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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Noah Weisleder in JoVE

 JoVE General

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems


JoVE 2717 6/30/2011

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Department of Physiology and Biophysics, Robert Wood Johnson Medical School

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Other articles by Noah Weisleder on PubMed

Bcl-2 Overexpression Corrects Mitochondrial Defects and Ameliorates Inherited Desmin Null Cardiomyopathy

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2004  |  Pubmed ID: 14715896

One of the hallmarks of cardiomyopathy and heart failure is pronounced and progressive cardiomyocyte death. Understanding the mechanisms involved in cardiomyocyte cell death is a topic of great interest for treatment of cardiac disease. Mice null for desmin, the muscle-specific member of the intermediate filament gene family, develop cardiomyopathy characterized by extensive cardiomyocyte death, fibrosis, calcification, and eventual heart failure. The earliest ultrastructural defects are observed in mitochondria. In the present study, we have demonstrated that these mitochondrial abnormalities are the primary cause of the observed cardiomyopathy and that these defects can be ameliorated by overexpression of bcl-2 in desmin null heart. Overexpression of bcl-2 in the desmin null heart results in correction of mitochondrial defects, reduced occurrence of fibrotic lesions in the myocardium, prevention of cardiac hypertrophy, restoration of cardiomyocyte ultrastructure, and significant improvement of cardiac function. Furthermore, we have found that loss of desmin also diminishes the capacity of mitochondria to resist exposure to calcium, a defect that can be partially restored by bcl-2 overexpression. These results point to a unique function for desmin in protection of mitochondria from calcium exposure that can be partially rescued by overexpression of bcl-2. We show that bcl-2 cardiac overexpression has provided significant improvement of an inherited form of cardiomyopathy, revealing the potential for bcl-2, and perhaps other genes in the family, as therapeutic agents for heart disease of many types, including inherited forms.

Cardiomyocyte-specific Desmin Rescue of Desmin Null Cardiomyopathy Excludes Vascular Involvement

Journal of Molecular and Cellular Cardiology. Jan, 2004  |  Pubmed ID: 14734054

Mice deficient in desmin, the muscle-specific member of the intermediate filament gene family, display defects in all muscle types and particularly in the myocardium. Desmin null hearts develop cardiomyocyte hypertrophy and dilated cardiomyopathy (DCM) characterized by extensive myocyte cell death, calcific fibrosis and multiple ultrastructural defects. Several lines of evidence suggest impaired vascular function in desmin null animals. To determine whether altered capillary function or an intrinsic cardiomyocyte defect is responsible for desmin null DCM, transgenic mice were generated to rescue desmin expression specifically to cardiomyocytes. Desmin rescue mice display a wild-type cardiac phenotype with no fibrosis or calcification in the myocardium and normalization of coronary flow. Cardiomyocyte ultrastructure is also restored to normal. Markers of hypertrophy upregulated in desmin null hearts return to wild-type levels in desmin rescue mice. Working hearts were perfused to assess coronary flow and cardiac power. Restoration of a wild-type cardiac phenotype in a desmin null background by expression of desmin specifically within cardiomyocyte indicates that defects in the desmin null heart are due to an intrinsic cardiomyocytes defect rather than compromised coronary circulation.

Structural and Functional Roles of Desmin in Mouse Skeletal Muscle During Passive Deformation

Biophysical Journal. May, 2004  |  Pubmed ID: 15111414

Mechanical interactions between desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein alpha-actinin, even at very high strains and stresses. These data indicate that desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires desmin. Passive mechanical analysis revealed that desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of alpha-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of desmin during passive fiber loading.

Alterations in the Heart Mitochondrial Proteome in a Desmin Null Heart Failure Model

Journal of Molecular and Cellular Cardiology. Mar, 2005  |  Pubmed ID: 15733906

Desmin, the major muscle-specific intermediate filament (IF) protein, is essential for mitochondrial behavior and function and maintenance of healthy muscle. Mice null for desmin develop dilated cardiomyopathy characterized by extensive cardiomyocyte death, fibrosis, calcification and eventual heart failure. We sought to investigate the heart mitochondrial proteome of wild type and desmin null mice in order to understand the cardiac and skeletal myopathy phenotype of desmin deficiency. The proteins were analyzed by 2-D electrophoresis, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Three hundred and eighty different gene products were identified, about 50% of which were enzyme subunits. Cytoskeletal and muscle-specific proteins, calcium-binding proteins, proteins with various other functions and about 70 unknown, hypothetical or poorly described gene products, were also identified. We have observed differences in most metabolic pathways, in apoptosis, calcium homeostasis, calcification and fibrosis and in different signaling pathways linked or not to mitochondrial function. The most significant changes were observed in ketone body and acetate metabolism, NADH shuttle proteins, amino-acid metabolism proteins and respiratory enzymes. Several of these changes are consistent with the known phenotype of desmin deficiency.

