Murine Myoblast Cell Line C2C12 Research Articles

HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes

HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop have not been clearly elucidated. Here, we sought to elucidate these mechanisms using the murine myoblast cell line C2C12 and disease-specific human induced pluripotent stem cells (hiPSCs). Myotubes differentiated from HOIL-1L-KO C2C12 cells exhibited deteriorated differentiation and mitotic cell accumulation. CMs differentiated from patient-derived hiPSCs had an abnormal morphology with a larger size and were excessively multinucleated compared with CMs differentiated from control hiPSCs. Further analysis of hiPSC-derived CMs showed that HOIL-1L deficiency caused cell cycle alteration and mitotic cell accumulation. These results demonstrate that abnormal cell maturation possibly contribute to the development of myopathy and DCM. In conclusion, HOIL-1L is an important intrinsic regulator of cell cycle-related myotube and CM maturation and cell proliferation.

Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in C2C12 myoblasts by modulating the β-catenin/NF-κB signaling pathway

A myogenetic oligodeoxynucleotide, iSN04, is the 18-base single-stranded DNA that acts as an anti-nucleolin aptamer. iSN04 has been reported to restore myogenic differentiation by suppressing inflammatory responses in myoblasts isolated from patients with diabetes or healthy myoblasts exposed to cancer-releasing factors. Thus, iSN04 is expected to be a nucleic acid drug for the muscle wasting associated with chronic diseases. The present study investigated the anti-inflammatory mechanism of iSN04 in the murine myoblast cell line C2C12. Tumor necrosis factor-α (TNF-α) or Toll-like receptor (TLR) ligands (Pam3CSK4 and FSL-1) induced nuclear translocation and transcriptional activity of nuclear factor-κB (NF-κB), resulting in upregulated expression of TNF-α and interleukin-6. Pre-treatment with iSN04 significantly suppressed these inflammatory responses by inhibiting the nuclear accumulation of β-catenin induced by TNF-α or TLR ligands. These results demonstrate that antagonizing nucleolin with iSN04 downregulates the inflammatory effect mediated by the β-catenin/NF-κB signaling pathway in C2C12 cells. In addition, the anti-inflammatory effects of iSN04 were also observed in the rat smooth muscle cell line A10 and the murine adipocyte-like fibroblast cell line 3T3-L1, suggesting that iSN04 may be useful in preventing inflammation induced by metabolic disorders.

Ablation of VDR Induced Atrophy of Skeletal Muscle via Locally Affecting the Renin‐Angiotensin System

Objective Our previous studies demonstrated that vitamin D is a vital protector against tissue injuries by locally suppressing renin-angiotensin system (RAS). It is known that vitamin D deficiency is associated with a range of muscle disorders, such as muscle atrophy. This study aimed to investigate the biological role of vitamin D receptor (VDR) in skeletal muscle and the underlying mechanism using a mice model with deletion of VDR associated with dexamethasone-induced muscle atrophy. Materials and Methods The wild type and VDR(-/-) mice were treated with vehicle and dexamethasone (i.p., once daily) for 10 consecutive days. The murine myoblast cell line C2C12 and the skeletal muscle satellite cells were applied as in vitro models in this study. Results Treatment with the angiotensin II (Ang II) type 1 receptor blocker olmesartan eliminated Ang II-induced atrophy of myotube cells, reflected by alleviating myotube cell atrophy, suggesting that Ang II could aggravate atrophy of skeletal muscle. The ablation of VDR in muscle satellite cells worsened the phenotype of muscle atrophy induced by Ang II. After challenging to dexamethasone, VDR-null mice exhibited more severe amyotrophy compared with wild-type counterparts, manifested by significant decrease in muscle wet weight coefficient of the tibialis anterior muscle and gastrocnemius as well as the reduction of limb grip and weight-loading swimming time. Similarly, the dystrophin fluorescence staining and the haematoxylin eosin (H&E) staining showed much more decrease in cross-sectional area of skeletal muscle in the VDR-null mice in a comparison with the counterparts. MAFbx and MuRF1 are two E3 ubiquitin ligases that are important regulators for ubiquitin-mediated protein degradation in skeletal muscle, while myogenic differentiation-1 (MyoD) and myosin heavy chain (MHC) are necessary to maintain myocytes proliferation and differentiation. VDR ablation significantly accelerated the up-regulation of MAFbx and MuRF1 proteins and down-regulated protein expression of MyoD and MHC in skeletal muscle tissue of mice with muscle atrophy. Conclusion These results provided evidences that VDR might attenuate skeletal muscle atrophy at least in part by suppressing the renin-angiotensin system.

