Proteins Of Dystrophin-glycoprotein Complex Research Articles

Glycative stress inhibits hypertrophy and impairs cell membrane integrity in overloaded mouse skeletal muscle.

Glycative stress, characterized by the formation and accumulation of advanced glycation end products (AGEs) associated with protein glycation reactions, has been implicated in inducing a decline of muscle function. Although the inverse correlation between glycative stress and muscle mass and strength has been demonstrated, the underlying molecular mechanisms are not fully understood. This study aimed to elucidate how glycative stress affects the skeletal muscle, particularly the adaptive muscle response to hypertrophic stimuli and its molecular mechanism. Male C57BL/6NCr mice were randomly divided into the following two groups: the bovine serum albumin (BSA)-treated and AGE-treated groups. Mice in the AGE-treated group were intraperitoneally administered AGEs (0.5mg/g) once daily, whereas those in the BSA-treated group received an equal amount of BSA (0.5mg/g) as the vehicle control. After 7days of continuous administration, the right leg plantaris muscle of mice in each group underwent functional overload treatment by synergist ablation for 7days to induce muscle hypertrophy. In in vitro studies, cultured C2C12 myocytes were treated with AGEs (1mg/mL) to examine cell adhesion and cell membrane permeability. Continuous AGE administration increased the levels of fluorescent AGEs, Nε-(carboxymethyl) lysine, and methylglyoxal-derived hydroimidazolone-1 in both plasma and skeletal muscle. Plantaris muscle weight, muscle fibre cross-sectional area, protein synthesis rate, and the number of myonuclei increased with functional overload in both groups; however, the increase was significantly reduced by AGE treatment. Some muscles of AGE-treated mice were destroyed by functional overload. Proteomic analysis was performed to explore the mechanisms of muscle hypertrophy suppression and myofibre destruction by AGEs. When principal component analysis was performed on 4659 data obtained by proteomic analysis, AGE treatment was observed to affect protein expression only in functionally overloaded muscles. Enrichment analysis of the 436 proteins extracted using the K-means method further identified a group of proteins involved in cell adhesion. Consistent with this finding, dystrophin-glycoprotein complex proteins and cell adhesion-related proteins were confirmed to increase with functional overload; however, this was attenuated by AGE treatment. Additionally, the treatment of C2C12 muscle cells with AGEs inhibited their ability to adhere and increased cell membrane permeability. This study indicates that glycative stress may be a novel pathogenic factor in skeletal muscle dysfunctions by causing loss of membrane integrity and preventing muscle mass gain.

Variants in DTNA cause a mild, dominantly inherited muscular dystrophy.

DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, β, δ and γ-sarcoglycans, and α and β-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Reduced plasticity and microtubule densification in muscular dystrophy-related cardiomyopathy

The dystrophin-glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular basement membrane, thereby providing structural support for the sarcolemma. Many patients with muscular dystrophies, particularly those with defects in cardiomyopathies with chamber dilation and myocardial dysfunction. Heart failure is the major cause of death for muscular dystrophy patients; however, the molecular pathomechanism remains unknown. Here, I show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced the glycosylation of α-dystroglycan and the expression of DGC proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that the physiological contribution of DGC proteins in the heart increases after 6 mo of age. In addition, Fktn-deficient mice maintain normal cardiac function at young age, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. Young Fktn-deficient mice did not show a compensative hypertrophic response to hemodynamic stress and quickly developed heart failure with chamber dilation and contractile dysfunction. In these mice, Ca2+-calcineurin signaling was already elevated under physiological conditions, and MEF2-HDAC axes essential for the hypertrophic response were unable to function under stress conditions. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. Microarray analysis in control and Fktn-deficient hearts suggest that elimination of Fktn impacts the expression profile of Golgi-related genes, and that the pathological mechanism of cardiac dysfunction induced by Fktn elimination partly overlaps with that of neurodegenerative disease. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and, thus, the results may lead to strategies for intervention.

Contraction-Induced Loss of Plasmalemmal Electrophysiological Function Is Dependent on the Dystrophin Glycoprotein Complex.