Uncontrolled Calcium Sparks Act As a Dystrophic Signal for Mammalian Skeletal Muscle

Nature Cell Biology. May, 2005  |  Pubmed ID: 15834406

Most excitable cells maintain tight control of intracellular Ca(2+) through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca(2+) release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca(2+) sparks in intact mammalian skeletal muscle. Spontaneous Ca(2+) 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 Ca(2+) sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca(2+) spark response. Thus, membrane-deformation-induced Ca(2+) sparks have an important role in physiological and pathophysiological regulation of Ca(2+) signalling, and uncontrolled Ca(2+) spark activity in connection with chronic activation of store-operated Ca(2+) entry may function as a dystrophic signal in mammalian skeletal muscle.

Enhanced Resistance to Fatigue and Altered Calcium Handling Properties of Sarcalumenin Knockout Mice

Physiological Genomics. Sep, 2005  |  Pubmed ID: 15998745

Sarcalumenin is a Ca2+-binding protein located in the sarcoplasmic reticulum of striated muscle cells, the physiological function of which has not been fully determined yet. Using sarcalumenin knockout (sar(-/-)) mice, we showed that sar ablation altered store-operated Ca2+ entry (SOCE) and enhanced muscle fatigue resistance. Sar(-/-) mice fatigued less with treadmill exercise, and intact isolated soleus and extensor digitorum longus muscles from sar(-/-) mice were more resistant to intermittent fatiguing stimulation than those from wild-type mice. Enhanced SOCE was observed in the sar(-/-) muscles. Biochemical analysis revealed that sar(-/-) muscles contained significantly elevated expression of mitsugumin 29 (MG29), a synaptophysin-related membrane protein located in the triad junction of skeletal muscle. Because the ablation of mg29 has been shown to cause increased fatigability and dysfunction of SOCE, the enhanced SOCE activity seen in sar(-/-) muscle may be correlated with the increased expression of MG29. Our data suggest that systemic ablation of sarcalumenin caused enhanced resistance to muscle fatigue by compensatory changes in Ca2+ regulatory proteins that effect SOCE.

Uncoupling Store-operated Ca2+ Entry and Altered Ca2+ Release from Sarcoplasmic Reticulum Through Silencing of Junctophilin Genes

Biophysical Journal. Jun, 2006  |  Pubmed ID: 16565048

Junctophilin (JP) mediates the close contact between cell surface and intracellular membranes in muscle cells ensuring efficient excitation-contraction coupling. Here we demonstrate that disruption of triad junction structure formed by the transverse tubular (TT) invagination of plasma membrane and terminal cisternae of sarcoplasmic reticulum (SR) by reduction of JP expression leads to defective Ca2+ homeostasis in muscle cells. Using adenovirus with small hairpin interference RNA (shRNA) against both JP1 and JP2 genes, we could achieve acute suppression of JPs in skeletal muscle fibers. The shRNA-treated muscles exhibit deformed triad junctions and reduced store-operated Ca2+ entry (SOCE), which is likely due to uncoupled retrograde signaling from SR to TT. Knockdown of JP also causes a reduction in SR Ca2+ storage and altered caffeine-induced Ca2+ release, suggesting an orthograde regulation of the TT membrane on the SR Ca2+ release machinery. Our data demonstrate that JPs play an important role in controlling overall intracellular Ca2+ homeostasis in muscle cells. We speculate that altered expression of JPs may underlie some of the phenotypic changes associated with certain muscle diseases and aging.

Ca2+ Sparks As a Plastic Signal for Skeletal Muscle Health, Aging, and Dystrophy

Acta Pharmacologica Sinica. Jul, 2006  |  Pubmed ID: 16787561

Ca2+ sparks are the elementary units of intracellular Ca2+ signaling in striated muscle cells revealed as localized Ca2+ release events from sarcoplasmic reticulum (SR) by confocal microscopy. While Ca2+ sparks are well defined in cardiac muscle, there has been a general belief that these localized Ca2+ release events are rare in intact adult mammalian skeletal muscle. Several laboratories determined that Ca2+ sparks in mammalian skeletal muscle could only be observed in large numbers when the sarcolemmal membranes are permeabilized or the SR Ca2+ content is artificially manipulated, thus the cellular and molecular mechanisms underlying the regulation of Ca2+ sparks in skeletal muscle remain largely unexplored. Recently, we discovered that membrane deformation generated by osmotic stress induced a robust Ca2+ spark response confined in close spatial proximity to the sarcolemmal membrane in intact mouse muscle fibers. In addition to Ca2+ sparks, prolonged Ca2+ transients, termed Ca2+ bursts, are also identified in intact skeletal muscle. These induced Ca2+ release events are reversible and repeatable, revealing a plastic nature in young muscle fibers. In contrast, induced Ca2+ sparks in aged muscle are transient and cannot be re-stimulated. Dystrophic muscle fibers display uncontrolled Ca2+ sparks, where osmotic stress-induced Ca2+ sparks are not reversible and they are no longer spatially restricted to the sarcolemmal membrane. An understanding of the mechanisms that underlie generation of osmotic stress-induced Ca2+ sparks in skeletal muscle, and how these mechanisms are altered in pathology, will contribute to our understanding of the regulation of Ca2+ homeostasis in muscle physiology and pathophysiology.

