Differentiation Of Murine Myoblasts Research Articles

NRF2 Regulates Viability, Proliferation, Resistance to Oxidative Stress, and Differentiation of Murine Myoblasts and Muscle Satellite Cells.

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2tKO). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2tKO mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment.

Myoblast 3D bioprinting to burst in vitro skeletal muscle differentiation.

Skeletal muscle regeneration is one of the major areas of interest in sport medicine as well as trauma centers. Three‐dimensional (3D) bioprinting (BioP) is nowadays widely adopted to manufacture 3D constructs for regenerative medicine but a comparison between the available biomaterial‐based inks (bioinks) is missing. The present study aims to assess the impact of different hydrogels on the viability, proliferation, and differentiation of murine myoblasts (C2C12) encapsulated in 3D bioprinted constructs aided to muscle regeneration. We tested three different commercially available hydrogels bioinks based on: (1) gelatin methacrylate and alginate crosslinked by UV light; (2) gelatin methacrylate, xanthan gum, and alginate‐fibrinogen; (3) nanofibrillated cellulose (NFC)/alginate‐fibrinogen crosslinked with calcium chloride and thrombin. Constructs embedding the cells were manufactured by extrusion‐based BioP and C2C12 viability, proliferation, and differentiation were assessed after 24 h, 7, 14, 21, and 28 days in culture. Although viability, proliferation, and differentiation were observed in all the constructs, among the investigated bioinks, the best results were obtained by using NFC/alginate‐fibrinogen‐based hydrogel from 7 to 14 days in culture, when the embedded myoblasts started fusing, forming at day 21 and day 28 multinucleated myotubes within the 3D bioprinted structures. The results revealed an extensive myotube alignment all over the linear structure of the hydrogel, demonstrating cell maturation, and enhanced myogenesis. The bioprinting strategies that we describe here denote a strong and endorsed approach for the creation of in vitro artificial muscle to improve skeletal muscle tissue engineering for future therapeutic applications.

Regulation of m6A RNA Methylation and Its Effect on Myogenic Differentiation in Murine Myoblasts

Regulation of m6A RNA Methylation and Its Effect on Myogenic Differentiation in Murine Myoblasts

Regulation of m^(6)A RNA Methylation and Its Effect on Myogenic Differentiation in Murine Myoblasts

N^(6)-methyladenosine (m^(6)A) has been identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, and plays important biological roles in the regulation of cellular metabolic processes. However, its role in myogenic differentiation is unclear. Here, we altered the m^(6)A RNA methylation level by overexpression of METTL3, and explored the effect of m^(6)A RNA methylation on myogenic differentiation of murine myoblasts in vitro. The m6A RNA methylation level is regulated by exogenous methylation inhibitor cycloleucine (Cyc) and methyl donor betaine (Bet). Therefore, chemical reagents of Cyc and Bet were used to test the regulatory effect of m^(6)A RNA methylation on myogenic differentiation. Results showed that METTL3 and Bet positively regulated the m^(6)A RNA methylation levels, and Cyc negatively regulated m^(6)A RNA methylation levels. In addition, m^(6)A methylation positively regulated myogenic differentiation in murine myoblasts. These findings provide insight in the mechanisms underlying the effect of m^(6)A RNA methylation on myogenesis.

Bradykinin mediates myogenic differentiation in murine myoblasts through the involvement of SK1/Spns2/S1P2 axis

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P2 receptor are involved in the action exerted by BK, since pharmacological inhibition/antagonism or specific down-regulation significantly alter BK-induced myogenic differentiation. Moreover, the molecular mechanism initiated by BK involves a rapid translocation of SK1 to plasma membrane, analyzed by time-lapse immunofluorescence analysis. The present study highlights the role of SK1/Spns2/S1P receptor 2 signaling axis in BK-induced myogenic differentiation, thus confirming the crucial involvement of this pathway in skeletal muscle cell biology.

Transient Ca2+ depletion from the endoplasmic reticulum is critical for skeletal myoblast differentiation.

Endoplasmic reticulum (ER) stress is a cellular condition in which unfolded proteins accumulate in the ER because of various but specific causes. Physiologic ER stress occurs transiently during myoblast differentiation, and although its cause remains unknown, it plays a critical role in myofiber formation. To examine the mechanism underlying ER stress, we monitored ER morphology during differentiation of murine myoblasts. Novel ER-derived structures transiently appeared prior to myoblast fusion both in vitro and in vivo. Electron microscopy studies revealed that these structures consisted of pseudoconcentric ER cisternae with narrow lumens. Similar structures specifically formed by pharmacologically induced ER Ca(2+) depletion, and inhibition of ER Ca(2+) efflux channels in differentiating myoblasts considerably suppressed ER-specific deformation and ER stress signaling. Thus, we named the novel structures stress-activated response to Ca(2+) depletion (SARC) bodies. Prior to SARC body formation, stromal interaction molecule 1 (STIM1), an ER Ca(2+) sensor protein, formed ER Ca(2+) depletion-specific clusters. Furthermore, myoblast differentiation manifested by myoblast fusion did not proceed under the same conditions as inhibition of ER Ca(2+) depletion. Altogether, these observations suggest that ER Ca(2+) depletion is a prerequisite for myoblast fusion, causing both physiologic ER stress signaling and SARC body formation.