Weakness and atrophy are key features of Duchenne muscular dystrophy (DMD). Dystrophin is one of the many proteins within the dystrophin glycoprotein complex (DGC) that maintains plasmalemmal integrity and cellular homeostasis. The dystrophin-deficient mdx mouse is also predisposed to weakness, particularly when subjected to eccentric (ECC) contractions due to electrophysiological dysfunction of the plasmalemma. Here, we determined if maintenance of plasmalemmal excitability during and after a bout of ECC contractions is dependent on intact and functional DGCs rather than, solely, dystrophin expression. Wild-type (WT) and dystrophic mice (mdx, mL172H and Sgcb−/− mimicking Duchenne, Becker and Limb-girdle Type 2E muscular dystrophies, respectively) with varying levels of dystrophin and DGC functionality performed 50 maximal ECC contractions with simultaneous torque and electromyographic measurements (M-wave root-mean-square, M-wave RMS). ECC contractions caused all mouse lines to lose torque (p<0.001); however, deficits were greater in dystrophic mouse lines compared to WT mice (p<0.001). Loss of ECC torque did not correspond to a reduction in M-wave RMS in WT mice (p=0.080), while deficits in M-wave RMS exceeded 50% in all dystrophic mouse lines (p≤0.007). Moreover, reductions in ECC torque and M-wave RMS were greater in mdx mice compared to mL172H mice (p≤0.042). No differences were observed between mdx and Sgcb−/− mice (p≥0.337). Regression analysis revealed ≥98% of the variance in ECC torque loss could be explained by the variance in M-wave RMS in dystrophic mouse lines (p<0.001) but not within WT mice (R2=0.211; p=0.155). By comparing mouse lines that had varying amounts and functionality of dystrophin and other DGC proteins, we observed that (1) when all DGCs are intact, plasmalemmal action potential generation and conduction is maintained, (2) deficiency of the DGC protein β-sarcoglycan is as disruptive to plasmalemmal excitability as is dystrophin deficiency and, (3) some functionally intact DGCs are better than none. Our results highlight the significant role of the DGC plays in maintaining plasmalemmal excitability and that a collective synergism (via each DGC protein) is required for this complex to function properly during ECC contractions.

Phosphorylation of α-syntrophin is responsible for its subcellular localization and interaction with dystrophin in muscle cells

Syntrophin is a well-known adaptor protein that links intracellular proteins with the dystrophin-glycoprotein complex (DGC) at the sarcolemma. However, little is known about the underlying mechanism that regulates the intracellular localization of α-syntrophin and its interaction with dystrophin. In this study, we demonstrate that α-syntrophin phosphorylation determines its intracellular localization and interaction with dystrophin in muscle cells. α-Syntrophin, a predominant isoform in skeletal muscles, directly interacts with ion channels, enzymes, receptors, and DGC proteins. Despite α-syntrophin being a potential signaling molecule, most studies focus on its function as a dystrophin-associated protein. However, we previously reported that α-syntrophin has a variety of DGC-independent functions to modulate cell migration, differentiation, survival, and protein stability. According to the results of the in vitro phosphorylation assays using subcellular fractions, the phosphorylated α-syntrophin accumulated only at the plasma membrane, and this event occurred regardless of dystrophin expression. However, the α-syntrophin interacting with dystrophin at the membrane was not in a phosphorylated state. We also identified that protein kinase C (PKC) was involved in the phosphorylation of α-syntrophin, which restricted α-syntrophin to interact with dystrophin. In conclusion, we demonstrate that the phosphorylation of α-syntrophin by PKC regulates its intracellular localization and interaction with dystrophin.

Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure

Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.

Exogenous expression of the glycosyltransferase LARGE1 restores \u03b1-dystroglycan matriglycan and laminin binding in rhabdomyosarcoma

Backgroundα-Dystroglycan is the highly glycosylated component of the dystrophin-glycoprotein complex (DGC) that binds with high-affinity to extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains via a unique heteropolysaccharide [-GlcA-beta1,3-Xyl-alpha1,3-]n called matriglycan. Changes in expression of components of the DGC or in the O-glycosylation of α-dystroglycan result in muscular dystrophy but are also observed in certain cancers. In mice, the loss of either of two DGC proteins, dystrophin or α-sarcoglycan, is associated with a high incidence of rhabdomyosarcoma (RMS). In addition, glycosylation of α-dystroglycan is aberrant in a small cohort of human patients with RMS. Since both the glycosylation of α-dystroglycan and its function as an ECM receptor require over 18 post-translational processing enzymes, we hypothesized that understanding its role in the pathogenesis of RMS requires a complete analysis of the expression of dystroglycan-modifying enzymes and the characterization of α-dystroglycan glycosylation in the context of RMS.MethodsA series of cell lines and biopsy samples from human and mouse RMS were analyzed for the glycosylation status of α-dystroglycan and for expression of the genes encoding the responsible enzymes, in particular those required for the addition of matriglycan. Furthermore, the glycosyltransferase LARGE1 was ectopically expressed in RMS cells to determine its effects on matriglycan modifications and the ability of α-dystroglycan to function as a laminin receptor.ResultsImmunohistochemistry and immunoblotting of a collection of primary RMS tumors show that although α-dystroglycan is consistently expressed and glycosylated in these tumors, α-dystroglycan lacks matriglycan and the ability to bind laminin. Similarly, in a series of cell lines derived from human and mouse RMS, α-dystroglycan lacks matriglycan modification and the ability to bind laminin. RNAseq data from RMS cell lines was analyzed for expression of the genes known to be involved in α-dystroglycan glycosylation, which revealed that, for most cell lines, the lack of matriglycan can be attributed to the downregulation of the dystroglycan-modifying enzyme LARGE1. Ectopic expression of LARGE1 in these cell cultures restored matriglycan to levels comparable to those in muscle and restored high-affinity laminin binding to α-dystroglycan.ConclusionsCollectively, our findings demonstrate that a lack of matriglycan on α-dystroglycan is a common feature in RMS due to the downregulation of LARGE1, and that ectopic expression of LARGE1 can restore matriglycan modifications and the ability of α-dystroglycan to function as an ECM receptor.