Muscle Aging is Associated with Compromised Ca2+ Spark Signaling and Segregated Intracellular Ca2+ Release

The Journal of Cell Biology. Aug, 2006  |  Pubmed ID: 16943181

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

The Journal of Biological Chemistry. Nov, 2006  |  Pubmed ID: 16945924

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) 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 Ca(2+) 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.

Systemic Ablation of RyR3 Alters Ca2+ Spark Signaling in Adult Skeletal Muscle

Cell Calcium. Dec, 2007  |  Pubmed ID: 17412417

Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle.

Mutations in JPH2-encoded Junctophilin-2 Associated with Hypertrophic Cardiomyopathy in Humans

Journal of Molecular and Cellular Cardiology. Jun, 2007  |  Pubmed ID: 17509612

Junctophilin-2 (JPH2) is a cardiac specific member of the junctophilins, a newly characterized family of junctional membrane complex proteins important in physically approximating the plasmalemmal L-type calcium channel and the sarcoplasmic reticulum ryanodine receptor for calcium-induced calcium release. JPH2 knockout mice showed disrupted calcium transients, altered junctional membrane complex formation, cardiomyopathy, and embryonic lethality. Furthermore, JPH2 gene expression is down-regulated in murine cardiomyopathy models. To this end, we explored JPH2 as a novel candidate gene for the pathogenesis of hypertrophic cardiomyopathy (HCM) in humans. Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of JPH2 was performed on DNA obtained from 388 unrelated patients with HCM. HCM-associated JPH2 mutations were engineered and functionally characterized using immunocytochemistry, cell morphometry measurements, and live cell confocal calcium imaging. Three novel HCM-susceptibility mutations: S101R, Y141H and S165F, which localize to key functional domains, were discovered in 3/388 unrelated patients with HCM and were absent in 1000 ethnic-matched reference alleles. Functionally, each human mutation caused (i) protein reorganization of junctophilin-2, (ii) perturbations in intracellular calcium signaling, and (iii) marked cardiomyocyte hyperplasia. The molecular and functional evidence implicates defective junctophilin-2 and disrupted calcium signaling as a novel pathogenic mechanism for HCM and establishes HCM as the first human disease associated with genetic defects in JPH2. Whether susceptibility for other cardiomyopathies, such as dilated cardiomyopathy, can be conferred by mutations in JPH2 warrants investigation.

TRIC Channels Are Essential for Ca2+ Handling in Intracellular Stores

Nature. Jul, 2007  |  Pubmed ID: 17611541

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.

The Tail-anchoring Domain of Bfl1 and HCCS1 Targets Mitochondrial Membrane Permeability to Induce Apoptosis

Journal of Cell Science. Aug, 2007  |  Pubmed ID: 17666431

Many Bcl2 family proteins target intracellular membranes by their C-terminal tail-anchor domain. Bfl1 is a bi-functional Bcl2 family protein with both anti- and pro-apoptotic activities and contains an amphipathic tail-anchoring peptide (ATAP; residues 147-175) with unique properties. Here we show that ATAP targets specifically to mitochondria, and induces caspase-dependent apoptosis that does not require Bax or Bak. Mutagenesis studies revealed that lysine residues flanking the ATAP sequence are involved in targeting of the peptide to the mitochondrial membrane, and charged residues that contribute to the amphipathic nature of ATAP are critical for its pro-apoptotic function. The ATAP sequence is present in another tumor suppressor gene, HCCS1, which contains an additional mitochondria-targeting signal (MTS) close to the ATAP. We propose that both ATAP and MTS could be used as therapeutic peptides to induce cell death in the treatment of cancer cells.

