Treatment Of Myoblasts Research Articles

Mitochondrial Oxidative Stress and Mitophagy Activation Contribute to TNF-Dependent Impairment of Myogenesis

Many muscular pathologies are associated with oxidative stress and elevated levels of the tumor necrosis factor (TNF) that cause muscle protein catabolism and impair myogenesis. Myogenesis defects caused by TNF are mediated in part by reactive oxygen species (ROS), including those produced by mitochondria (mitoROS), but the mechanism of their pathological action is not fully understood. We hypothesized that mitoROS act by triggering and enhancing mitophagy, an important tool for remodelling the mitochondrial reticulum during myogenesis. We used three recently developed probes-MitoTracker Orange CM-H2TMRos, mito-QC, and MitoCLox-to study myogenesis in human myoblasts. Induction of myogenesis resulted in a significant increase in mitoROS generation and phospholipid peroxidation in the inner mitochondrial membrane, as well as mitophagy enhancement. Treatment of myoblasts with TNF 24 h before induction of myogenesis resulted in a significant decrease in the myoblast fusion index and myosin heavy chain (MYH2) synthesis. TNF increased the levels of mitoROS, phospholipid peroxidation in the inner mitochondrial membrane and mitophagy at an early stage of differentiation. Trolox and SkQ1 antioxidants partially restored TNF-impaired myogenesis. The general autophagy inducers rapamycin and AICAR, which also stimulate mitophagy, completely blocked myogenesis. The autophagy suppression by the ULK1 inhibitor SBI-0206965 partially restored myogenesis impaired by TNF. Thus, suppression of myogenesis by TNF is associated with a mitoROS-dependent increase in general autophagy and mitophagy.

Ferritin-dependent cellular autophagy pathway promotes ferroptosis in beef during cold storage

Ferroptosis plays a pivotal role in regulating various physiological processes and quality of post-mortem muscle. However, the molecular mechanisms underlying ferroptosis remain unclear. The study investigated how ferroptosis was induced in beef during cold storage. Results showed that the expression of autophagy-related genes, LC3, ATG5, ATG7, and NCOA4 in beef during cold storage promoted the degradation of ferritin heavy chains. Ferritin evoked ferroptosis by releasing free iron, inducing reactive oxygen species (ROS) accumulation and inhibiting the glutathione (GSH)-glutathione peroxidase 4 (GPX4) pathway. Furthermore, treatment of myoblasts with GSK 2656157 (autophagy inhibitor) showed that ferritin degradation was lower in the GSK 2656157-treated myoblasts than in the control, while GSH content and GPX4 activity were higher than the control (P < 0.05), and the contents of free iron, ROS and malondialdehyde, and apoptosis were lower than the control (P < 0.05). These results suggest that ferroptosis is induced by degradation of ferritin via the autophagic pathway.

Effect of All-trans Retinoic Acid on Panniculus Carnosus Muscle Regeneration in Fetal Mouse Wound Healing.

Background:The dermal panniculus carnosus (PC) muscle is critical for wound contraction in lower mammals and is a useful model of muscle regeneration owing to its high cellular metabolic turnover. During wound healing in mice, skin structures, including PC, are completely regenerated up to embryonic day (E) 13, but PC is only partially regenerated in fetuses or adult animals after E14. Nevertheless, the mechanisms underlying wound repair for complete regeneration in PC have not been fully elucidated. We hypothesized that retinoic acid (RA) signaling, which is involved in muscle differentiation, regulates PC regeneration.Methods:Surgical injury was induced in ICR mice on E13 and E14. RA receptor alpha (RARα) expression in tissue samples from embryos was evaluated using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. To evaluate the effects of RA on PC regeneration, beads soaked in all-trans RA (ATRA) were implanted in E13 wounds, and tissues were observed. The effects of RA on myoblast migration were evaluated using a cell migration assay.Results:During wound healing, RARα expression was enhanced at the cut surface in PCs of E13 wounds but was attenuated at the cut edge of E14 PCs. Implantation of ATRA-containing beads inhibited PC regeneration on E13 in a concentration-dependent manner. Treatment of myoblasts with ATRA inhibited cell migration.Conclusions:ATRA inhibits PC regeneration, and decreased RARα expression in wounds after E14 inhibits myoblast migration. Our findings may contribute to the development of therapies to promote complete wound regeneration, even in the muscle.