Two Isoforms of Eya3 Influence Muscle Cell Differentiation and Become Dysregulated in Striated Muscle Diseases

Alternative splicing is a ubiquitous gene regulatory mechanism by which diverse transcripts are derived from a finite genome. During development, complex splicing transitions occur, leading to varied transcript profiles in fetal and adult tissues. Like gene expression, splicing often becomes dysregulated in cancer, neurological, and muscular diseases. One barrier to therapeutic targeting of alternative splicing is that the function of splice isoforms, both physiologically and pathologically, are vastly understudied. Here, we begin to address this knowledge gap by describing an evolutionarily conserved alternative splicing event in eyes absent homolog 3 (Eya3) gene for the first time. The short isoform of Eya3 is a dual threonine- and tyrosine-phosphatase that is known to promote cell growth and chemotherapeutic resistance. By analyzing RNA-sequencing data from various mouse tissues, we identified the long isoform of Eya3, which contains an additional 138nt, representing a 46 amino acid in-frame insertion immediately following the threonine phosphatase domain. Using reverse transcription-PCR (RT-PCR) of mouse, human, and rabbit tissues, we discovered that Eya3 splicing is tissue-specific, with the long isoform expressed exclusively in the heart, skeletal muscle, and brain. Additionally, we found that during heart and skeletal muscle development, expression of the long Eya3 isoform gradually increases relative to the expression of the short isoform. In myotonic dystrophy type 1 patient hearts, and failing mouse hearts, the short Eya3 isoform is re-expressed, illustrating that Eya3 splicing is dysregulated in striated muscle diseases. To understand Eya3 splicing in further molecular detail, we utilized the well-established C2C12 murine myoblast cell line. Differentiation of myoblasts to myotubes robustly recapitulates the Eya3 splicing transition observed during tissue development, making them an appropriate cell culture system for our work. Depletion of Eya3 via siRNA transfection inhibited myoblast proliferation and fusion. Interestingly, blocking the endogenous Eya3 alternative splicing transition with morpholino antisense oligonucleotides also suppressed normal differentiation as determined by immunohistochemical staining and confocal microscopy. These results indicate that Eya3, and its splice isoforms, play an important role in early muscle development. In summary, our studies uncover a developmentally-regulated, tissue-specific, and disease-relevant splicing event in Eya3. Moving forward, we seek to determine the molecular mechanism by which Eya3 isoforms regulate normal muscle development and to evaluate Eya3 as a potential therapeutic target.

Cytotoxicity and genotoxicity of Vernonia condensata Baker aqueous extracts in an Allium cepa test system and C2C12 cell culture

Vernonia condensata Baker is a medicinal species widely used by the Brazilian population for its analgesic, hepatoprotective and digestive properties. This study aimed to investigate the cytotoxic and genotoxic effects of V. condensata aqueous extracts using Allium cepa test system and in vitro micronucleus test. Fresh V. condensata leaves were collected, dried and ground, and aqueous extracts were prepared at concentrations of 1500; 3000; and 6000 µg/ml of dry extract. In the A. cepa test, ten repetitions were used per treatment. Distilled water was used as negative control and sodium azide (2M) as positive control. The onion bulbs were rooted in distilled water for 72 h and then exposed to the extracts for 24 h. The roots were collected and fixed in Carnoy’s solution (3:1) for 24 h. Smear slides were prepared of the root apical meristems and 2000 cells/concentration was analyzed, measuring the mitotic index, micronuclei frequency and mitotic abnormalities of A. cepa . In vitro cytotoxicity and genotoxicity were also performed using the murine myoblast cell line C2C12. In vivo data demonstrated that an increase in the concentration of V. condensata aqueous extracts caused cytotoxic and genotoxic effects in meristematic cells of A. cepa . A concentration of 1500 µg/ml showed no cytotoxic or genotoxic effects, while 3000 µg/ml resulted in mitotic abnormalities and a decline in the mitotic index, indicating cytotoxicity. The 6000 µg/ml aqueous extract showed cytotoxicity and genotoxicity, increasing mitotic abnormalities and micronuclei frequencies in the A. cepa test. An IC 50 value of 15.610 µg/mL was obtained for C2C12 cell line and the doses of 6000 µg/mL and 12000 µg/mL of Vernonia extract induced genotoxicity. The data of the present study reveals that V. condensate extract should be used with parsimony, once that it can bring unwanted side effects, which can be dangerous.

