High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.     

Insulin-like 6 Is Induced by Muscle Injury and Functions as a Regenerative Factor

The insulin-like family of factors are involved in the regulation of a variety of physiological processes, but the function of the family member termed insulin-like 6 (Insl6) in skeletal muscle has not been reported. We show that Insl6 is a myokine that is up-regulated in skeletal muscle downstream of Akt signaling and in regenerating muscle in response to cardiotoxin (CTX)-induced injury. In the CTX injury model, myofiber regeneration was improved by the intramuscular or systemic delivery of an adenovirus expressing Insl6. Skeletal muscle-specific Insl6 transgenic mice exhibited normal muscle mass under basal conditions but elevated satellite cell activation and enhanced muscle regeneration in response to CTX injury. The Insl6-mediated regenerative response was associated with reductions in muscle cell apoptosis and reduced serum levels of creatine kinase M. Overexpression of Insl6 stimulated proliferation and reduced apoptosis in cultured myogenic cells. Conversely, knockdown of Insl6 reduced proliferation and increased apoptosis. These data indicate that Insl6 is an injury-regulated myokine that functions as a myogenic regenerative factor.     

Description of a utrophin associated protein complex in lipid raft domains of human artery smooth muscle cells

The dystrophin-associated protein complex (DAPC) is a multimeric complex that links the extracellular matrix to the actin cytoskeleton, and in some cases dystrophin can be substituted by its autosomal homologue utrophin to form the utrophin-associated protein complex (UAPC). Both complexes maintain the stability of plasma membrane during contraction process and play an important role in transmembrane signaling. Mutations in members of the DAPC are associated with muscular dystrophy and dilated cardiomyopathy. In a previous study with human umbilical cord vessels, we observed that utrophin colocalize with caveolin-1 (Cav-1) which proposed the presence of UAPC in the plasma membrane of vascular smooth muscle (VSM). In the current study, we demonstrated by immunofluorescence analysis, co-immunoprecipitation assays, and subcellular fractionation by sucrose gradients, the existence of an UAPC in lipid raft domains of human umbilical artery smooth muscle cells (HUASMC). This complex is constituted by utrophin, β-DG, ε-SG, α-smooth muscle actin, Cav-1, endothelial nitric oxide synthase (eNOS) and cavin-1. It was also observed the presence of dystrophin, utrophin Dp71, β-SG, δ-SG, δ-SG3 and sarcospan in non-lipid raft fractions. Furthermore, the knockdown of α/β-DG was associated with the decrease in both the synthesis of nitric oxide (NO) and the presence of the phosphorylated (active) form of eNOS; and with a reduction in the downstream activation of some cGMP signaling transduction pathway components. Together these results show the presence of an UAPC complex in HUASMC that may participate in the activity regulation of eNOS and in the vascular function.     

Genetic disruption of calcineurin improves skeletal muscle pathology and cardiac disease in a mouse model of limb-girdle muscular dystrophy

Calcineurin (Cn) is a Ca(2+)/calmodulin-dependent serine/threonine phosphatase that regulates differentiation-specific gene expression in diverse tissues, including the control of fiber-type switching in skeletal muscle. Recent studies have implicated Cn signaling in diminishing skeletal muscle pathogenesis associated with muscle injury or disease-related muscle degeneration. For example, use of the Cn inhibitor cyclosporine A has been shown to delay muscle regeneration following toxin-induced injury and inhibit regeneration in the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy. In contrast, transgenic expression of an activated mutant of Cn in skeletal muscle was shown to increase utrophin expression and reduce overall disease pathology in mdx mice. Here we examine the effect of altered Cn activation in the context of the delta-sarcoglycan-null (scgd(-/-)) mouse model of limb-girdle muscular dystrophy. In contrast to results discussed in mdx mice, genetic deletion of a loxP-targeted calcineurin B1 (CnB1) gene using a skeletal muscle-specific cre allele in the scgd(-/-) background substantially reduced skeletal muscle degeneration and histopathology compared with the scgd(-/-) genotype alone. A similar regression in scgd-dependent disease manifestation was also observed in calcineurin Abeta (CnAbeta) gene-targeted mice in both skeletal muscle and heart. Conversely, increased Cn expression using a muscle-specific transgene increased cardiac fibrosis, decreased cardiac ventricular shortening, and increased muscle fiber loss in the quadriceps. Our results suggest that inhibition of Cn may benefit select types of muscular dystrophy.     

Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages

Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor-α and interleukin-1β, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.     