C-type natriuretic peptide stimulates chicken myoblast differentiation through NPRB/NPRC receptors and metabolism pathway

Skeletal muscle development is closely related with the amount of meat production and its quality in chickens. Natriuretic peptides (NPs) play an important role in myotube formation and fat oxidation of skeletal muscle in animals. The effect of C-type natriuretic peptide (CNP), an important member of the NPs, and its underlying molecular mechanisms in skeletal muscle are incompletely understood. Treatment of myoblasts with CNP led to enhanced proliferation/differentiation and significantly upregulated (P<0.05) mRNA expression of the CNP receptors natriuretic peptide receptor B (NPRB) and the clearance receptor C (NPRC). In cells exposed to CNP, 142 differentially expressed genes (84 up-regulation and 58 down-regulation) (P<0.05) were identified by RNA-sequencing compared with those in control cells. Sixteen genes were significantly enriched (P<0.05) in the metabolic pathway, and six of them (phospholipase C β4, phospholipase C β2, phosphoglycerate mutase 1, creatine kinase B, peroxiredoxin 6 and CD38) were closely related to skeletal muscle development and differentially expressed. In conclusion, CNP stimulated differentiation of myoblasts by upregulating expression of the NPRB and NPRC receptors and enriching key genes in the metabolic pathway.

Preliminary study of the gene expression of sulfation and degradation enzymes for chondroitin sulfate in glycerol-treated C2C12 myoblast cells.

In this study, we induced chemical damage of C2C12 myoblasts that had differentiated into myotubes with glycerol, and four sulfation enzymes for chondroitin sulfate (CS) [carbohydrate sulfotransferase (Chst) 12, Chst15 and Chst3 and uronyl 2-O-sulfotransferase (UST)] and two CS degradation enzymes [hyaluronidase (Hyal) 1 and Hyal2] were examined for changes in gene expression. Treatment of myoblasts with 5% glycerol significantly increased the expression levels of the sulfation enzymes Chst12 and Chst15 and the degradation enzymes Hyal1 and Hyal2. However, the expression levels of the other two genes (Chst3 and Ust) showed no change. Differences in the expression levels of these enzymes may help to understand the difference in responsiveness of myoblasts to glycerol after muscle injury in vivo or in vitro.

Chemotherapeutic drugs negatively affect myoblast proliferation and myotube anabolism by reducing rDNA transcription

Cancer cachexia negatively affects quality of life and contributes to increased morbidity and mortality in a wide variety of cancer types. Emerging evidence suggests that, in addition to tumor derived factors, chemotherapeutic drugs may also play a role in muscle wasting. The goal of this study was to determine the cellular mechanisms underpinning skeletal muscle depletion by chemotherapy drugs. Treatment of myoblasts or myotubes with Paclitaxel (PTX) or Doxorubicin (DXR) reduced cell viability in a dose dependent manner with myoblasts displaying more susceptibility than myotubes regardless of the drug. At non‐lethal doses, PTX or DXR induced significant reductions in myoblast proliferation (47%, p&lt;0.01 and 43%, p&lt;0.05 respectively). In myoblasts, PTX and DXR caused a reduction in rDNA transcription of 24% (p&lt;0.0001) and 19% (p&lt;0.0005) relative to control and a concomitant reduction in rRNA of 16% (p&lt;0.0001) and 18% (p&lt;0.0001), respectively. In myotubes, PTX or DXR reduced rDNA transcription by 21% (p&lt;0.0001) and 30% (p&lt;0.0001) compared to control, and consequently rRNA was 20% (p&lt;0.01) and 17% (p&lt;0.05) lower after the respective drug treatments. The reduction in rRNA reduced translational capacity in myotubes as evidenced by lower global protein synthesis of 52% (p&lt;0.001) and 47% (p&lt;0.001) following exposure to PTX or DXR. These results indicate that chemotherapeutic agents may affect muscle mass by impairing the ability of myogenic cells to proliferate, and by reducing the capacity for protein synthesis in myofibers via a reduction in rDNA transcription.Support or Funding InformationSupported by NIH grant AR073385