Brazilian green propolis promotes the cytoprotective expression of heme oxygenase-1 against oxidative stress injury in murine myoblast cells

Background: Sarcopenia is a progressive skeletal muscle disorder characterized by the progressive loss of muscle mass and function, resulting in physical disability and mortality. Although sarcopenia impacts a large proportion of elderly individuals, no effective treatment for this disease has yet been identified. The excessive production of reactive oxygen species (ROS) can damage tissues and promote aging, and the daily use of dietary antioxidants can be effective for maintaining skeletal muscle health. Propolis, a natural substance that is collected by honey bees, has been used as traditional medicine, and many reports have described its antioxidative properties. However, how propolis exhibits cytoprotective effects and antioxidative effects in skeletal muscles remains unclear. The purpose of this study was to investigate the antioxidative effects of ethanol-extracted Brazilian green propolis (EEBP, from Baccharis dracunculifolia) and its three constituents using an in vitro myoblast cell model.Methods: Murine myoblast C2C12 cells were treated with either EEBP or its constituents, including caffeic acid, trans-ferulic acid, and p-coumaric acid, in the presence of 100 or 300 mM H2O2 to induce oxidative stress injury. The cell death ratio and cell viability were assessed by Hoechst 33342 and propidium iodide staining and the WST-8 assay, respectively. Simultaneously, intracellular ROS production was measured by CM-H2DCFDA [5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester] assay. Finally, immunoblotting was performed in myoblast cell lysates to assess the expression level of an antioxidative enzyme, heme oxygenase-1 (HO-1).Results: We demonstrated that EEBP significantly reduced H2O2-induced cell death at a concentration of 3 µg/ml in myoblasts. Additionally, caffeic acid at 100 µM improved cell viability under oxidative stress conditions, but not trans-ferulic acid or p-coumaric acid. Both EEBP and caffeic acid inhibited the H2O2-induced increase in ROS production. Finally, HO-1 expression was increased by treatment with either EEBP or caffeic acid. The increase in HO-1 expression induced by H2O2 was enhanced in the presence of EEBP and caffeic acid.Conclusions: These findings indicated that EEBP has protective effects against oxidative damage in C2C12 murine myoblast cell line. Caffeic acid is an EEBP constituent that contributes to cytoprotective activity. EEBP may act as an inducer of HO-1 to prevent oxidative stress-induced myoblast death.Keywords: C2C12 murine myoblast cells, heme oxygenase-1, oxidative stress, propolis, reactive oxygen species

FLNC Expression Level Influences the Activity of TEAD-YAP/TAZ Signaling.

Filamin C (FLNC), being one of the major actin-binding proteins, is involved in the maintenance of key muscle cell functions. Inherited skeletal muscle and cardiac disorders linked to genetic variants in FLNC have attracted attention because of their high clinical importance and possibility of genotype-phenotype correlations. To further expand on the role of FLNC in muscle cells, we focused on detailed alterations of muscle cell properties developed after the loss of FLNC. Using the CRISPR/Cas9 method we generated a C2C12 murine myoblast cell line with stably suppressed Flnc expression. FLNC-deficient myoblasts have a significantly higher proliferation rate combined with an impaired cell migration capacity. The suppression of Flnc expression leads to inability to complete myogenic differentiation, diminished expression of Myh1 and Myh4, alteration of transcriptional dynamics of myogenic factors, such as Mymk and Myog, and deregulation of Hippo signaling pathway. Specifically, we identified elevated basal levels of Hippo activity in myoblasts with loss of FLNC, and ineffective reduction of Hippo signaling activity during myogenic differentiation. The latter was restored by Flnc overexpression. In summary, we confirmed the role of FLNC in muscle cell proliferation, migration and differentiation, and demonstrated for the first time the direct link between Flnc expression and activity of TEAD-YAP\\TAZ signaling. These findings support a role of FLNC in regulation of essential muscle processes relying on mechanical as well as signaling mechanisms.

