Interactions between myoblasts and macrophages under high glucose milieus result in inflammatory response and impaired insulin sensitivity

BACKGROUND Skeletal muscle handles about 80% of insulin-stimulated glucose uptake and become the major organ occurring insulin resistance(IR).Many studies have confirmed the interactions between macrophages and skeletal muscle regulated the inflammation and regeneration of skeletal muscle.However,despite of the decades of research,whether macrophages infiltration and polarization in skeletal muscle under high glucose(HG)milieus results in the development of IR is yet to be elucidated.C2C12 myoblasts are well-established and excellent model to study myogenic regulation and its responses to stimulation.Further exploration of macrophages'role in myoblasts IR and the dynamics of their infiltration and polarization is warranted.AIM To evaluate interactions between myoblasts and macrophages under HG,and its effects on inflammation and IR in skeletal muscle.METHODS We detected the polarization status of macrophages infiltrated to skeletal muscles of IR mice by hematoxylin and eosin and immunohistochemical staining.Then,we developed an in vitro co-culture system to study the interactions between myoblasts and macrophages under HG milieus.The effects of myoblasts on macrophages were explored through morphological observation,CCK-8 assay,Flow Cytometry,and enzyme-linked immunosorbent assay.The mediation of macrophages to myogenesis and insulin sensitivity were detected by morphological observation,CCK-8 assay,Immunofluorescence,and 2-NBDG assay.RESULTS The F4/80 and co-localization of F4/80 and CD86 increased,and the myofiber size decreased in IR group(P<0.01,g=6.26).Compared to Mc group,F4/80+CD86+CD206-cells,tumor necrosis factor-α(TNFα),inerleukin-1β(IL-1β)and IL-6 decreased,and IL-10 increased in McM group(P<0.01,g>0.8).In McM+HG group,F4/80+CD86+CD206-cells,monocyte chemoattractant protein 1,TNFα,IL-1βand IL-6 were increased,and F4/80+CD206+CD86-cells and IL-10 were decreased compared with Mc+HG group and McM group(P<0.01,g>0.8).Compered to M group,myotube area,myotube number and E-MHC were increased in MMc group(P<0.01,g>0.8).In MMc+HG group,myotube area,myotube number,E-MHC,GLUT4 and glucose uptake were decreased compared with M+HG group and MMc group(P<0.01,g>0.8).CONCLUSION Interactions between myoblasts and macrophages under HG milieus results in inflammation and IR,which support that the macrophage may serve as a promising therapeutic target for skeletal muscle atrophy and IR.

Effects of areca nut consumption on cell differentiation of osteoblasts, myoblasts, and fibroblasts

Areca nut is used worldwide as a hallucinogenic addicting drug along the tropical belt.Arecoline,a toxic compound,is the most important alkaloid in areca nuts.The adverse effects of oral uptake and chewing of areca nut are well known.For example,the possibility of cancer caused by chewing areca nuts is widely discussed.Chewing areca nut has other adverse effects on other organs,including abnormal cell differentiation,oral cancer,and several other diseases.The use of areca nut is also associated with low birthweight.Skeletal musculature is the largest organ in the body and is attached to the bones.During embryo development,the differentiation of bone and muscle cells is critical.In this article,we reviewed the effects of areca nut and arecoline on embryonic cell differentiation,particularly osteoblasts,myoblasts,and fibroblasts.

Micro RNA transcriptome of skeletal muscle during yak development reveals that miR-652 regulates myoblasts differentiation and survival by targeting ISL1