α-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts

Syntrophins are adaptor proteins that link intracellular signaling molecules to the dystrophin based scaffold. In this study, we investigated the function of syntrophins in cell migration, one of the early steps in myogenic differentiation and in regeneration of adult muscle. Hepatocyte growth factor (HGF) stimulates migration and lamellipodia formation in cultured C2 myoblasts. In the migrating cells, syntrophin concentrated in the rear-lateral region of the cell, opposite of the lamellipodia, instead of being diffusely present throughout the cytoplasm of non-migrating cells. When the expression of α-syntrophin, the major syntrophin isoform of skeletal muscle, was reduced by transfection with the α-syntrophin-specific siRNA, HGF stimulation of lamellipodia formation was prevented. Likewise, migration of myoblasts from α-syntrophin knockout mice could not be stimulated by HGF. However, HGF-induced migration was restored in myoblasts isolated from a transgenic mouse expressing α-syntrophin only in muscle cells. Treatment of C2 myoblasts with inhibitors of PI3-kinase not only reduced the rate of cell migration, but also impaired the accumulation of syntrophins in the rear-lateral region of the migrating cells. Phosphorylation of Akt was reduced in the α-syntrophin siRNA-treated C2 cells. These results suggest that α-syntrophin is required for HGF-induced migration of myoblasts and for proper PI3-kinase/Akt signaling.     

Recovery of skeletal muscle mass after extensive injury: positive effects of increased contractile activity

The present study was designed to test the hypothesis that increasing physical activity by running exercise could favor the recovery of muscle mass after extensive injury and to determine the main molecular mechanisms involved. Left soleus muscles of female Wistar rats were degenerated by notexin injection before animals were assigned to either a sedentary group or an exercised group. Both regenerating and contralateral intact muscles from active and sedentary rats were removed 5, 7, 14, 21, 28 and 42 days after injury (n = 8 rats/group). Increasing contractile activity through running exercise during muscle regeneration ensured the full recovery of muscle mass and muscle cross-sectional area as soon as 21 days after injury, whereas muscle weight remained lower even 42 days postinjury in sedentary rats. Proliferator cell nuclear antigen and MyoD protein expression went on longer in active rats than in sedentary rats. Myogenin protein expression was higher in active animals than in sedentary animals 21 days postinjury. The Akt-mammalian target of rapamycin (mTOR) pathway was activated early during the regeneration process, with further increases of mTOR phosphorylation and its downstream effectors, eukaryotic initiation factor-4E-binding protein-1 and p70(s6k), in active rats compared with sedentary rats (days 7-14). The exercise-induced increase in mTOR phosphorylation, independently of Akt, was associated with decreased levels of phosphorylated AMP-activated protein kinase. Taken together, these results provided evidence that increasing contractile activity during muscle regeneration ensured early and full recovery of muscle mass and suggested that these beneficial effects may be due to a longer proliferative step of myogenic cells and activation of mTOR signaling, independently of Akt, during the maturation step of muscle regeneration.     

Captopril treatment induces hyperplasia but inhibits myonuclear accretion following severe myotrauma in murine skeletal muscle

The role of ANG II in skeletal muscle and satellite cell regulation is largely unknown. Cardiotoxin (CTX) was used to investigate whether muscle injury activates a local ANG II signaling system. Following injury, immunohistochelmistry (IHC) analysis revealed a robust increase in the intensity of angiotensinogen and angiotensin type 1 (AT(1)) receptor expression. As regeneration proceeded, however, AT(1) and angiotensinogen were downregulated. Nuclear accretion and fiber formation were also assessed during muscle regeneration in mice treated with captopril (an angiotensin-converting enzyme inhibitor). When ANG II formation was blocked through the use of captopril, we observed a significantly reduced accretion of nuclei into myofibers (-25%), while tibialis anterior total fiber number was significantly increased +37%. This phenotype appeared to be due to alterations in satellite cell differentiation kinetics; captopril treatment led to sustained mRNA expression of markers associated with quiescence and proliferation (Myf5, Pax7) and simultaneously delayed or inhibited the expression of myogenin. IHC staining supported these findings, revealing that captopril treatment resulted in a strong trend (P = 0.06) for a decrease in the proportion of myogenin-positive myoblasts. Furthermore, these observations were associated with a delay in muscle fiber maturation; captopril treatment resulted in sustained expression of embryonic myosin heavy chain. Collectively, these findings demonstrate that localized skeletal muscle angiotensin signaling is important to muscle fiber formation, myonuclear accretion, and satellite cell function.     

Angiotensin Type 2 Receptor Signaling in Satellite Cells Potentiates Skeletal Muscle Regeneration

Patients with advanced congestive heart failure (CHF) or chronic kidney disease (CKD) often have increased angiotensin II (Ang II) levels and cachexia. Ang II infusion in rodents causes sustained skeletal muscle wasting and decreases muscle regenerative potential through Ang II type 1 receptor (AT1R)-mediated signaling, likely contributing to the development of cachexia in CHF and CKD. However, the potential role of Ang II type 2 receptor (AT2R) signaling in skeletal muscle physiology is unknown. We found that AT2R expression was increased robustly in regenerating skeletal muscle after cardiotoxin (CTX)-induced muscle injury in vivo and differentiating myoblasts in vitro, suggesting that the increase in AT2R played an important role in regulating myoblast differentiation and muscle regeneration. To determine the potential role of AT2R in muscle regeneration, we infused C57BL/6 mice with the AT2R antagonist PD123319 during CTX-induced muscle regeneration. PD123319 reduced the size of regenerating myofibers and expression of the myoblast differentiation markers myogenin and embryonic myosin heavy chain. On the other hand, AT2R agonist {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 infusion potentiated CTX injury-induced myogenin and embryonic myosin heavy chain expression and increased the size of regenerating myofibers. In cultured myoblasts, AT2R knockdown by siRNA suppressed myoblast differentiation marker expression and myoblast differentiation via up-regulation of phospho-ERK1/2, and ERK inhibitor treatment completely blocked the effect of AT2R knockdown. These data indicate that AT2R signaling positively regulates myoblast differentiation and potentiates skeletal muscle regenerative potential, providing a new therapeutic target in wasting disorders such as CHF and CKD.     