Positive effects of conjugated linoleic acid (CLA) on the PGC1-α expression under the inflammatory conditions induced by TNF-α in the C2C12 cell line

The aim of the current study was to evaluate the effects of conjugated linoleic acid (CLA) on the expression of the genes involved in the reduction of inflammatory conditions induced by tumor necrosis factor alpha (TNF-α). To fulfill this, the C2C12 murine myoblast cell line was used. The experimental treatments consisted of 50 and 100 µM of CLA with 10, 15 and 20 ng/mL of TNF-α. Our results indicated that the concentrations of 50, 100 and 150 µM of CLA at 24 and 48 h increased the cell survival rate (p < 0.05), as compared with the control group. The effect of pre-treating CLA before the inflammation induced by TNF-α showed that the amount of cell death caused by TNF-α was inhibited in both 50 and 100 µM concentrations of CLA (p < 0.05). Modulatory effects of CLA on PGC1-α and Fndc5 transcript levels showed that the PGC1-α and Fndc5 expression levels were significantly increased 48 h after treating with 50 and 100 µM of CLA (p < 0.05). Overall, these findings indicate that using CLA could considerably improve the inflammatory condition induced by TNF-α.

Biomechanical effects on microRNA expression in skeletal muscle differentiation

We examined whether mechanical stretch affected expression of muscle-specific microRNAs (miRNAs) that regulate proliferation (miR-133a) or differentiation (miR-1, -206). Real-time quantitative RT-PCR was used to assess miRNA regulation in the murine myoblast cell line C2C12 exposed to stretch regimens that promote either proliferation (high stretch: 17%, 1 Hz) or differentiation (moderate stretch: 10%, 0.5 Hz) after adding media that promotes differentiation. Controls consisted of myoblasts cultured under static conditions. While miRNA expression was not affected by high stretch, a significant effect of stretch (P

Toxoplasma gondii Impairs Myogenesis in vitro, With Changes in Myogenic Regulatory Factors, Altered Host Cell Proliferation and Secretory Profile.

Toxoplasma gondii is the causative agent of toxoplasmosis, a parasitic disease with a wide global prevalence. The parasite forms cysts in skeletal muscle cells and neurons, although no evident association with inflammatory infiltrates has been typically found. We studied the impact of T. gondii infection on the myogenic program of mouse skeletal muscle cells (SkMC). The C2C12 murine myoblast cell line was infected with T. gondii tachyzoites (ME49 strain) for 24 h followed by myogenic differentiation induction. T. gondii infection caused a general decrease in myotube differentiation, fusion and maturation, along with decreased expression of myosin heavy chain. The expression of Myogenic Regulatory Factors Myf5, MyoD, Mrf4 and myogenin was modulated by the infection. Infected cultures presented increased proliferation rates, as assessed by Ki67 immunostaining, whereas neither host cell lysis nor apoptosis were significantly augmented in infected dishes. Cytokine Bead Array indicated that IL-6 and MCP-1 were highly increased in the medium from infected cultures, whereas TGF-β1 was consistently decreased. Inhibition of the IL-6 receptor or supplementation with recombinant TGF-β failed to reverse the deleterious effects caused by the infection. However, conditioned medium from infected cultures inhibited myogenesis in C2C12 cells. Activation of the Wnt/β-catenin pathway was impaired in T. gondii-infected cultures. Our data indicate that T. gondii leads SkMCs to a pro-inflammatory phenotype, leaving cells unresponsive to β-catenin activation, and inhibition of the myogenic differentiation program. Such deregulation may suggest muscle atrophy and molecular mechanisms similar to those involved in myositis observed in human patients.

MicroRNA-95 promotes myogenic differentiation by down-regulation of aminoacyl-tRNA synthase complex-interacting multifunctional protein 2.