Placenta-derived mesenchymal stromal cells and their exosomes exert therapeutic effects in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a degenerative lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Cell therapy using different cell types, including mesenchymal stromal cells (MSCs), has been considered as a potential approach for the treatment of DMD. MSCs can be obtained from autologous sources such as bone marrow and adipose tissues or from allogeneic placenta and umbilical cord. The safety and therapeutic impact of these cells has been demonstrated in pre-clinical and clinical studies and their functions are attributed to paracrine effects that are mediated by secreted cytokines and extracellular vesicles. Here, we studied the therapeutic effects of placenta-derived MSCs (PL-MSCs) and their secreted exosomes using mouse and human myoblasts from healthy controls, Duchenne patients and mdx mice. Treatment of myoblasts with conditioned medium or exosomes secreted by PL-MSCs increased the differentiation of these cells and decreased the expression of fibrogenic genes in DMD patient myoblasts. In addition, these treatments also increased the expression of utrophin in these cells. Using a quantitative miR-29c reporter, we demonstrated that the PL-MSC effects were partly mediated by the transfer of exosomal miR-29c. Intramuscular transplantation of PL-MSCs in mdx mice resulted in decreased creatine kinase levels. PL-MSCs significantly decreased the expression of TGF-β and the level of fibrosis in the diaphragm and cardiac muscles, inhibited inflammation and increased utrophin expression. In vivo imaging analyses using MSCs labeled with gold nanoparticles or fluorescent dyes demonstrated localization of the cells in the muscle tissues up to 3 weeks post treatment. Altogether, these results demonstrate that PL-MSCs and their secreted exosomes have important clinical applications in cell therapy of DMD partly via the targeted delivery of exosomal miR-29c.

Association of lamin A/C with muscle gene-specific promoters in myoblasts

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome–lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin–chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina–chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.

Inhibition of Drp1-dependent mitochondrial division impairs myogenic differentiation

Mitochondria are dynamic organelles forming a tubular network that is continuously fusing and dividing to control their morphology and functions. Recent literature has shed new light on a potential link between the dynamic behavior of mitochondria and muscle development. In this study, we investigate the role of mitochondrial fission factor dynamin-related protein 1 (Drp1) in myogenic differentiation. We found that differentiation of C2C12 myoblasts induced by serum starvation was accompanied by a gradual increase in Drp1 protein expression (to ∼350% up to 3 days) and a fast reduction of Drp1 phosphorylation at Ser-637 (to ∼30%) resulting in translocation of Drp1 protein from the cytosol to mitochondria. During differentiation, treatment of myoblasts with mitochondrial division inhibitor (mdivi-1), a specific inhibitor of Drp1 GTPase activity, caused extensive formation of elongated mitochondria, which coincided with increased apoptosis evidenced by both enhanced caspase-3 activity and increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Furthermore, the mdivi-1-treated myotubes (day 3 in differentiation media) showed a reduction in mitochondrial DNA content, mitochondrial mass, and membrane potential in a dose-dependent manner indicating defects in mitochondrial biogenesis during myogenic differentiation. Most interestingly, mdivi-1 treatment significantly suppressed myotube formation in both C2C12 cells and primary myoblasts. Likewise, stable overexpression of a dominant negative mutant Drp1 (K38A) dramatically reduced myogenic differentiation. These data suggest that Drp-1-dependent mitochondrial division is a necessary step for successful myogenic differentiation, and perturbation of mitochondrial dynamics hinders normal mitochondrial adaptations during muscle development. Therefore, in the present study, we report a novel physiological role of mitochondrial dynamics in myogenic differentiation.