Skeletal muscle-specific ablation of gamma(cyto)-actin does not exacerbate the mdx phenotype

We previously documented a ten-fold increase in gamma(cyto)-actin expression in dystrophin-deficient skeletal muscle and hypothesized that increased gamma(cyto)-actin expression may participate in an adaptive cytoskeletal remodeling response. To explore whether increased gamma(cyto)-actin fortifies the cortical cytoskeleton in dystrophic skeletal muscle, we generated double knockout mice lacking both dystrophin and gamma(cyto)-actin specifically in skeletal muscle (ms-DKO). Surprisingly, dystrophin-deficient mdx and ms-DKO mice presented with comparable levels of myofiber necrosis, membrane instability, and deficits in muscle function. The lack of an exacerbated phenotype in ms-DKO mice suggests gamma(cyto)-actin and dystrophin function in a common pathway. Finally, because both mdx and ms-DKO skeletal muscle showed similar levels of utrophin expression and presented with identical dystrophies, we conclude utrophin can partially compensate for the loss of dystrophin independent of a gamma(cyto)-actin-utrophin interaction.     

Myelin-associated glycoprotein (Siglec-4) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides

Myelin-associated glycoprotein (MAG, Siglec-4) is a quantitatively minor membrane component expressed preferentially on the innermost myelin wrap, adjacent to the axon. It stabilizes myelin-axon interactions by binding to complementary ligands on the axolemma. MAG, a member of the Siglec family of sialic acid-binding lectins, binds specifically to gangliosides GD1a and GT1b, which are the major sialoglycoconjugates on mammalian axons. Mice with a disrupted Galgt1 gene lack UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and fail to express complex brain gangliosides, including GD1a and GT1b, instead expressing a comparable amount of the simpler gangliosides GM3, GD3, and O-acetyl-GD3. Galgt1-null mice produce similar amounts of total myelin compared to wild-type mice, but as the mice age, they exhibit axon degeneration and dysmyelination with accompanying motor behavioral deficits. Here we report that Galgt1-null mice display progressive and selective loss of MAG from the brain. At 1.5 months of age, MAG expression was similar in Galgt1-null and wild-type mice. However, by 6 months of age MAG was decreased approximately 60% and at 12 months of age approximately 70% in Galgt1-null mice compared to wild-type littermates. Expression of the major myelin proteins (myelin basic protein and proteolipid protein) was not reduced in Galgt1-null mice compared to wild type. MAG mRNA expression was the same in 12-month-old Galgt1-null compared to wild-type mice, an age at which MAG protein expression was markedly reduced. We conclude that the maintenance of MAG protein levels depends on the presence of complex gangliosides, perhaps due to enhanced stability when MAG on myelin binds to its complementary ligands, GD1a and GT1b, on the apposing axon surface.     

Rescue of dystrophic skeletal muscle by PGC-1α involves a fast to slow fiber type shift in the mdx mouse

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.     

Functional analysis of α1,3/4-fucosyltransferase VI in human hepatocellular carcinoma cells

The α1,3/4-fucosyltransferases (FUT) subfamily are key enzymes in cell surface antigen synthesis during various biological processes. A novel role of FUTs in tumorigenesis has been discovered recently, however, the underlying mechanism remains largely unknown. Here, we characterized FUT6, a member of α1,3/4-FUT subfamily, in human hepatocellular carcinoma (HCC). In HCC tissues, the expression levels of FUT6 and its catalytic product SLe(x) were significantly up-regulated. Overexpression of FUT6 in HCC cells enhanced S-phase cell population, promoted cell growth and colony formation ability. Moreover, subcutaneously injection of FUT6-overexpressing cells in nude mice promoted cell growth in vivo. In addition, elevating FUT6 expression markedly induced intracellular Akt phosphorylation, and suppressed the expression of the cyclin-dependent kinases inhibitor p21. Bath application of the PI3K inhibitor blocked FUT6-induced Akt phosphorylation, p21 suppression and cell proliferation. Our results suggest that FUT6 plays an important role in HCC growth by regulating the PI3K/Akt signaling pathway.     

Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.     

Age-dependent decline in mouse lung regeneration with loss of lung fibroblast clonogenicity and increased myofibroblastic differentiation

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration.