customized 96-gene PCR array, and measurements of intracellular calcium levels. MLO-Y4 CM and PGE(2), but not sclerostin and MCP-3, induced acceleration of myogenesis of C2C12 myoblasts that was linked with significant modifications in intracellular calcium homeostasis. This finding should further stimulate the pursuit of new patents to explore the use of PGE(2) and the new concept of bone-muscle crosstalk for the development and application of inventions designed to treat muscle diseases characterized by enhanced muscle wasting, such as sarcopenia.

Compromised store-operated Ca2+ entry in aged skeletal muscle

In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca2+ entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store‐operated Ca2+ entry (SOCE) in aged mouse skeletal muscle. Using Mn2+ quenching of Fura‐2 fluorescence and confocal‐microscopic imaging of Ca2+ movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26–27 months) as compared with those from young (2–5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin‐29, a synaptophysin‐related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin‐29 expression and reduced SOCE may contribute to the diminished intracellular Ca2+ homeostatic capacity generally associated with muscle aging.

Kruppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation

The ability of skeletal muscle to enhance lipid utilization during exercise is a form of metabolic plasticity essential for survival. Conversely, metabolic inflexibility in muscle can cause organ dysfunction and disease. Although the transcription factor Kruppel-like factor 15 (KLF15) is an important regulator of glucose and amino acid metabolism, its endogenous role in lipid homeostasis and muscle physiology is unknown. Here we demonstrate that KLF15 is essential for skeletal muscle lipid utilization and physiologic performance. KLF15 directly regulates a broad transcriptional program spanning all major segments of the lipid-flux pathway in muscle. Consequently, Klf15-deficient mice have abnormal lipid and energy flux, excessive reliance on carbohydrate fuels, exaggerated muscle fatigue, and impaired endurance exercise capacity. Elucidation of this heretofore unrecognized role for KLF15 now implicates this factor as a central component of the transcriptional circuitry that coordinates physiologic flux of all three basic cellular nutrients: glucose, amino acids, and lipids.

Endocrine Crosstalk Between Muscle and Bone

The musculoskeletal system is a complex organ comprised of the skeletal bones, skeletal muscles, tendons, ligaments, cartilage, joints, and other connective tissue that physically and mechanically interact to provide animals and humans with the essential ability of locomotion. This mechanical interaction is undoubtedly essential for much of the diverse shape and forms observed in vertebrates and even in invertebrates with rudimentary musculoskeletal systems such as fish. It makes sense from a historical point of view that the mechanical theories of musculoskeletal development have had tremendous influence of our understanding of biology, because these relationships are clear and palpable. Less visible to the naked eye or even to the microscope is the biochemical interaction among the individual players of the musculoskeletal system. It was only in recent years that we have begun to appreciate that beyond this mechanical coupling of muscle and bones, these 2 tissues function at a higher level through crosstalk signaling mechanisms that are important for the function of the concomitant tissue. Our brief review attempts to present some of the key concepts of these new concepts and is outline to present muscles and bones as secretory/endocrine organs, the evidence for mutual genetic and tissue interactions, pathophysiological examples of crosstalk, and the exciting new directions for this promising field of research aimed at understanding the biochemical/molecular coupling of these 2 intimately associated tissues.

Defective maintenance of intracellular Ca2+ homeostasis is linked to increased muscle fatigability in the MG29 null mice

ABSTRACTMitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of mg29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca2+-uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and mg29 knockout mice. Compared with the control muscle, submaximal Ca2+-uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca2+ inside the SR was less in the mutant muscle, due to increased leakage process of Ca2+ movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca2+/caffeine -induced Ca2+ release to myoplasmic Ca2+. Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca2+ uptake, modification of Ca2+-induced Ca2+ release (CICR), and a reduction in total SR Ca2+ content.