The growth and development of skeletal muscle also determine the meat production of yak, ultimately affecting the economic benefits. Hence, improving growth performance is a top priority in the yak industry. Skeletal muscle development is a complex process involving the regulation of several genes, including microRNAs(miRNAs). However,the transcription of miRNAs in yak skeletal muscle during prenatal to postnatal stages is unknown. We used small RNA sequencing(small RNA-Seq) to determine the global miRNAs of longissimus dorsi muscle from yak(the samples were collected from three fetuses and three adults). Totally 264 differently expressed miRNAs(|log2(fold change)|>1and P-value≤0.05) were detected between the two groups. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis showed that differently expressed miRNAs-targeted genes participated in pathways associated with muscle development, such as MAPK, PI3K-Akt, and Hippo signaling pathways, etc. MiR-652, which was up-regulated in the fetal group, was transfected into C2C12 myoblasts to examine its role. miR-652 promoted(P≤0.05)proliferation and differentiation, but inhibited(P≤0.001) apoptosis at early period. Furthermore, miR-652 reduced(P≤0.001) the proportion of C2C12 myoblasts in the G1 phase while increasing(P≤0.01) the proportion of cells in the S and G2 phases. Dual-luciferase reporter assays indicated that ISL1 served as a target of miR-652. In general, these findings expand our understanding of yak skeletal muscle miRNAs, and suggested that miR-652 probably regulated myogenesis by regulating ISL1.

Culture and Identification of Myoblasts Isolated from Duck Embryos

Using embryonic myoblasts to research the formation and de-velopmental mechanisms of skeletal muscle is becoming a research hotspot. This study aimed to establish a method of isolation, culture and identification of my-oblasts in duck embryos. [Method] Pectoral and leg muscle samples were isolated from the embryos of Gaoyou duck at 13 d of hatching, then disassociated with col-lagenase and trypsin and purified via differential adhesion. The isolated cells were cultured in vitro and detected for the expression of Pax7 protein using immunofluo-rescence technique. [Result] Myoblasts were obtained successful y both from pectoral and leg muscles in duck embryos and these cells proliferated strongly and differen-tiated wel . Immunofluorescence staining showed that more than 95% cells could express Pax7 protein. [Conclusion] In summary, we report the successful establish-ment of a complete system for the isolation, purification, identification and culture of myoblasts from duck embryos.

Change of p16^(INK4a) and PNCA Protein Expression in Myocardium after Injection of hIGF-1 Gene Modified Skeletal Myoblasts into Post-infarction Rats

This study examined the change of p16^INK4a and PNCA protein expression in myocardium after injection of hIGF-1 gene modified skeletal myoblasts into post-infarction rats. HIGF-1 gene modified skeletal myoblasts （hIGF-1-myoblasts） were injected into hind limb muscles of 18 post-infraction rats （experimental group）. Primary-myoblasts were injected into 18 post-infraction rats （control group） and 12 non-infarction rats （sham group）. Expression of p16INK4a and PCNA pro- tein in myocardiums were separately detected immunocytochemically 1, 2 and 4 weeks after the inuection. The level of hIGF-1 and rIGF-1 protein in serum and myocardium were detected by en- zyme-linked immunosorbent assay （ELISA）. Compared with the sham group, the percentage of p^16INK4a and PCNA positive cells reached a peak after 1 week in the control group and the experimental group （P〈0.01）. Moreover, the percentage of p16^INK4a-positive cells in the experimental group was lower than in control group whereas the percentage of PCNA-positive cells was lower in the control group than in the experimental group （P〈0.01）. The percentage of p16^INK4a-positive cells in the experimental group and the percentage of PCNA-positive cells in the control group were close to that in the sham group from the 2nd week （P〉0.05）. ELISA analysis disclosed that the myocardium level of rIGF-1 protein increased gradually in the controls and especially in the experimental group （P〈0.01）. The serum level of rIGF-1 decreased significantly in post-infraction rats, but these conditions were improved in the experimental group （P〈0.01）. The hIGF-1 protein in serum and myocar- dium were detected from the 1st week to the 4th week in the experimental group. Statistical analysis revealed significant associations of myocardium level of hIGF-1 protein with expression of p^16INK4a and PCNA protein （r=–0.323, P〈0.05; r=0.647, P〈0.01）. It is concluded that genetically hIGF-1-myoblast provides a means for constant synthesis and release of hIGF-1. It could not only improve the expression of rIGF-1 and PCNA protein in myocardium, but also suppress the expression of p16^INK4a protein for 30 days in post-infraction rats. Myoblasts-mediated IGF-1 gene therapy may provide a new alternative for the clinical treatment of heart failure.