Role of vitamin D on the expression of glucose transporters in L6 myotubes

Altered expression of glucose transporters is a major characteristic of diabetes. Vitamin D has evolved widespread interest in the pathogenesis and prevention of diabetes. The present study was designed to investigate the effect of vitamin D in the overall regulation of muscle cell glucose transporter expression. L6 cells were exposed to type 1 and type 2 diabetic conditions and the effect of calcitriol (1,25, dihydroxy cholicalciferol) on the expression of glucose transporters was studied by real time polymerase chain reaction (RT-PCR). There was a significant decrease in glucose transporter type 1 (GLUT1), GLUT4, vitamin D receptor (VDR), and IR expression in type 1 and 2 diabetic model compared to control group. Treatment of myoblasts with 10-7 M calcitriol for 24 h showed a significant increase in GLUT1, GLUT4, VDR, and insulin receptor (IR) expression. The results indicate a potential antidiabetic function of vitamin D on GLUT1, GLUT4, VDR, and IR by improving receptor gene expression suggesting a role for vitamin D in regulation of expression of the glucose transporters in muscle cells.

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110β and PKCδ in myoblasts

Context: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. Objective: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. Methods: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. Results: Treatment of myoblasts with exogenous LPA revealed that PI3K p110β mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. Conclusion: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110β, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.

Myostatin facilitates slow and inhibits fast myosin heavy chain expression during myogenic differentiation

Skeletal muscles in the limb and body trunk are composed of heterogeneous myofibers expressing different isoforms of myosin heavy chain (Myh), including type I (slow, Myh7), IIA (intermediate, Myh2), IIX (fast, Myh1), and IIB (very fast, Myh4). While the contraction force and speed of a muscle are known to be determined by the relative abundance of myofibers expressing each Myh isoform, it is unclear how specific combinations of myofiber types are formed and regulated at the cellular and molecular level. We report here that myostatin (Mstn) positively regulates slow but negatively regulates fast Myh isoforms. Mstn was expressed at higher levels in the fast muscle myoblasts and myofibers than in the slow muscle counterparts. Interestingly, Mstn knockout led to a shift of Myh towards faster isoforms, suggesting an inhibitory role of Mstn in fast Myh expression. Consistently, when induced to differentiate, Mstn null myoblasts formed myotubes preferentially expressing fast Myh. Conversely, treatment of myoblasts with a recombinant Mstn protein upregulated Myh7 but downregulated Myh4 gene expression in newly formed myotubes. Importantly, both Mstn antibody and soluble activin type 2B receptor inhibited slow Myh7 and promoted fast Myh4 expression, indicating that myostatin acts through canonical activin receptor to regulate the expression of Myh genes. These results demonstrate a role of myostatin in the specification of myofiber types during myogenic differentiation.

EPA decreases mRNA and protein for endocannabinoid receptors in myoblasts

Our research objective is to characterize the actions of n‐3 PUFA on mRNA and protein levels for endocannabinoid (EC) receptors (CB1 and CB2) and the EC synthesizing and degrading enzymes in muscle. In two experiments, C2C12 myoblasts were cultured until 50% confluency. Cells were assigned to two groups: one maintained with standard growth media (proliferating) and the other with differentiating media (differentiated). After 48 h, both groups were treated with 100μM of either eicosapenaenoic (EPA), docosahexaenoic (DHA) and arachidonic acids (AA), or bovine serum albumin (BSA) control for 24 h. RNA was then isolated from cells and subjected to real time PCR analysis. Compared to the BSA, the amount of CB2 in EPA and DHA treated cells was lower in differentiated cells. The EPA treated proliferating cells had lower CB1 and CB2 compared to the AA and BSA control. CB1 protein was lower in differentiated cells treated with EPA. The synthesizing enzymes, NAPE‐PLD and DAGL, of the endogenous cannabinoids anandamide (AEA) and 2‐ arachidonylgycerol (2‐AG); respectively, mRNA was lower in differentiated cell treated with DHA and EPA. These data suggest that DHA and EPA may potentially inhibit EC signaling in C2C12 by reducing the agonists (AEA and 2‐AG) and their receptors. Thus treatment of myoblasts with DHA or EPA results in lower mRNA for EC receptors and endogenous cannabinoid synthesizing enzymes in proliferating C2C12.Grant Funding Source : Diet and Health Initiative (UConn)