MicroRNA-95 (miR-95) is well known for its ability to promote the proliferation of a variety of cancer cells, but its function in skeletal muscle development has not been reported so far. Our laboratory has recently generated genetically engineered Meishan pigs containing a loss-of-function myostatin (MSTN) mutant (MSTN-/-). These MSTN-/- pigs grow and develop normally but show clear double muscle phenotype as observed in Belgian cattle. We observed that the expression of miR-95 was up-regulated in the longissimus dorsi from MSTN-/- Meishan pigs at day 65 during embryo development. In this study, we investigated the role of miR-95 in the myogenic differentiation using a murine myoblast cell line C2C12. Our results revealed that miR-95 may play a very important role in regulating the expression of myogenic differentiation marker genes myosin heavy chain (MHC) and myogenin. By use of bioinformatical analysis and luciferase reporter gene assay, aminoacyl-tRNA synthase complex-interacting multifunctional protein 2 (AIMP2) gene was identified as a miR-95 target gene involved in myogenic differentiation. Our results indicated that higher miR-95 expression level leads to lower level of AIMP2 protein expression. When the endogenous expression of AIMP2 is inhibited by siRNA, the expression levels of myogenic differentiation marker genes MHC and myogenin increased, implying that AIMP2 negatively regulates myogenic differentiation. Taken together, it is likely that miR-95 promotes myogenic differentiation in C2C12 myoblasts and may play a positive functional role in skeletal muscle development by down regulating the expression of AIMP2 at protein level.

Glypican-based drug releasing titania implants to regulate BMP2 bioactivity as a potential approach for craniosynostosis therapy

Advances in molecular biology and nanomedicine based therapies hold promise to obviate the need of multiple surgical interventions (associated with current management) in craniosynostosis by preventing bone re-ossification. One such adjunctive therapy involves application of glypicans 1 and 3 (GPC1 and GPC3) that are BMP inhibitors implicated in downregulating the BMP2 activity in prematurely fusing sutures. Electrochemically anodized Titania nanotube (TNT) arrays have been recognized as a promising localized, long-term drug delivery platform for bone-related therapies. This study presents the application of nanoengineered TNT/Ti implants loaded with recombinant glypicans for craniosynostosis therapy. By using Dual luciferase Reporter assay, we tested the biofunctionality of eluted glypicans from the TNT/Ti implants for BMP2 bioactivity regulation in C2C12 murine myoblast cell line. BMP2 activity was inhibited significantly for up to 15days by the glypicans released from polymer-coated TNT/Ti implants, indicating their potential application in adjunctive craniosynostosis treatment.

Engineering muscle cell alignment through 3D bioprinting

Processing of hydrogels represents a main challenge for the prospective application of additive manufacturing (AM) to soft tissue engineering. Furthermore, direct manufacturing of tissue precursors with a cell density similar to native tissues has the potential to overcome the extensive in vitro culture required for conventional cell-seeded scaffolds seeking to fabricate constructs with tailored structural and functional properties. In this work, we present a simple AM methodology that exploits the thermoresponsive behavior of a block copolymer (Pluronic® ) as a means to obtain good shape retention at physiological conditions and to induce cellular alignment. Pluronic/alginate blends have been investigated as a model system for the processing of C2C12 murine myoblast cell line. Interestingly, C2C12 cell model demonstrated cell alignment along the deposition direction, potentially representing a new avenue to tailor the resulting cell histoarchitecture during AM process. Furthermore, the fabricated constructs exhibited high cell viability, as well as a significantly improved expression of myogenic genes vs. conventional 2D cultures. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2582-2588, 2017.

Chitosan/poly-octanoic acid 2-thiophen-3-yl-ethyl ester blends as a scaffold to maintain myoblasts regeneration potential in vitro.