Novel long noncoding RNAs (lncRNAs) in myogenesis: a miR-31 overlapping lncRNA transcript controls myoblast differentiation.

Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels.

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels.

Biologically derived soft conducting hydrogels using heparin-doped polymer networks.

The emergence of flexible and stretchable electronic components expands the range of applications of electronic devices. Flexible devices are ideally suited for electronic biointerfaces because of mechanically permissive structures that conform to curvilinear structures found in native tissue. Most electronic materials used in these applications exhibit elastic moduli on the order of 0.1–1 MPa. However, many electronically excitable tissues exhibit elasticities in the range of 1–10 kPa, several orders of magnitude smaller than existing components used in flexible devices. This work describes the use of biologically derived heparins as scaffold materials for fabricating networks with hybrid electronic/ionic conductivity and ultracompliant mechanical properties. Photo-cross-linkable heparin–methacrylate hydrogels serve as templates to control the microstructure and doping of in situ polymerized polyaniline structures. Macroscopic heparin-doped polyaniline hydrogel dual networks exhibit impedances as low as Z = 4.17 Ω at 1 kHz and storage moduli of G′ = 900 ± 100 Pa. The conductivity of heparin/polyaniline networks depends on the oxidation state and microstructure of secondary polyaniline networks. Furthermore, heparin/polyaniline networks support the attachment, proliferation, and differentiation of murine myoblasts without any surface treatments. Taken together, these results suggest that heparin/polyaniline hydrogel networks exhibit suitable physical properties as an electronically active biointerface material that can match the mechanical properties of soft tissues composed of excitable cells.

Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation.

BackgroundBetaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood.MethodsWe examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy.ResultsWe performed a dose–response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length.In addition, we investigated BET role on myoblasts proliferation and differentiation. During proliferation, BET did not modify C2C12 proliferative rate, but promoted myogenic induction, enhancing MyoD protein content and cellular elongation. During differentiation, BET caused an increase of muscle-specific markers and IGF-1 R protein levels.ConclusionsOur findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment.

Heme oxygenase-1 inhibits murine myoblast differentiation and may induce tumor development

Heme oxygenase-1 inhibits murine myoblast differentiation and may induce tumor development

Inhibition of mammalian muscle differentiation by excretory secretory products of muscle larvae of Trichinella spiralis in vitro

The excretory-secretory products (ESP) released by muscle stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ESP modulate nurse cell formation remain unclear. In the present study, the ability of ESP of muscle larvae of T. spiralis (ML-ESP) to influence the proliferation and differentiation of murine myoblasts and the mechanisms were evaluated in vitro using C2C12 myoblast cell line, which were incubated for various times under grow or differentiation culture medium containing various concentrations of ML-ESP. The results indicated that ML-ESP promoted myoblast proliferation in a dose-dependent manner and increased the expression of the cell-cycle regulator cyclin D1 as well as that of proliferating cell nuclear antigen (PCNA). Conversely, ML-ESP inhibited the differentiation of these cells, which was evidenced by a reduction in the levels of MHC and MRFs expression (MyoD and myogenin) as well as that of p21. In addition, ML-ESP also inhibited the phosphorylation of p38 MAPK in differentiating C2C12 myoblast. Taken together, these results imply that certain critical mediators contained in ML-ESP inhibit myogenesis through enhancing skeletal myoblasts proliferation and down-regulating the expression of MRFs as well as involving p38 MAPK signalling pathway, which provides insight into the mechanisms utilised by T. spiralis to interfere normal wound repair in infected muscle cells and affect nurse cell formation.

A novel β1 integrin isoform produced by alternative splicing: unique expression in cardiac and skeletal muscle

The mRNA's of several integrin subunits are alternatively spliced in the region encoding cytoplasmic domains, that may potentially provide alternative integrin-cytoskeleton interactions and transmembrane signaling pathways. We identified a novel cytoplasmic tail variant of the human β1 subunit by reverse transcriptase polymerase chain reaction. This fourth β1 variant, named β1D, is specific for skeletal and cardica muscle. The determined genomic organization of the 3′-region of the human β1 gene reveals that β1D is produced by alternative splicing of mRNA. In addition, we show that the expression of β1D is developmentally regulated during murine myoblast differentiation, suggesting a role for β1D in myogenesis.