Induced Differentiation of Adipose-derived Stromal Cells into Myoblasts

This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells(ADSCs) into myoblasts,which may provide a new strategy for tissue engineering in patients with stress urinary incontinence(SUI).ADSCs,isolated and cultured ex vivo,were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine(5-aza),5% FBS,and 5% horse serum.Cellular morphology was observed under an inverted microscope.Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy.Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation.Reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting were used to detect mRNA and protein expression,respectively,of sarcomeric and desmin smooth muscle proteins.The results showed that ADSCs were mainly of a spindle or polygon shape.Flow cytometry analysis revealed that ADSCs did not express CD34,CD45,and CD106 but high levels of CD44 and CD90,which confirmed that the cultured cells were indeed ADSCs.After induction with a 5-aza-containing solution,morphological changes in ADSCs,including irregular cell size,were observed.Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface.Many organelles were observed and the cytoplasm was found to contain many mitochondria,rough endoplasmic reticulum(rER),and myofilament-like structures.Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process,with the highest expression level found on day 28 of induction.RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells,but no significant difference between the two groups of cells in sarcomeric protein expression.It was concluded that under specific induction setting,ADSCs can be induced to differentiate into myoblasts,providing a potential new option in stem cell transplantation therapy for SUI.

Regulation of Hypoxic Response Elements on the Expression of Vascular Endothelial Growth Factor Gene Transfected to Rat Skeletal Myoblasts under Hypoxic Environment

The regulation of hypoxic response elements on the expression of vascular endothelial growth factor （VEGF） gene transfected to primary cultured rat skeletal myoblasts under hypoxic environment was investigated. pEGFP-C3-9HRE-CMV-VEGF vector was constructed with molecular biology technique and transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, Western blot and fluorescence microscope under different oxygen concentrations and different hypoxia time. The results showed that in hypoxia group, the VEGF gene bands were seen and with the decrease of oxygen concentrations and prolongation of hypoxia time, the expression of VEGF mRNA was obviously increased. Under hypoxic environment, the expression of VEGF protein in the transfected myoblasts was significantly increased. EGFP was expressed only under hypoxic environment but not under normoxic environment. It was concluded that hypoxia promoter could be constructed with HRE and regulate the expression of VEGF gene under hypoxic and normoxic environment, which could enhance the re- liability of gene therapy.

Modified methods for culturing myoblasts of rats: Combination of multi-enzymatic digestion and double purification

BACKGROUND： With developments of tissue engineering and genetic engineering, we aim to culture myoblasts, which are characterized by high purity, high quality and high production, for wide application in neural regeneration researches. OBJECTIVE： To modify traditional dissociation method in order to obtain myoblasts, which are characterized by high purity, high quality and high production, and explore the biological properties under in vitro culture. DESIGN： Observational study. SETTING： Basic Institute of Academy of Military Medical Sciences of Chinese PLA. MATERIALS： Four neonatal Wistar rats of 5 days old, both genders and mean body mass of 10 g were selected in this study. The main reagents and devices were detailed as follows： DMEM medium （Gibco Company）, fetus bovine serum （FBS, Hycolne Company）, collagenase Ⅱ （Sigma Company）, trypsin （Sigma Company）, dispase Ⅱ （Sigma Company）, desmin antibody （Fuzhou Maixin Company）, antibody Ⅱ and ABC kit （Wuhan Baster Biotechnology Company）, desk centrifuge （KUBATO, Japan）, and inverted phase contrast microscope （LEICA DMIRB, Germany）. METHODS： The experiment was carried out in the Basic Institute of Academy of Military Medical Sciences of Chinese PLA from June to October 2006. Neonatal rats were sacrificed under sterile condition to obtain skeletal muscles of limbs, which were washed with cold PBS （containing benzylpenicillin and estreptomicina）, and muscular tissue was sheared into pieces. Then, those muscular pieces were added with mixed digestive enzyme （containing 2 g/L collagenase Ⅱ ＋ 5 g/L dispase Ⅱ ＋ 0.28 g/L CaCl2） as twice volume as pieces, dealt with mechanical pipetting for 5 minutes and cultured in CO2 incubator for 10 minutes. The operation was done for three times and the muscular pieces were digested for 45 minutes in total. Moreover, cells were suspended again in order to obtain myoblasts from skeletal muscle of neonatal rats. In addition, myoblasts were purified with differential attachment technique and enzyme digestion so as to observe morphological characteristics and growth, draw growth curve, analyze surface structure under scanning electron microscope, and evaluate with Desmin immunohistochemical staining. MAIN OUTCOME MEASURES： Morphological characteristics and growth ofmyoblasts cultured in vitro. RESULTS： ①Growth of myoblasts of skeletal muscle： Primary cells had well growth, mature and differentiation. The positive rate of Desmin was 94% and purification of cells was ideal. Growth curve of cells demonstrated that myoblasts which were characterized by high purification started proliferation plentiful through transient growth lag phase （about at one or two days after inoculation）. If myoblasts were not dealt with any interventions, they might become sarcotubule gradually at 3 - 5 days after proliferative phase. During this period, myoblasts maintained a monocaryon-bipolarity state under inverted phase contrast microscope. Furthermore, the growth of cells was the strongest and reproductive activity was the most powerful. This suggested that myotube started to form; in addition, muscle fiber of contractility might form under a well culturing condition. ②Immunocytochemical stain with desmin antibody： Interzonal fiber of desmin from myoblasts showed strongly positive reaction. Positive staining existed in cytoplasm had a high nucleus-cytoplasm ratio. However, myoblasts showed negative or mildly positive reaction. CONCLUSION： It is ideal for modified multi-enzymatic digestion and double purification method to dissociate and purify myoblasts of skeletal muscle; meanwhile, these two methods are both the effective ways to provide convenient conditions to obtain seed cells for neural regeneration researches.