Satellite cells are a small cell population that function as muscle-specific adult stem cells. When muscle damage occurs, these cells are able to activate, proliferate, and ultimately fuse with each other in order to form new myofibers or fuse with existing ones. For tissue engineering applications, obtaining a sufficient number of myoblasts prior transplantation that maintains their regenerative capacity is critical. This can be obtained by in vitro expansion of autologous satellite cells. However, once plated, the self-renewal and regenerative capacity of myoblasts is rapidly lost, obtaining low yields per biopsy. For this purpose, we evaluated in vitro culture of the murine myoblast cell line C2C12 and mouse primary myoblasts with chitosan and chitosan/poly-octanoic acid 2-thiophen-3-yl-ethyl ester blends (poly(OTE)). The films of chitosan/poly(OTE) blends were heterogeneous and slightly rougher than chitosan and poly(OTE) films. Poly(OTE) presence improved myoblast adhesion in both cell types and prevented complete differentiation, but maintaining their differentiation potential in vitro. We identified that the polymer blend chitosan/poly(OTE) could be a suitable substrate to culture satellite cells/myoblasts in vitro preventing differentiation prior transplantation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 118-130, 2017.

Chromatin-wide and transcriptome profiling integration uncovers p38α MAPK as a global regulator of skeletal muscle differentiation.

BackgroundExtracellular stimuli induce gene expression responses through intracellular signaling mediators. The p38 signaling pathway is a paradigm of the mitogen-activated protein kinase (MAPK) family that, although originally identified as stress-response mediator, contributes to establishing stem cell differentiation fates. p38α is central for induction of the differentiation fate of the skeletal muscle stem cells (satellite cells) through not fully characterized mechanisms.MethodsTo investigate the global gene transcription program regulated by p38α during satellite cell differentiation (myogenesis), and to specifically address whether this regulation occurs through direct action of p38α on gene promoters, we performed a combination of microarray gene expression and genome-wide binding analyses. For experimental robustness, two myogenic cellular systems with genetic and chemical loss of p38α function were used: (1) satellite cells derived from mice with muscle-specific deletion of p38α, and (2) the C2C12 murine myoblast cell line cultured in the absence or presence of the p38α/β inhibitor SB203580. Analyses were performed at cell proliferation and early differentiation stages.ResultsWe show that p38α binds to a large set of active promoters during the transition of myoblasts from proliferation to differentiation stages. p38α-bound promoters are enriched with binding motifs for several transcription factors, with Sp1, Tcf3/E47, Lef1, FoxO4, MyoD, and NFATc standing out in all experimental conditions. p38α association with chromatin correlates very well with high levels of transcription, in agreement with its classical function as an activator of myogenic differentiation. Interestingly, p38α also associates with genes repressed at the onset of differentiation, thus highlighting the relevance of p38-dependent chromatin regulation for transcriptional activation and repression during myogenesis.ConclusionsThese results uncover p38α association and function on chromatin at novel classes of target genes during skeletal muscle cell differentiation. This is consistent with this MAPK isoform being a transcriptional regulator.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0074-x) contains supplementary material, which is available to authorized users.

Genetically modified human myoblasts with eNOS may improve regenerative ability of myogenic stem cells to infarcted heart

Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium. To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene. Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants. Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05). The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.

Abstract 2630: Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration.