Immuno-proteomic Approach to Excitation--contraction Coupling in Skeletal and Cardiac Muscle: Molecular Insights Revealed by the Mitsugumins

Cell Calcium. Jan, 2008  |  Pubmed ID: 18061662

A comprehensive understanding of excitation-contraction (E-C) coupling in skeletal and cardiac muscle requires that all the major components of the Ca(2+) release machinery be resolved. We utilized a unique immuno-proteomic approach to generate a monoclonal antibody library that targets proteins localized to the skeletal muscle triad junction, which provides a structural context to allow efficient E-C coupling. Screening of this library has identified several mitsugumins (MG); proteins that can be localized to the triad junction in mammalian skeletal muscle. Many of these proteins, including MG29 and junctophilin, are important components in maintaining the structural integrity of the triad junction. Other triad proteins, such as calumin, play a more direct role in regulation of muscle Ca(2+) homeostasis. We have recently identified a family of trimeric intracellular cation-selective (TRIC) channels that allow for K(+) movement into the endoplasmic or sarcoplasmic reticulum to counter a portion of the transient negative charge produced by Ca(2+) release into the cytosol. Further study of TRIC channel function and other novel mitsugumins will increase our understanding of E-C coupling and Ca(2+) homoeostasis in muscle physiology and pathophysiology.

Altered Ca2+ Sparks in Aging Skeletal and Cardiac Muscle

Ageing Research Reviews. Jul, 2008  |  Pubmed ID: 18272434

Ca2+ sparks are the fundamental units that comprise Ca2+-induced Ca2+ release (CICR) in striated muscle cells. In cardiac muscle, spontaneous Ca2+ sparks underlie the rhythmic CICR activity during heart contraction. In skeletal muscle, Ca2+ sparks remain quiescent during the resting state and are activated in a plastic fashion to accommodate various levels of stress. With aging, the plastic Ca2+ spark signal becomes static in skeletal muscle, whereas loss of CICR control leads to leaky Ca2+ spark activity in aged cardiomyocytes. Ca2+ spark responses reflect the integrated function of the intracellular Ca2+ regulatory machinery centered around the triad or dyad junctional complexes of striated muscles, which harbor the principal molecular players of excitation-contraction coupling. This review highlights the contribution of age-related modification of the Ca2+ release machinery and the effect of membrane structure and membrane cross-talk on the altered Ca2+ spark signaling during aging of striated muscles.

Caveolae/caveolin-1 Are Important Modulators of Store-operated Calcium Entry in Hs578/T Breast Cancer Cells

Journal of Pharmacological Sciences. Feb, 2008  |  Pubmed ID: 18296864

Caveolin-1 is a principal component of caveolae, invaginations of the plasma membrane that are enriched in cholesterol and sphingolipids. The expression of caveolin-1 has been shown to be tightly correlated to the progression of breast cancer tumors. However, the consequences of altered caveolin-1 expression during tumor progression still remain unclear. Modification of caveolin-1 expression modulates store-operated Ca(2+) entry (SOCE) in various cell types. SOCE is a ubiquitous Ca(2+) entry pathway that previous studies have linked to apoptosis and tumor progression in prostate cancer cells. In this study, we tested the effect of altering caveolin-1 expression on SOCE in Hs578/T breast cancer cells. Through overexpression of caveolin-1 and small hairpin RNA (shRNA) knockdown, we generated four stable cell lines that have 3 different caveolin-1 protein levels. Cav-1 overexpression could increase SOCE activity, while knockdown of caveolin-1 significantly reduced SOCE activity. These functional consequences were correlated with changes in caveolae number in Hs578/T cells. Our results suggest alteration of SOCE by caveolin-1 expression changes could be one of the mechanisms contributing to the progression of breast cancer.

Compromised Store-operated Ca2+ Entry in Aged Skeletal Muscle

Aging Cell. Aug, 2008  |  Pubmed ID: 18505477

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 Ca(2+) entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store-operated Ca(2+) entry (SOCE) in aged mouse skeletal muscle. Using Mn(2+) quenching of Fura-2 fluorescence and confocal-microscopic imaging of Ca(2+) 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 Ca(2+) homeostatic capacity generally associated with muscle aging.

A Missense Mutation in Desmin Tail Domain Linked to Human Dilated Cardiomyopathy Promotes Cleavage of the Head Domain and Abolishes Its Z-disc Localization

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Sep, 2008  |  Pubmed ID: 18539904

A missense mutation (Ile 451 to Met) at the tail domain of the muscle-specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. The Ile451Met mutation is located inside a conserved motif in the desmin tail domain, believed to have a potential role in the lateral packing of type III intermediate filaments. Nevertheless, the role of the type III intermediate filament tail domain remains elusive. To further study the role of this domain in the function of cardiomyocytes and in the development of cardiomyopathy, we generated transgenic mice expressing the mutant desmin(I451M) in the cardiac tissue. Analysis of hearts from transgenic animals revealed that mutant desmin loses its Z-disc localization but it can still associate with the intercalated discs, which, however, have an altered architecture, resembling other examples of dilated cardiomyopathy. This is the first report demonstrating a critical role of the desmin head and tail domains in the formation of the IF scaffold around Z discs. It is further suggested that in cardiomyocytes, an interplay between desmin tail and head domains is taking place, which potentially protects the amino terminus of desmin from specific proteases. The fact that the association with intercalated discs seems unchanged suggests that this association must take place through the desmin tail, in contrast to the head domain that is most possibly involved in the Z-disc binding.