Regulation of the dystrophin-associated glycoprotein complex composition by the metabolic properties of muscle fibres

The dystrophin-glycoprotein complex (DGC) links the muscle cytoskeleton to the extracellular matrix and is responsible for force transduction and protects the muscle fibres from contraction induced damage. Mutations in components of the DGC are responsible for muscular dystrophies and congenital myopathies. Expression of DGC components have been shown to be altered in many myopathies. In contrast we have very little evidence of whether adaptive changes in muscle impact on DGC expression. In this study we investigated connection between muscle fibre phenotype and the DGC. Our study reveals that the levels of DGC proteins at the sarcolemma differ in highly glycolytic muscle compared to wild-type and that these changes can be normalised by the super-imposition of an oxidative metabolic programme. Importantly we show that the metabolic properties of the muscle do not impact on the total amount of DGC components at the protein level. Our work shows that the metabolic property of a muscle fibre is a key factor in regulating the expression of DGC proteins at the sarcolemma.

Absence of γ-sarcoglycan alters the response of p70S6 kinase to mechanical perturbation in murine skeletal muscle

BackgroundThe dystrophin glycoprotein complex (DGC) is located at the sarcolemma of muscle fibers, providing structural integrity. Mutations in and loss of DGC proteins cause a spectrum of muscular dystrophies. When only the sarcoglycan subcomplex is absent, muscles display severe myofiber degeneration, but little susceptibility to contractile damage, suggesting that disease occurs not by structural deficits but through aberrant signaling, namely, loss of normal mechanotransduction signaling through the sarcoglycan complex. We extended our previous studies on mechanosensitive, γ-sarcoglycan-dependent ERK1/2 phosphorylation, to determine whether additional pathways are altered with the loss of γ-sarcoglycan.MethodsWe examined mechanotransduction in the presence and absence of γ-sarcoglycan, using C2C12 myotubes, and primary cultures and isolated muscles from C57Bl/6 (C57) and γ-sarcoglycan-null (γ-SG-/-) mice. All were subjected to cyclic passive stretch. Signaling protein phosphorylation was determined by immunoblotting of lysates from stretched and non-stretched samples. Calcium dependence was assessed by maintaining muscles in calcium-free or tetracaine-supplemented Ringer’s solution. Dependence on mTOR was determined by stretching isolated muscles in the presence or absence of rapamycin.ResultsC2C12 myotube stretch caused a robust increase in P-p70S6K, but decreased P-FAK and P-ERK2. Neither Akt nor ERK1 were responsive to passive stretch. Similar but non-significant trends were observed in C57 primary cultures in response to stretch, and γ-SG-/- cultures displayed no p70S6K response. In contrast, in isolated muscles, p70S6K was mechanically responsive. Basal p70S6K activation was elevated in muscles of γ-SG-/- mice, in a calcium-independent manner. p70S6K activation increased with stretch in both C57 and γ-SG-/- isolated muscles, and was sustained in γ-SG-/- muscles, unlike the transient response in C57 muscles. Rapamycin treatment blocked all of p70S6K activation in stretched C57 muscles, and reduced downstream S6RP phosphorylation. However, even though rapamycin treatment decreased p70S6K activation in stretched γ-SG-/- muscles, S6RP phosphorylation remained elevated.Conclusionsp70S6K is an important component of γ-sarcoglycan-dependent mechanotransduction in skeletal muscle. Our results suggest that loss of γ-sarcoglycan uncouples the response of p70S6K to stretch and implies that γ-sarcoglycan is important for inactivation of this pathway. Overall, we assert that altered load-sensing mechanisms exist in muscular dystrophies where the sarcoglycans are absent.