MicroRNA bta-miR-365-3p inhibits proliferation but promotes differentiation of primary bovine myoblasts by targeting the activin A receptor type Ⅰ

Background: MicroRNAs act as post-transcriptional regulators that repress translation or degrade mRNA transcripts.Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically btamiR-365-3 p, in muscle biology, we investigated its functions in regulating primary bovine myoblast proliferation and differentiation.Results: Firstly, we found that bta-miR-365-3 p was predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting results showed that overexpression of btamiR-365-3 p significantly reduced the expression levels of cyclin D1(CCND1), cyclin dependent kinase 2(CDK2) and proliferating cell nuclear antigen(PCNA) but stimulated the expression levels of muscle differentiation markers, i.e.,MYOD1, MYOG at both mRNA and protein level. Moreover, downregulation of bta-miR-365-3 p increased the expression of CCND1, CDK2 and PCNA but decreased the expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining results showed that increased levels of bta-miR-365-3 p suppressed cell proliferation but promoted myotube formation, whereas decreased levels of bta-miR-365-3 p resulted in the opposite consequences. Finally, we identified that activin A receptor type I(ACVR1) could be a direct target of bta-miR-365-3 p. It was demonstrated that bta-miR-365-3 p can bind to the 3'UTR of ACVR1 gene to regulate its expression based on dual luciferase gene reporter assays.Consistently, knock-down of ACVR1 was associated with decreased expressions of CDK2, CCND1 and PCNA but increased expression of MYOG and MYOD1 both at mRNA and protein level.Conclusion: Collectively, these data suggested that bta-miR-365-3 p represses proliferation but promotes differentiation of bovine myoblasts through several biological mechanisms involving downregulation of ACVR1.

Exosomes derived from inflammatory myoblasts promote M1 polarization and break the balance of myoblast proliferation/differentiation