206 MODULATION OF MICROENVIRONMENT BY GROWTH FACTORS REGULATES IN VIVO GROWTH OF SKELETAL MYOBLASTS

You have accessJournal of UrologyStem Cell Research1 Apr 2012206 MODULATION OF MICROENVIRONMENT BY GROWTH FACTORS REGULATES IN VIVO GROWTH OF SKELETAL MYOBLASTS Akihiro Yanagiuchi, Hideaki Miyake, and Masato Fujisawa Akihiro YanagiuchiAkihiro Yanagiuchi Kobe, Japan More articles by this author , Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , and Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.259AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES With advances in the field of tissue engineering, transplantation of myogenic stem cells, called myoblasts, have been extensively investigated as a therapeutic option in muscle-related diseases. Such an approach seems to be extremely promising for the treatment of stress urinary incontinence, since myoblasts have the ability to undergo differentiation and fusion to form myofibers, leading to sphincter regeneration; however, to date, transplantation of myoblasts by direct intramuscular injection in vivo have shown limited efficiency in the muscle integration of donor cells. The objective of this study was to investigate the optimal microenvironment for efficient myoblast transplantation in vivo focusing on the interaction between myoblasts and growth factors. METHODS The effects of coculture with growth factors, including basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I) and platelet-derived growth factor (PDGF), on in vitro growth, migration and proteolytic activity of mouse skeletal myoblasts were investigated. Myoblasts were coinjected with growth factors into the subcutis and bladder wall of nude mice, and its impact on the growth patterns of myoblasts in vivo was assessed. RESULTS Dose-dependent stimulation of in vitro myoblast growth was observed following treatment with each of the four kinds of growth factors, but bFGF induced the most remarkable increase in the growth of myoblasts. Treatment of myoblasts with all kinds of growth factors also resulted in a dose-dependent increase in the in vitro migration of myoblasts, and PDGF exerted the most prominent effect on myoblast migration. Increased secretion of matrix metalloproteinase-9 (MMP-9) in myoblasts induced by growth factors was proportional to their increased migration capacity, which was partially inhibited by SB-3CT, a specific inhibitor of MMP-9. The in vivo growth of myoblasts was significantly enhanced by coinjection with all kinds of growth factors into both the subcutis and bladder wall; however, this effect was most remarkable 1 and 2 weeks after coinjection with bFGF and PDGF, respectively. Furthermore, synergistic in vivo growth of myoblasts was observed by coinjection of both bFGF and PDGF compared with that achieved with either agent alone. CONCLUSIONS These findings suggest that modulation of the microenvironment using growth factors, particularly bFGF and PDGF, could provide the optimal condition for efficacious myoblast transplantation in vivo. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e86 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akihiro Yanagiuchi Kobe, Japan More articles by this author Hideaki Miyake Kobe, Japan More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Myostatin inhibits proliferation but not differentiation of trout myoblasts

The muscle growth in mammals is regulated by several growth factors including myostatin (MSTN), a member of the transforming growth factor-beta (TGF-beta) superfamily. To date, it is unknown in fish whether MSTN could have any effect on proliferation or differentiation of myogenic cells. Using culture of trout satellite cells, we showed that mstn1a and mstn1b mRNA are expressed in myoblasts and that their expression decreased in differentiating myoblasts. We also demonstrated that a treatment with huMSTN decreased the proliferation of IGF1-stimulated myoblasts in a dose-dependent manner. By contrast, treatment of myoblasts with 100nM of huMSTN for three days, did not affect the percentage of positive cells for myogenin neither the percentage of nuclei in myosin positive cells. Moreover, our results clearly indicated that huMSTN treatment had no effect on MyoD and myogenin protein levels, which suggests that huMSTN did not strongly affect MyoD activity.In conclusion, we showed that huMSTN inhibited proliferation but not differentiation of trout myoblasts, probably resulting from a lack of huMSTN effect on MyoD activity. Altogether, these results show high interspecies differences in the function of MSTN.

Regulation of myotube formation by the actin-binding factor drebrin

BackgroundMyogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.MethodsRNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells.ResultsIn a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated.ConclusionsOur findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.

Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor β (TGF-β) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-β on CTGF expression. We found that myoblasts treated with LPA and TGF-β1 produced an additive effect on CTGF expression. In the absence of TGF-β, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-β receptor type II (TGF-βRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-βRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-βRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-βRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-βRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-β are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.