Effects of a high fat diet and Metformin on skeletal muscle membrane proteins and fiber size in young mice

A prolonged high fat diet (HFD) is known to impair insulin signaling in skeletal muscle. Specifically, HFD reduces caveolin‐3, Akt activation, and GLUT‐4 translocation to the membrane. Type II diabetes results in the loss of sarcolemmal proteins of the dystrophin‐glycoprotein complex (DGC), particularly dystrophin and nNOS. However, the effect of HFD on DGC proteins is unknown. We hypothesized that HFD would cause translocation of DGC proteins away from the membrane. We further postulated that Metformin, an oral anti‐diabetic drug, would alleviate translocation of DGC proteins. To test our hypotheses, 8 week old C57Bl‐6 mice were fed a high fat diet for 14 weeks (n=8). After 9 weeks, mice were also given either PBS (n=4) or Metformin (n=4) for the remaining 5 weeks. Soleus muscle fiber cross sectional area (CSA) and DGC protein expression were quantified using immunohistochemistry assays. Surprisingly, we found a marked increase in fiber CSA that was normalized by Metformin. HFD resulted in a small decrease of dystrophin membrane expression with no change in nNOS, while the Metformin group expressed normal levels. These findings indicate that the DGC may undergo only minor functional changes in young mice fed a short term HFD, which are similar findings seen with muscle fiber hypertrophy. We postulate that intact DGC proteins may help preserve insulin signaling during short term HFD. Our future research will therefore focus on understanding how insulin signaling (e.g., IRS‐1, mTOR) might be preserved, and the point at which intact muscle insulin pathways in young mice become perturbed with prolonged HFD. This work was supported in part by ADA grant 1–10‐JF‐54 and AHA 12BGIA9050003 (to C.W).

Age‐related changes in dystrophin–glycoprotein complex and in utrophin are not correlated with intrinsic laryngeal muscles protection in mdx mice

In this study we investigate whether dystrophic intrinsic laryngeal muscles (ILM) from aged mdx mice show alterations in dystrophin-glycoprotein complex (DGC) components.Immunofluorescence and immunoblotting analyses of beta-sarcoglycan, beta-dystroglycan, and utrophin showed that aged ILM had a similar pattern of changes in aged affected muscles (diaphragm and limb), suggesting that aging leads to changes in utrophin and DGC proteins in dystrophic ILM that cannot be correlated with their protection from dystrophic change.

P22 Clinical and pathological heterogeneity in partial merosin deficiency

P22 Clinical and pathological heterogeneity in partial merosin deficiency

MLC1 trafficking and membrane expression in astrocytes: Role of caveolin-1 and phosphorylation

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.

Correlation of dystrophin–glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscle

The dystrophin-glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow-type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast-type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue-resistant characteristics of skeletal muscle. We measured the expression of dystrophin and beta1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. The expression of dystrophin and beta1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast-type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Our results support the idea that DGC and FAC are among the factors that explain the fatigue-resistant property not only of slow-type but also of fast-type skeletal muscles.

Dystrophin glycoprotein complex‐associated Gβγ subunits activate phosphatidylinositol‐3‐kinase/Akt signaling in skeletal muscle in a laminin‐dependent manner

Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein (Galphabetagamma) to bind, changing the activation state of the Gsalpha subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. Gbetagamma, released when Gsalpha is activated, is known to bind phosphatidylinositol-3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gbetagamma, using immunoprecipitation and immunoblotting, and purified Gbetagamma. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to alpha-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain Gbetagamma also caused PI3K and Akt activation. These results show that DGC-Gbetagamma is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin beta1 compared to normal muscle. This integrin binds laminin, Gbetagamma, and PI3K. Collectively, these suggest that PI3K is an important target for the Gbetagamma, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-Gbetagamma-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Upregulating integrin beta1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease.

Skeletal muscle syntrophin interactors revealed by yeast two-hybrid assay.