BACKGROUND Acute muscle injuries are one of the most common injuries in sports.Severely injured muscles are prone to re-injury due to fibrotic scar formation caused by prolonged inflammation.How to regulate inflammation and suppress fibrosis is the focus of promoting muscle healing.Recent studies have found that myoblasts and macrophages play important roles in the inflammatory phase following muscle injury;however,the crosstalk between these two types of cells in the inflammatory environment,particularly the exosome-related mechanisms,had not been well studied.AIM To evaluate the effects of exosomes from inflammatory C2C12 myoblasts(IFC2C12-Exos)on macrophage polarization and myoblast proliferation/differentiation.METHODS A model of inflammation was established in vitro by lipopolysaccharide stimulation of myoblasts.C2C12-Exos were isolated and purified from the supernatant of myoblasts by gradient centrifugation.Multiple methods were used to identify the exosomes.Gradient concentrations of IF-C2C12-Exos were added to normal macrophages and myoblasts.PKH67 fluorescence tracing was used to identify the interaction between exosomes and cells.Microscopic morphology,Giemsa stain,and immunofluorescence were carried out for histological analysis.Additionally,ELISA assays,flow cytometry,and western blot were conducted to analyze molecular changes.Moreover,myogenic proliferation was assessed by the BrdU test,scratch assay,and CCK-8 assay.RESULTS We found that the PKH-67-marked C2C12-Exos can be endocytosed by both macrophages and myoblasts.IF-C2C12-Exos induced M1 macrophage polarization and suppressed the M2 phenotype in vitro.In addition,these exosomes also stimulated the inflammatory reactions of macrophages.Further-more,we demonstrated that IF-C2C12-Exos disrupted the balance of myoblast proliferation/differentiation,leading to enhanced proliferation and suppressed fibrogenic/myogenic differentiation.CONCLUSION IF-C2C12-Exos can induce M1 polarization,resulting in a sustained and aggravated inflammatory environment that impairs myoblast differentiation,and leads to enhanced myogenic proliferation.These results demonstrate why prolonged inflammation occurs after acute muscle injury and provide a new target for the regulation of muscle regeneration.

Anti-Diabetic Activity of an Extract of Syzygium Jambolanum - A Review

Syzygium jambolanum is a promising natural treatment for diabetes.The potential benefits of S jambolanum for diabetes include lowering blood sugar levels,increasing insulin sensitivity,protecting pancreatic beta cells,and slowing the absorption of glucose into the bloodstream.The anti-diabetic activity of the crude extract of S jambolanum was evaluated in L6 myotubes and the lipid deposition in tissue was measured using Nile red Staining.Nile red staining confirmed that a considerable quantity of lipids had been deposited in the tissue treated with a crude extract of S jambolanum,comparable to the quantity of lipids deposited with a standard drug known as Rosiglitazone.This study analyzed the anti-diabetic activity of a crude extract of S jambolanum to understand its potential as a feedstock for extracting bioactive constituents to screen for bioactive molecules in the treatment of diabetes.

High expression of human clotting factor IX cDNA in myoblasts C2C12 cells and C3H mice

Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the poten-tial to be optimized as a safe and effective therapeutic modality for hemophilia B.

MicroR NA-21a enhances the expression of fibrosis-associated genes in TGF-β1 treated rat H9C2 myoblasts

Background H9c2 cell line is mononucleated myoblast derived from embryonic rat heart tissue. Activities of TGF-β1, MMP-2 and MMP-9 increase in H9c2 cells after treatment with fibrosis stimuli. MicroRNA （miRNA）, a kind of endogenous small non-coding RNA, participates in cardiac fibrosis. In the present study, expressions of fibrosis-related genes and microRNAs in TGF-β1treated H9c2 cells were investigated. Methods Expressions of fibrosis-associated genes, including Co13al, α-SMA, FN1, CTGF and TSP-1, were measured in TGF-β1 treated H9c2 cells by quantitative reverse transcription and PCR （qRT- PCR）. Level of α-SMA in H9c2 cells was demonstrated by fluorescence immunohistochemistry （FIHC） assay. Expressions of mature miR-16, -21a, -29b in H9c2 cells were determined by qRT-PCR assay. Activations of Smad3 and NF-kB signaling in TGF-β1-treated H9c2 cells were studied by dual luciferase assay. Expressions of Col3al, α-SMA, FN1, CTGF and TSP-1 were detected in H9c2 cells with adenovirus-mediated overexpression of miR-21a. Results qRT-PCR assay showed that ot-SMA, FN1, CTGF, TSP-1, but not Co13al, were up-regulated in TGF-β1treated H9c2 cells. FIHC result also revealed that α-SMA was increased in TGF-β1-treated H9c2 cells. Consistently, dual luciferase assay showed that Smad3 and NF-kB signaling proteins were activated in TGF-β1-treated H9c2 cells, miR-21a, but not miR- 16 and -29b, was significantly up-regulated. Additionally, over-expression of miR-21a significantly increases mRNA expressions of α-SMA, FN1, CTGF and TSP-1 in H9c2 cells. Conclusions MiR-21a is up-regulated in TGF-β1 treated H9c2 cells, and may contribute to up-regulations of fibrosis-associated genes.