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and is thought to arise from primitive mesenchymal cells committed to skeletal muscle differentiation. Less than 30% of patients with metastatic disease survive even with the most intensive treatment regimens. Approximately 50% of patients with RMS have intermediate risk and the event-free survival of the intermediate risk patients is approximately 70%. However, even within this group, the survival of patients with metastatic embryonal tumors is much poorer (∼50%). Thus understanding the molecular mechanism of RMS metastasis and the development of therapeutic strategies to inhibit RMS metastasis is critical for these patients. We have reported that zinc finger transcription factor CASZ1 functions as a tumor suppressor gene in neuroblastoma. In these studies we found that CASZ1 regulates genes involved in cell movement and many pertaining to skeletal/muscle system development. To understand whether the induction of muscle developmental genes by CASZ1 was an anomaly associated with pathology, we evaluated the expression and regulation of CASZ1 in normal myoblasts. CASZ1 expression significantly increased when the C2C12 murine myoblast cell line differentiated upon serum withdrawal. Knockdown of CASZ1 in myoblasts causes a 30% increase in cell migration (p&amp;lt;0.02) as determined using an Essen IncuCyte scratch-wound healing assay. Furthermore knock-down of CASZ1 in C2C12 cells leads to a 50% decrease in expression of the skeletal muscle development gene myogenin while over-expression of CASZ1 leads to 2-fold increase in myogenin mRNA. Since CASZ1 regulates aspects of normal muscle development, we hypothesized that its dysregulation in RMS may impact tumor progression. To test this hypothesis, we first evaluated the expression of CASZ1 in primary RMS tumors. We found that CASZ1 mRNA level in embryonal RMS patients’ samples is ∼1.5 lower than in normal muscle or alveolar RMS (p&amp;lt;0.001). To investigate the role of CASZ1 in embryonal RMS cell growth and migration, we developed a Tet-On CASZ1 expression system using the RD RMS cell line. Overexpression of CASZ1 does not affect RD cell proliferation based on MTS and IncuCyte cell confluence assays. Over-expression of CASZ reduces cell motility by 20% (p&amp;lt;0.01) using an Essen IncuCyte scratch-wound healing assay. Knockdown of CASZ1 in RD cells increases cell migration by 40% (p&amp;lt;0.01) but does not affect cell proliferation. However when CASZ1 is expressed in the presence of a muscle inducing differentiation cue, 12-O-tetradecanoylphorbol-13-acetate, TPA, the proliferation of RD cells is suppressed an additional 40% (p&amp;lt;0.01). Taken together, our results suggest that regulation of myogenin expression and cell migration by CASZ1 in C2C12 maybe integral to proper myogenic differentiation, and dysregulation of CASZ1 during early myogenesis may contribute to embryonal RMS tumorigenesis and metastasis. Citation Format: Zhihui Liu, Wilson Chan, Carol J. Thiele. Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2630. doi:10.1158/1538-7445.AM2013-2630 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Comparison of satellite cell-derived myoblasts and C2C12 differentiation in two- and three-dimensional cultures: changes in adhesion protein expression

Changes in the expression of adhesion proteins involved in myoblast differentiation were investigated in monolayer (two-dimensional) and 3D (three-dimensional) cell cultures. The expression of integrin alpha3 subunit, integrin beta1 subunit, ADAM12 (a disintegrin and metalloproteinase 12), tetraspanins CD9 and CD81 and M-cadherin were examined in the murine myoblast cell line C2C12 and in a primary culture of rat satellite cells. Myoblasts in monolayer and 3D cultures showed significant differences in their morphology and cytoskeletal organization. All of the studied proteins participated in myoblast fusion in each culture examined, but differences in their levels of expression were observed. Satellite cell-derived myoblasts exhibited higher expression of adhesion protein mRNAs than C2C12 cells. Also, C2C12 cells from a 3D culture showed slightly higher expression of adhesion protein transcripts than the same cells cultured as a monolayer. Significantly, the levels of adhesion protein mRNAs were found to change in parallel in all cell culture types. Despite this finding, it is important that differences between satellite cell-derived myoblasts and cell line C2C12 grown in monolayer and 3D cultures are taken into account when studying processes of myoblast differentiation in vitro.

Leukemia inhibitory factor-dependent increase in myoblast cell number is associated with phosphotidylinositol 3-kinase-mediated inhibition of apoptosis and not mitosis

Leukemia inhibitory factor (LIF) is an important regulator of skeletal muscle regeneration and has been suggested to be mitogenic for myogenic cells because it has been shown to increase the quantity of myoblast cells grown in culture over extended periods of time. Using the established C2C12 murine myoblast cell line, we observed that LIF treatment did not significantly increase the rate at which myoblasts synthesise DNA under conditions which increased cell quantity by 73% above control, whilst the known mitogen fibroblast growth factor-2 significantly increased DNA synthesis under these conditions. Consequently, we examined the capacity of LIF to prevent apoptotic cell death. LIF treatment significantly reduced staurosporine-induced apoptotic DNA fragmentation by 37% compared to control and also reduced the proteolytic activation of caspase-3 by 40% compared to control. This effect of LIF was completely abolished by addition of the phosphatidylinositol 3-kinase inhibitor wortmannin, indicating that the phosphatidylinositol 3-kinase signalling pathway, previously shown to be linked to LIF-dependent increases in cell number, is necessary in mediating the anti-apoptotic effects of LIF. LIF treatment was also associated with increased levels of Bcl-xL and XIAP transcripts compared to control. Therefore, we suggest that the role of LIF in skeletal muscle regeneration and myogenesis is that of a survival factor rather than a mitogen.