Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophy via paracrine signaling

Adipose-derived stem cells have been shown to promote peripheral nerve regeneration through the paracrine secretion of neurotrophic factors. However, it is unclear whether these cells can promote myogenic differentiation in muscular dystrophy. Adipose-derived stem cells （6 × 106） were injected into the gastrocnemius muscle of mdx mice at various sites. Dystrophin expression was found in the muscle fibers. Phosphorylation levels of Akt, mammalian target of rapamycin （mTOR）, eIF-4E binding protein 1 and $6 kinase 1 were increased, and the Akt/mTOR pathway was activated. Simultaneously, myogenin levels were increased, whereas cleaved caspase 3 and vimentin levels were decreased. Necrosis and fibrosis were reduced in the muscle fibers. These findings suggest that adipose-derived stem cells promote the re- generation and survival of muscle cells by inhibiting apoptosis and fibrosis, thereby alleviating muscle damage in muscular dystrophy.

Ryanodine receptor RyR1-mediated elevation of Ca^(2+)concentration is required for the late stage of myogenic differentiation and fusion

Background:Cytosolic Ca^(2+)plays vital roles in myogenesis and muscle development.As a major Ca^(2+)release channel of endoplasmic reticulum(ER),ryanodine receptor 1(RyR1)key mutations are main causes of severe congenital myopathies.The role of RyR1 in myogenic differentiation has attracted intense research interest but remains unclear.Results:In the present study,both RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts were employed to explore the role of RyR1 in myogenic differentiation,myotube formation as well as the potential mechanism of RyR1-related myopathies.We observed that RyR1 expression was dramatically increased during the late stage of myogenic differentiation,accompanied by significantly elevated cytoplasmic Ca^(2+)concentration.Inhibition of RyR1 by siRNA-mediated knockdown or chemical inhibitor,dantrolene,significantly reduced cytosolic Ca^(2+)and blocked multinucleated myotube formation.The elevation of cytoplasmic Ca^(2+)concentration can effectively relieve myogenic differentiation stagnation by RyR1 inhibition,demonstrating that RyR1 modulates myogenic differentiation via regulation of Ca^(2+)release channel.However,RyR1-knockout-induced Ca^(2+)leakage led to the severe ER stress and excessive unfolded protein response,and drove myoblasts into apoptosis.Conclusions:Therefore,we concluded that Ca^(2+)release mediated by dramatic increase in RyR1 expression is required for the late stage of myogenic differentiation and fusion.This study contributes to a novel understanding of the role of RyR1 in myogenic differentiation and related congenital myopathies,and provides a potential target for regulation of muscle characteristics and meat quality.

Factors influencing myogenic differentiation of adipose-derived stem cells and their application in muscle regeneration

Skeletal muscle regeneration mainly depends on muscle satellite cells;however,these cells are not sufficient for supporting repair and regeneration in volumetric muscle loss(VML),Duchenne muscular dystrophy,and other muscle injuries or muscle diseases.As such,much work has been conducted in recent years to search for myogenic stem cells.Adipose-derived stem cells(ADSCs)have a wide range of sources,rapid growth,and multi-directional differentiation potential,and have become vital candidates for muscle regeneration.Multiple factors influence the myogenic differentiation capacity of ADSCs.This paper reviews the regulatory aspects and possible factors that have been identified in recent years to affect myogenic differentiation of ADSCs.Based on these factors,gene editing,and perfusion concepts,a method was proposed to achieve maximal differentiation efficiency of ADSCs.This study focused on the application of ADSCs in muscle regeneration and disease.Based on the importance of myogenic differentiation of ADSCs for the repair and regeneration of muscle damage,this study provides a basis for future research surrounding the efficient induction of myogenic differentiation of ADSCs in vitro.

3D bioprinting of conductive hydrogel for enhanced myogenic differentiation

Recently,hydrogels have gained enormous interest in three-dimensional(3D)bioprinting toward developing functional substitutes for tissue remolding.However,it is highly challenging to transmit electrical signals to cells due to the limited electrical conductivity of the bioprinted hydrogels.Herein,we demonstrate the 3D bioprinting-assisted fabrication of a conductive hydrogel scaffold based on poly-3,4-ethylene dioxythiophene(PEDOT)nanoparticles(NPs)deposited in gelatin methacryloyl(GelMA)for enhanced myogenic differentiation of mouse myoblasts(C2C12 cells).Initially,PEDOT NPs are dispersed in the hydrogel uniformly to enhance the conductive property of the hydrogel scaffold.Notably,the incorporated PEDOT NPs showed minimal influence on the printing ability of GelMA.Then,C2C12 cells are successfully encapsulated within GelMA/PEDOT conductive hydrogels using 3D extrusion bioprinting.Furthermore,the proliferation,migration and differentiation efficacies of C2C12 cells in the highly conductive GelMA/PEDOT composite scaffolds are demonstrated using various in vitro investigations of live/dead staining,F-actin staining,desmin and myogenin immunofluorescence staining.Finally,the effects of electrical signals on the stimulation of the scaffolds are investigated toward the myogenic differentiation of C2C12 cells and the formation of myotubes in vitro.Collectively,our findings demonstrate that the fabrication of the conductive hydrogels provides a feasible approach for the encapsulation of cells and the regeneration of the muscle tissue.