Modulation of the microenvironment by growth factors regulates the in vivo growth of skeletal myoblasts

To investigate the optimal microenvironment for efficient myoblast transplantation in vivo. The effects of co-culture with growth factors, including basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor-I) and platelet-derived growth factor (PDGF), on in vitro growth, migration and proteolytic activity of mouse skeletal myoblasts were investigated. Myoblasts were co-injected with growth factors into the subcutis and bladder wall of nude mice, and its impact on the growth patterns of myoblasts in vivo assessed. There was dose-dependent stimulation of in vitro myoblast growth after treatment with each of the four growth factors, but bFGF induced the most marked increase in the growth of myoblasts. Treatment of myoblasts with all types of growth factors also resulted in a dose-dependent increase in the in vitro migration of myoblasts, and PDGF had the most prominent effect on myoblast migration. Increased secretion of matrix metalloproteinase-9 (MMP-9) in myoblasts induced by growth factors was proportional to their increased migration capacity, which was partly inhibited by SB-3CT, an inhibitor of MMP-9. The in vivo growth of myoblasts was significantly enhanced by co-injection with all types of growth factor into both the subcutis and bladder wall, but this effect was most marked 1 and 2 weeks after co-injection with bFGF and PDGF, respectively. Furthermore, there was synergistic in vivo growth of myoblasts by co-injection of both bFGF and PDGF compared with that achieved with either agent alone. These findings suggest that modulation of the microenvironment using growth factors, particularly bFGF and PDGF, could provide the optimum condition for effective myoblast transplantation in vivo.

Prostaglandin F2α promotes muscle cell survival and growth through upregulation of the inhibitor of apoptosis protein BRUCE

During skeletal muscle growth and regeneration, the majority of differentiating myoblasts undergoes cell-cell fusion to form multinucleated myofibers, whereas a proportion of myoblasts undergoes apoptosis. The treatment of myoblasts with prostaglandin F2alpha (PGF2alpha) during myogenesis in vitro leads to the formation of large myotubes, but the mechanism by which PGF2alpha promotes myotube growth has not been investigated. Here, we demonstrate that PGF2alpha reduces cell death during myogenesis in vitro and in vivo. In addition, we show that PGF2alpha increases expression of the inhibitor of apoptosis protein (IAP) BRUCE through a pathway dependent on the nuclear factor of activated T cell 2 transcription factor. Importantly, PGF2alpha-mediated reduction in muscle cell death is dependent on BRUCE, and overexpression of BRUCE is sufficient to promote muscle cell survival and growth. These results establish a previously unrecognized link between NFAT signaling and regulation of IAP expression and are the first to identify a signaling pathway that increases BRUCE expression. In addition, our results provide evidence that increasing the pool of muscle cells available for fusion by inhibiting cell death enhances myotube growth.

Voltage-dependent Na+ channel phenotype changes in myoblasts. Consequences for cardiac repair☆

Cellular cardiomyoplasty using skeletal myoblasts is a promising therapy for myocardial infarct repair. Once transplanted, myoblasts grow, differentiate and adapt their electrophysiological properties towards more cardiac-like phenotypes. Voltage-dependent Na(+) channels (Na(v)) are the main proteins involved in the propagation of the cardiac action potential, and their phenotype affects cardiac performance. Therefore, we examined the expression of Na(v) during proliferation and differentiation in skeletal myocytes. We used the rat neonatal skeletal myocyte cell line L6E9. Proliferation of L6E9 cells induced Na(v)1.4 and Na(v)1.5, although neither protein has an apparent role in cell growth. During myogenesis, Na(v)1.5 was largely induced. Electrophysiological and pharmacological properties, as well as mRNA expression, indicate that cardiac-type Na(v)1.5 accounts for almost 90% of the Na(+) current in myotubes. Unlike in proliferation, this protein plays a pivotal role in myogenesis. The adoption of a cardiac-like phenotype is further supported by the increase in Na(v)1.5 colocalization in caveolae. Finally, we demonstrate that the treatment of myoblasts with neuregulin further increased Na(v)1.5 in skeletal myocytes. Our results indicate that skeletal myotubes adopt a cardiac-like phenotype in cell culture conditions and that the expression of Na(v)1.5 acts as an underlying molecular mechanism.