MG53 Regulates Membrane Budding and Exocytosis in Muscle Cells

The Journal of Biological Chemistry. Jan, 2009  |  Pubmed ID: 19029292

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.

MG53 Nucleates Assembly of Cell Membrane Repair Machinery

Nature Cell Biology. Jan, 2009  |  Pubmed ID: 19043407

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 Ca(2+) facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca(2+)-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.

The Amino-terminal Peptide of Bax Perturbs Intracellular Ca2+ Homeostasis to Enhance Apoptosis in Prostate Cancer Cells

American Journal of Physiology. Cell Physiology. Feb, 2009  |  Pubmed ID: 19091958

During apoptosis, proteolytic cleavage of Bax at the amino terminus generates a truncated Bax of approximately 18 kDa (p18Bax) and an amino-terminal peptide of approximately 3 kDa (p3Bax). Whereas extensive studies have shown that p18Bax behaves like a BH3 protein with enhanced pro-apoptotic function over that of the full-length Bax (p21Bax), little is known about the function of p3Bax in apoptosis. We have previously shown that Bax and Ca2+ play a synergistic role in amplifying apoptosis signaling and that store-operated Ca2+ entry (SOCE) contributes to Bax-mediated apoptosis in prostate cancer cells. Here we test whether p3Bax can contribute to regulation of Ca2+ signaling during apoptosis through use of a membrane-penetrating peptide to facilitate delivery of recombinant p3Bax into NRP-154 cells, a prostate epithelial cell line with tumorigenic capacity. We find that human immunodefficiency virus transactivator of transcription protein (TAT)-p3Bax fusion peptide can enhance thapsigargin-induced apoptosis in NRP-154 cells, elevate SOCE activity, and increase inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores. Our data indicates that p3Bax can modulate the entry of extracellular Ca2+ and thus regulate the amplification of apoptosis in prostate cancer cells.

Membrane Repair Defects in Muscular Dystrophy Are Linked to Altered Interaction Between MG53, Caveolin-3, and Dysferlin

The Journal of Biological Chemistry. Jun, 2009  |  Pubmed ID: 19380584

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.

Mitsugumin 53 (MG53) Facilitates Vesicle Trafficking in Striated Muscle to Contribute to Cell Membrane Repair

Communicative & Integrative Biology. May, 2009  |  Pubmed ID: 19641737

Repair of the plasma membrane following damage is an important aspect of normal cellular physiology, and disruption of this process is observed in many pathologic states. In a recent series of publications, we resolved that Mitsugumin 53 (MG53) is a novel, muscle-specific member of the tripartite motif/RING B-box Coiled Coil (TRIM/RBCC) family of proteins (TRIM72) that contributes to vesicle trafficking in the course of normal cellular physiology. MG53 can bind phosphatidylserine (PS) with some specificity, and interacts with caveolin-3 (Cav-3) as part of its function in vesicle trafficking. As part of the response to membrane damage in muscle cells, MG53 acts in an oxidation-dependent manner to facilitate vesicle translocation to the sites of membrane injury where these vesicles are involved in patching the membrane. Here we discuss these findings and examine the implications of this work in the field of membrane repair. Further discussion is provided about potential therapeutic applications for MG53.

S165F Mutation of Junctophilin 2 Affects Ca2+ Signalling in Skeletal Muscle

The Biochemical Journal. Apr, 2010  |  Pubmed ID: 20095964

JPs (junctophilins) contribute to the formation of junctional membrane complexes in muscle cells by physically linking the t-tubule (transverse-tubule) and SR (sarcoplasmic reticulum) membranes. In humans with HCM (hypertrophic cardiomyopathy), mutations in JP2 are linked to altered Ca2+ signalling in cardiomyocytes; however, the effects of these mutations on skeletal muscle function have not been examined. In the present study, we investigated the role of the dominant-negative JP2-S165F mutation (which is associated with human HCM) in skeletal muscle. Consistent with the hypertrophy observed in human cardiac muscle, overexpression of JP2-S165F in primary mouse skeletal myotubes led to a significant increase in myotube diameter and resting cytosolic Ca2+ concentration. Single myotube Ca2+ imaging experiments showed reductions in both the excitation-contraction coupling gain and RyR (ryanodine receptor) 1-mediated Ca2+ release from the SR. Immunoprecipitation assays revealed defects in the PKC (protein kinase C)-mediated phosphorylation of the JP2-S165F mutant protein at Ser165 and in binding of JP2-S165F to the Ca2+ channel TRPC3 (transient receptor potential cation canonical-type channel 3) on the t-tubule membrane. Therefore both the hypertrophy and altered intracellular Ca2+ signalling in the JP2-S165F-expressing skeletal myotubes can be linked to altered phosphorylation of JP2 and/or altered cross-talk among Ca2+ channels on the t-tubule and SR membranes.

Repression of Cardiac Phospholamban Gene Expression is Mediated by Thyroid Hormone Receptor-{alpha}1 and Involves Targeted Covalent Histone Modifications

Endocrinology. Jun, 2010  |  Pubmed ID: 20392835

Phospholamban (PLB) is a critical regulator of Ca(2+) cycling in heart muscle cells, and its gene expression is markedly down-regulated by T(3). Nonetheless, little is known about the molecular mechanisms of T(3)-dependent gene silencing in cardiac muscle, and it remains unclear whether thyroid hormone receptors (TRs) directly bind at the PLB gene in vivo and facilitate transcriptional repression. To investigate the regulatory role of TRs in PLB transcription, we used a physiological murine heart muscle cell line (HL-1) that retains cardiac electrophysiological properties, expresses both TRalpha1 and TRbeta1 subtypes, and exhibits T(3)-dependent silencing of PLB expression. By performing RNA interference assays with HL-1 cells, we found that TRalpha1, but not TRbeta1, is essential for T(3)-dependent PLB gene repression. Interestingly, a PLB reporter gene containing only the core promoter sequences -156 to +64 displayed robust T(3)-dependent silencing in HL-1 cells, thus suggesting that transcriptional repression is facilitated by TRalpha1 via the PLB core promoter, a regulatory region highly conserved in mammals. Consistent with this notion, chromatin immunoprecipitation and in vitro binding assays show that TRalpha1 directly binds at the PLB core promoter region. Furthermore, addition of T(3) triggered alterations in covalent histone modifications at the PLB promoter that are associated with gene silencing, namely a pronounced decrease in both histone H3 acetylation and histone H3 lysine 4 methylation. Taken together, our data reveal that T(3)-dependent repression of PLB in cardiac myocytes is directly facilitated by TRalpha1 and involves the hormone-dependent recruitment of histone-modifying enzymes associated with transcriptional silencing.

Cardioprotection of Ischemia/reperfusion Injury by Cholesterol-dependent MG53-mediated Membrane Repair

Circulation Research. Jul, 2010  |  Pubmed ID: 20466981

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.

MG53 Constitutes a Primary Determinant of Cardiac Ischemic Preconditioning

Circulation. Jun, 2010  |  Pubmed ID: 20516375

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-3beta [GSK3beta] 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.

Increased Store-operated Ca2+ Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Biophysical Journal. Sep, 2010  |  Pubmed ID: 20816068

Store-operated Ca(2+) entry (SOCE) contributes to Ca(2+) handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn(2+) fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca(2+) store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39 degrees C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca(2+) permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca(2+) as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca(2+) entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca(2+) signaling in MH.

Amphipathic Tail-anchoring Peptide and Bcl-2 Homology Domain-3 (BH3) Peptides from Bcl-2 Family Proteins Induce Apoptosis Through Different Mechanisms

The Journal of Biological Chemistry. Mar, 2011  |  Pubmed ID: 21189256

Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosis-resistant nature of cancer cells.

Junctophilin-2 Expression Silencing Causes Cardiocyte Hypertrophy and Abnormal Intracellular Calcium-handling

Circulation. Heart Failure. Mar, 2011  |  Pubmed ID: 21216834

Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca(2+)) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca(2+) channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of downregulation of JPH2 expression on intracellular Ca(2+) handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca(2+) handling in a prohypertrophic manner.

Polymerase Transcriptase Release Factor (PTRF) Anchors MG53 Protein to Cell Injury Site for Initiation of Membrane Repair

The Journal of Biological Chemistry. Apr, 2011  |  Pubmed ID: 21343302

Plasma membrane repair is an essential process for maintenance of homeostasis at the cellular and tissue levels, whereas compromised repair capacity contributes to degenerative human diseases. Our recent studies show that MG53 is essential for muscle membrane repair, and defects in MG53 function are linked to muscular dystrophy and cardiac dysfunction. Here we report that polymerase I and transcript release factor (PTRF), a gene known to regulate caveolae membrane structure, is an indispensable component of the membrane repair machinery. PTRF acts as a docking protein for MG53 during membrane repair potentially by binding exposed membrane cholesterol at the injury site. Cells lacking expression of endogenous PTRF show defective trafficking of MG53 to membrane injury sites. A mutation in PTRF associated with human disease results in aberrant nuclear localization of PTRF and disrupts MG53 function in membrane resealing. Although RNAi silencing of PTRF leads to defective muscle membrane repair, overexpression of PTRF can rescue membrane repair defects in dystrophic muscle. Our data suggest that membrane-delimited interaction between MG53 and PTRF contributes to initiation of cell membrane repair, which can be an attractive target for treatment or prevention of tissue injury in human diseases.

Dysferlin, Annexin A1, and Mitsugumin 53 Are Upregulated in Muscular Dystrophy and Localize to Longitudinal Tubules of the T-system with Stretch

Journal of Neuropathology and Experimental Neurology. Apr, 2011  |  Pubmed ID: 21412170

Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.

A Versatile Single-plasmid System for Tissue-specific and Inducible Control of Gene Expression in Transgenic Mice

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Aug, 2011  |  Pubmed ID: 21518849

We describe a novel transgenic system for tissue-specific and inducible control of gene expression in mice. The system employs a tetracycline-responsive CMV promoter that controls transcription of a short-hairpin RNA (shRNA) that remains nonfunctional until an interrupting reporter cassette is excised by Cre recombinase. Insertion of Dicer and Drosha RNase processing sites within the shRNA allows generation of siRNA to knock down a target gene efficiently. Tissue-specific shRNA expression is achieved through the use of appropriate inducer mice with tissue-specific expression of Cre. We applied this system to regulate expression of junctophilins (JPs), genes essential for maintenance of membrane ultrastructure and Ca(2+) signaling in muscle. Transgenic mice with skeletal muscle-specific expression of shRNA against JP mRNAs displayed no basal change of JP expression before treatment with doxycycline (Dox), while inducible and reversible knockdown of JPs was achieved by feeding mice with Dox-containing water. Dox-induced knockdown of JPs led to abnormal junctional membrane structure and Ca(2+) signaling in adult muscle fibers, consistent with essential roles of JPs in muscle development and function. This transgenic approach can be applied for inducible and reversible gene knockdown or gene overexpression in many different tissues, thus providing a versatile system for elucidating the physiological gene function in viable animal models.

Redox-dependent Oligomerization Through a Leucine Zipper Motif is Essential for MG53-mediated Cell Membrane Repair

American Journal of Physiology. Cell Physiology. Jul, 2011  |  Pubmed ID: 21525429

We recently discovered that MG53, a muscle-specific tripartite motif (TRIM) family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery. Our data showed that disulfide bond formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that facilitates oligomerization of MG53 during cell membrane repair. Two leucine zipper motifs in the coiled-coil domain of MG53 (LZ1 - L176/L183/L190/V197 and LZ2 - L205/L212/L219/L226) are highly conserved across the different animal species. Chemical cross-linking studies show that LZ1 is critical for MG53 homodimerization, whereas LZ2 is not. Mutations of the conserved leucines into alanines in LZ1, not in LZ2, diminish the redox-dependent oligomerization of MG53. Live cell imaging studies demonstrate that the movement of green fluorescent protein (GFP)-tagged MG53 mutants (GFP-LA1 and GFP-LA2) is partially compromised in response to mechanical damage of the cell membrane, and the GFP-LA1/2 double mutant is completely ineffective in translocation toward the injury sites. In addition to the leucine zipper-mediated intermolecular interaction, redox-dependent cross talk between MG53 appears to be an obligatory step for cell membrane repair, since in vivo modification of cysteine residues with alkylating reagents can prevent the movement of MG53 toward the injury sites. Our data show that oxidation of the thiol group of Cys242 and leucine zipper-mediated interaction among the MG53 molecules both contribute to the nucleation process for MG53-mediated cell membrane repair.

Imaging Superoxide Flash and Metabolism-coupled Mitochondrial Permeability Transition in Living Animals

Cell Research. Sep, 2011  |  Pubmed ID: 21556035

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.

Store-operated Ca(2+) Entry (SOCE) Contributes to Normal Skeletal Muscle Contractility in Young but Not in Aged Skeletal Muscle

Aging. Jun, 2011  |  Pubmed ID: 21666285

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

Mitochondrial Calcium Uptake Regulates Rapid Calcium Transients in Skeletal Muscle During Excitation-contraction (E-C) Coupling

The Journal of Biological Chemistry. Sep, 2011  |  Pubmed ID: 21795684

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.

Disrupted Membrane Structure and Intracellular Ca²⁺ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

PloS One. 2011  |  Pubmed ID: 21984944

Efficient intracellular Ca²⁺ ([Ca²⁺]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca²⁺ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short -hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca²⁺ release. Voltage-induced Ca²⁺ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca²⁺ current and SR Ca²⁺ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca²⁺ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca²⁺ sparks' amplitude that was attributed to decreased total Ca²⁺ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca²⁺]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.