Arginine promotes myogenic differentiation and myotube formation through the elevation of cytoplasmic calcium concentration

This study aimed to explore the mechanism underlying arginine-promoted myogenesis of myoblasts.C2C12 cells were cultured with a medium containing 0.1,0.4,0.8,or 1.2 mmol/L arginine,respectively.Cell proliferation,viability,differentiation indexes,cytoplasmic Ca^(2+)concentration,and relative mRNA expression levels of myogenic regulatory factors(MRF)and key Ca2+channels were measured in the absence or presence of 2 chemical inhibitors,dantrolene(DAN,10μmol/L)and nisoldipine(NIS,10μmol/L),respectively.Results demonstrated that arginine promoted myogenic differentiation and myotube formation.Compared with the control(0.4 mmol/L arginine),1.2 mmol/L arginine upregulated the relative mRNA expression levels of myogenin(MyoG)and Myomaker at d 2 during myogenic induction(P<0.05).Cytoplasmic Ca^(2+)concentrations were significantly elevated by arginine supplementation at d 2 and 4(P<0.05).Relative mRNA expression levels of Ca^(2+)channels including the type 1 ryanodine recepto r(RyR1)and voltage-gated Ca^(2+)channel(Cav1.1)were upregulated by 1.2 mmol/L arginine during2-d myogenic induction(P<0.01).However,arginine-promoted myogenic potential of myoblasts was remarkably compromised by DAN and NIS,respectively(P<0.05).These findings evidenced that the supplementation of arginine promoted myogenic differentiation and myotube formation through increasing cytoplasmic Ca^(2+)concentration from both extracellular and sarcoplasmic reticulum Ca^(2+).

GLP inhibits heterochromatin clustering and myogenic differentiation by repressing MeCP2

Myogenic differentiation is accompanied by alterations in the chromatin states, which permit or restrict the transcriptional machinery and thus impact distinctive gene expression profiles. The mechanisms by which higher-order chromatin remodeling is associated with gene activation and silencing during differentiation is not fully understood. In this study, we provide evidence that the euchromatic lysine metbyltransferase GLP regulates heterocbromatin organization and myogenic differentiation. Interestingly, GLP represses expression of the methyl-binding protein MeCP2 that induces heterochromatin clustering during differentiation. Consequently, MeCP2 and HP1γ localization at major satellites are altered upon modulation of GLP expression. In GLP knockdown cells, depletion of MeCP2 restored both chromatin organization and myogenic differentiation. These results identify a novel regulatory axis between a histone methylation writer and DNA methylation reader, which is important for beterochromatin organization during differentiation.

Global transcriptomic analysis reveals Lnc-ADAMTS9 exerting an essential role in myogenesis through modulating the ERK signaling pathway

Background:Long non-coding RNAs(lncRNAs)are emerging key regulators involved in a variety of biological processes such as cell differentiation and development.The balance between myogenesis and adipogenesis is crucial for skeletal muscle homeostasis in humans and meat quality in farm animals.The present study aimed to reveal the global transcriptomic profiles of adipogenic(Adi-)and myogenic(Myo-)precursors derived from porcine skeletal muscle and identify lncRNAs involved in the modulation of myogenesis homeostasis in porcine skeletal muscle.Results:In this study,a total of 655 novel individual lncRNAs including differentially expressed 24 lncRNAs,and 755 differentially expressed mRNAs were identified(fold change≥2 or≤0.5 and adjusted P<0.05).Integrated results of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis accompanied by the variation of intracellular Ca2+concentration highlighted Lnc-ADAMTS9 involved in the modulation of myogenesis homeostasis in porcine skeletal muscle.Although Lnc-ADAMTS9 knock-down did not alter the mRNA expression of ADAMTS9,we demonstrated that Lnc-ADAMTS9 can promote myogenic proliferation and myogenic differentiation of myogenic precursors through inhibiting the ERK/MAPK signaling pathway.Conclusion:We deciphered a comprehensive catalog of mRNAs and lncRNAs that might be involved in the regulation of myogenesis and adipogenesis homeostasis in the skeletal muscle of pigs.The Lnc-ADAMTS9 exerts an essential role in myogenesis through the ERK signaling pathway.

Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

The extracellular matrix （ECM） performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 （Matn2） in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Math2 can also promote or suppress tumor growth.

3D-printed tissue repair patch combining mechanical support and magnetism for controlled skeletal muscle regeneration

Physical forces,such as magnetic and mechanical stimulation,are known to play a significant role in the regulation of cell response.In the present study,a biomimetic regeneration patch was fabricated using E-jet 3 D printing,which integrates mechanical and magnetic stimulation in a biocompatible"one-pot reaction"strategy when combined with a static magnetic field(SMF).The magneto-based therapeutic regeneration patch induced myoblasts to form aligned and multinucleated myotubes,regulated the expression of myogenic-related genes,and activated the p38αmitogen-activated protein kinase pathway via the initiation of myogenic differentiation.To validate the efficiency of the proposed strategy,the regeneration patch was implanted into mice and exposed to a suitable SMF,which resulted in significantly enhanced in vivo skeletal muscle regeneration.The findings demonstrated that appropriate external physical stimulation provides a suitable biophysical microenvironment that is conducive to tissue regeneration.The method used in the present study represents a promising technique to induce the regeneration of damaged skeletal muscle tis sue.

Natural flavonoid luteolin promotes the differentiation of porcine myoblasts through activation of PI3K/Akt/mTOR signaling

A key step in the manufacturing of cultured meat is to produce myotubes by induced myogenic differentiation.The development of effective,low-cost,and food-safe components that promote the in vitro differentiation of myoblasts is essential for the industrialization of cultured meat.Flavonoids are a class of plant secondary metabolites with various biological activities,but their effects on the regulation of myoblast behaviors are a lack of study.In this study,we selected four representative flavonoids including luteolin,chrysin,apigenin,and genistein,and investigated their effects on porcine myoblasts in the aspects of proliferation,migration,and differentiation.The results showed that four flavonoids all had relatively low cell toxicity but weak ability to promote the proliferation of porcine myoblasts.A positive effect of luteolin was observed in the migration and differentiation of porcine myoblasts,as indicated by improved migration rate and fusion index,as well as upregulated expression of Myogenin and MyHC.Pharmacological inhibition of PI3K activity attenuated the efficacy of luteolin on porcine myoblasts,and further analysis showed that luteolin increased the phosphorylation of PI3K,Akt,and mTOR,indicating the activation of the PI3K/Akt/mTOR signaling pathway.In conclusion,these findings showed that luteolin promoted the migration and differentiation of porcine myoblast via the activation of PI3K/Akt/mTOR signaling,providing biological evidence for its application in cultured meat production.

Injectable muscle-adhesive antioxidant conductive photothermal bioactive nanomatrix for efficiently promoting full-thickness skeletal muscle regeneration

The completed skeletal muscle regeneration resulted from severe injury and muscle-related disease is still a challenge.Here,we developed an injectable muscle-adhesive antioxidant conductive bioactive photothermo-responsive nanomatrix for regulating the myogenic differentiation and promoting the skeletal muscle regeneration in vivo.The multifunctional nanomatrix was composed of polypyrrole@polydopamine(PPy@PDA,342±5.6 nm)nanoparticles-crosslinked Pluronic F-127(F127)-polycitrate matrix(FPCP).The FPCP nanomatrix demonstrated inherent multifunctional properties including excellent photothermo-responsive and shear-thinning behavior,muscle-adhesive feature,injectable ability,electronic conductivity(0.48±0.03 S/m)and antioxidant activity and photothermal function.The FPCP nanomatrix displayed better photothermal performance with near-infrared irradiation,which could provide the photo-controlled release of protein(91%±2.6%of BSA was released after irradiated 3 times).Additionally,FPCP nanomatrix could significantly enhance the cell proliferation and myogenic differentiation of mouse myoblast cells(C2C12)by promoting the expressions of myogenic genes(MyoD and MyoG)and myosin heavy chain(MHC)protein with negligible cytotoxicity.Based on the multifunctional properties,FPCP nanomatrix efficiently promoted the full-thickness skeletal muscle repair and regeneration in vivo,through stimulating the angiogenesis and myotube formation.This study firstly indicated the vital role of multifunctional PPy@PDA nanoparticles in regulating myogenic differentiation and skeletal muscle regeneration.This work also suggests that rational design of bioactive matrix with multifunctional feature would greatly enhance the development of regenerative medicine.