Syntrophins are the cytoplasmic peripheral proteins of dystrophin glycoprotein complex, of which five (alpha l, beta 1, beta 2, gamma 1 and gamma 2) isoforms have been identified so far. Respective syntrophin isoforms are encoded by different genes but have similar domain structures. At the sarcolemma of skeletal muscle, the most abundant alpha l-syntrophin was shown to interact at its PDZ domain with many membrane proteins. Among them, the AQP4 interaction with alpha 1-syntrophin PDZ domain was demonstrated by a Tg mouse study, prompting us to investigate the interaction between mouse alpha l-syntrophin (BC018546: nt.267-492, PDZ domain) pEXP-AD502 as prey vector and mouse AQP4 (NM009700: nt.805-969) pDBLeu as bait vector by the yeast two-hybrid assay, resulting in a negative study. We further studied the binding partner of another sarcolemma located beta 1-syntrophin, and performed a yeast two-hybrid experiment. With human beta 1-syntrophin as bait and human skeletal muscle cDNA library as prey, we obtained one positive clone which turned out to be alpha-dystrobrevin. Although the interaction of human beta 1-syntrophin with alpha-dystrobrevin has already been shown by immunoprecipitation assay, we have here confirmed this interaction by a yeast two-hybrid experiment.

Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation

Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. The DGC is expressed in smooth muscle tissue, but its functional role is not fully established. We tested whether contractile phenotype maturation of human ASM is associated with accumulation of DGC proteins. We compared subconfluent, serum-fed cultures and confluent cultures subjected to serum deprivation, which express a contractile phenotype. Western blotting confirmed that beta-dystroglycan, beta-, delta-, and epsilon-sarcoglycan, and dystrophin abundance increased six- to eightfold in association with smooth muscle myosin heavy chain (smMHC) and calponin accumulation during 4-day serum deprivation. Immunocytochemistry showed that the accumulation of DGC subunits was specifically localized to a subset of cells that exhibit robust staining for smMHC. Laminin competing peptide (YIGSR, 1 microM) and phosphatidylinositol 3-kinase (PI3K) inhibitors (20 microM LY-294002 or 100 nM wortmannin) abrogated the accumulation of smMHC, calponin, and DGC proteins. These studies demonstrate that the accumulation of DGC is an integral feature for phenotype maturation of human ASM cells. This provides a strong rationale for future studies investigating the role of the DGC in ASM smooth muscle physiology in health and disease.

Complete Deletion of All α-Dystrobrevin Isoforms Does Not Reveal New Neuromuscular Junction Phenotype

The dystrophin glycoprotein complex (DGC) is critical for muscle stability, and mutations in DGC proteins lead to muscular dystrophy. The DGC also contributes to the maturation and maintenance of the neuromuscular junction (NMJ). The gene encoding the DGC protein alpha-dystrobrevin undergoes alternative splicing to produce at least five known isoforms. Isoform-specific antibody staining and reverse transcription PCR in mutant mice with a deletion of exon 3 of the alpha-dystrobrevin gene suggested the existence of a remaining synaptic isoform, which might be compensating for alpha-dystrobrevin function. To test this possibility and to more completely understand the synaptic function of alpha-dystrobrevin, we used a two-step homologous recombination strategy combined with in vivo Cre-mediated excision to generate mice with a large deletion of the alpha-dystrobrevin gene to disrupt all isoforms. However, these mice did not exhibit a more severe NMJ phenotype than that observed in the exon 3-deleted mice. Nonetheless, these mice not only eliminate possible compensation by remaining isoforms of alpha-dystrobrevin, but also offer a conditional allele that could be used to identify tissue-specific and developmental functions of alpha-dystrobrevin. This work also demonstrates a successful strategy to achieve deletion of a large genomic sequence, which can be a valuable tool for functional studies of genes encoding multiple isoforms that span a large genomic region.

Role of dystrophin and utrophin for assembly and function of the dystrophin glycoprotein complex in non-muscle tissue.

The dystrophin glycoprotein complex (DGC) is a multimeric protein assembly associated with either the X-linked cytoskeletal protein dystrophin or its autosomal homologue utrophin. In striated muscle cells, the DGC links the extracellular matrix to the actin cytoskeleton and mediates three major functions: structural stability of the plasma membrane, ion homeostasis, and transmembrane signaling. Mutations affecting the DGC underlie major forms of congenital muscle dystrophies. The DGC is prominent also in the central and peripheral nervous system and in tissues with a secretory function or which form barriers between functional compartments, such as the blood-brain barrier, choroid plexus, or kidney. A considerable molecular heterogeneity arises from cell-specific expression of its constituent proteins, notably short C-terminal isoforms of dystrophin. Experimentally, the generation of mice carrying targeted gene deletions affecting the DGC has clarified the interdependence of DGC proteins for assembly of the complex and revealed its importance for brain development and regulation of the 'milieu intérieur. Here, we focus on recent studies of the DGC in brain, blood-brain barrier and choroid plexus, retina, and kidney and discuss the role of dystrophin isoforms and utrophin for assembly of the complex in these tissues.