Store-operated Calcium Entry is Present in HL-1 Cardiomyocytes and Contributes to Resting Calcium

Biochemical and Biophysical Research Communications. Dec, 2011  |  Pubmed ID: 22079292

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.

Detection of Calcium Sparks in Intact and Permeabilized Skeletal Muscle Fibers

Methods in Molecular Biology (Clifton, N.J.). 2012  |  Pubmed ID: 22130850

Ca(2+) sparks are the elementary units of Ca(2+) signaling in striated muscle fibers that appear as highly localized Ca(2+) release events through ryanodine receptor (RyR) Ca(2+) release channels in the sarcoplasmic reticulum (SR). While these events are commonly observed in resting cardiac myocytes, they are rarely seen in resting skeletal muscle fibers. Since Ca(2+) spark analysis can provide extensive data on the Ca(2+) handling characteritsics of normal and diseased striated muscle, there has been interest in developing methods for observing Ca(2+) sparks in skeletal muscle. Previously, we discovered that stress generated by osmotic pressure changes induces a robust Ca(2+) spark response confined in close spatial proximity to the sarcolemmal membrane in wild-type intact mammalian muscles. Our studies showed these peripheral Ca(2+) sparks (PCS) were altered in dystrophic or aged skeletal muscles. Other methods to induce Ca(2+) sparks include permeabilization of the sarcolemmal membrane with detergents, such as saponin. In this chapter, we will discuss the methods for isolation of muscle fibers, the techniques for inducing Ca(2+) sparks in these isolated fibers, and provide guidance on the analysis of data from these experiments.

Enhancing Muscle Membrane Repair by Gene Delivery of MG53 Ameliorates Muscular Dystrophy and Heart Failure in δ-Sarcoglycan-deficient Hamsters

Molecular Therapy : the Journal of the American Society of Gene Therapy. Feb, 2012  |  Pubmed ID: 22314291

Muscular dystrophies (MDs) are caused by genetic mutations in over 30 different genes, many of which encode for proteins essential for the integrity of muscle cell structure and membrane. Their deficiencies cause the muscle vulnerable to mechanical and biochemical damages, leading to membrane leakage, dystrophic pathology, and eventual loss of muscle cells. Recent studies report that MG53, a muscle-specific TRIM-family protein, plays an essential role in sarcolemmal membrane repair. Here, we show that systemic delivery and muscle-specific overexpression of human MG53 gene by recombinant adeno-associated virus (AAV) vectors enhanced membrane repair, ameliorated pathology, and improved muscle and heart functions in δ-sarcoglycan (δ-SG)-deficient TO-2 hamsters, an animal model of MD and congestive heart failure. In addition, MG53 overexpression increased dysferlin level and facilitated its trafficking to muscle membrane through participation of caveolin-3. MG53 also protected muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2), and glycogen synthase kinase-3β (GSK-3β) and inhibiting proapoptotic protein Bax. Our results suggest that enhancing the muscle membrane repair machinery could be a novel therapeutic approach for MD and cardiomyopathy, as demonstrated here in the limb girdle MD (LGMD) 2F model.

Nonmuscle Myosin IIA Facilitates Vesicle Trafficking for MG53-mediated Cell Membrane Repair

The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jan, 2012  |  Pubmed ID: 22253476

Repair of injury to the plasma membrane is an essential mechanism for maintenance of cellular homeostasis and integrity that involves coordinated movement of intracellular vesicles to membrane injury sites to facilitate patch formation. We have previously identified MG53 as an essential component of the cell membrane repair machinery. In order for MG53 and intracellular vesicles to translocate to membrane injury sites, motor proteins must be involved. Here, we show that nonmuscle myosin type IIA (NM-IIA) interacts with MG53 to regulate vesicle trafficking during cell membrane repair. In cells that are deficient for NM-IIA expression, MG53 cannot translocate to acute injury sites, whereas rescue of NM-IIA expression in these cells can restore MG53-mediated membrane repair. Compromised cell membrane repair is observed in cells with RNAi-mediated knockdown of NM-IIA expression, or following pharmacological alteration of NM-IIA motor function. Together, our data reveal NM-IIA as a key cytoskeleton motor protein that facilitates vesicle trafficking during MG53-mediated cell membrane repair.-Lin, P., Zhu, H., Cai, C., Wang, X., Cao, C., Xiao, R., Pan, Z., Weisleder, N., Takeshima, H., Ma, J. Nonmuscle myosin IIA facilitates vesicle trafficking for MG53-mediated cell membrane repair.

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