Effects of transplanted myoblasts transfected with human growth hormone gene on improvement of ventricular function of rats

Background Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human growth hormone （hGH） has been shown to promote angiogensis and attenuate apoptosis in the experimental animal. This study was conducted to explore the effects of myoblast-based hGH gene therapy on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences between myoblast-based hGH gene therapy and myoblast therapy.Methods Myoblasts were isolated from several SD rats, cultured, purified, and transfected with plasmid pLghGHSN and pLgGFPSN. Radioimmunoassay （RIA） was used to detect the expression of hGH in these myoblasts. SD rats underwent the ligation of the left anterior descending coronary artery so as to establish a heart ischemia model. Thirty surviving rats that underwent ligation were randomly divided into 3 equal groups 2 weeks after left coronary artery occlusion： pLghGHSN group received myoblast infected with hGH gene transplantation; pLgGFPSN group received myoblast infected with GFP gene transplantation; control group： received cultured medium only. Four weeks after the injection the surviving rat underwent evaluation of cardiac function by echocardiography. The rats were killed and ventricular samples were undergone immunohistochemistry with hematoxylin-eosin and factor Ⅷ. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein. Reverse transcriptase-polymerase chain reaction （RT-PCR） was used to examine the mRNA expression of vascular endothelial growth factor （VEGF）, bax and Bcl-2. hGH expression in myocardium was examined by Western blot.Results Myoblast can be successfully isolated, cultured and transfected. The expression of hGH in transfected myoblast was demonstrated with RIA. Four weeks after therapy, the cardiac function was improved significantly in pLghGHSN group and pLgGFPSN group. Fractional shortening （FS） and ejection fraction （EF） in pLghGHSN group were elevated significantly compared with pLgGFPSN group and control group after therapy （FS： 36.9±5.3 vs 29.5±3.5, 21.8±2.9; EF： 56.9±4.3 vs 47.1±3.6, 38.4±4.8, P〈0.05）. Left ventricular end-diastolic dimension （LVEDD） and heart infracted size in pLghGHSN group were decreased significantly compared with pLgGFPSN group and control group after therapy （LVEDD： 5.9±0.3 vs 6.8±0.2, 8.6±0.3; heart infracted size： （34.5±4.2）% vs （40.0±3.9）%, （46.1±3.8）%, P〈0.05）; Green fluorescence was detected in cryosection of pLgGFPSN group. The capillary density of the pLgGFPSN group was significantly greater than those of the pLghGHSN group and control group （P〈0.05）. The mRNA expression of VEGF and Bcl-2/bax in pLghGHSN group was higher than in pLgGFPSN group or control group （P〈0.05）. The expression of hGH clene in myocardium tissue can be detected by Westem blot assay in pLghGHSN group.Conclusions Transplantation of heart cells transfected with hGH induced greater angiogenesis and effect of antiapoptosis than transplantation of cells transfected with GFP. Combined GH gene transfer and cell transplantation provided an effective strategy for improving postinfarction ventricular function.

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.

Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treat-ment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and mas-sive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42℃ for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and non- treated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analy-sis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treat-ment.

Identification of transition factors in myotube formation from proteome and transcriptome analyses

Muscle fibers are the main component of skeletal muscle and undergo maturation through the formation of myotubes.During early development,a population of skeletal muscle satellite cells(SSCs)proliferate into myoblasts.The myoblasts then undergo further differentiation and fusion events,leading to the development of myotubes.However,the mechanisms involved in the transition from SSCs to myotube formation remain unclear.In this study,we characterized changes in the proteomic and transcriptomic expression profiles of SSCs,myoblasts(differentiation for 2 d)and myotubes(differentiation for 10 d).Proteomic analysis identified SLMAP and STOM as potentially associated with myotube formation.In addition,some different changes in MyoD,MyoG,Myosin7 and Desmin occurred after silencing SLMAP and STOM,suggesting that they may affect changes in the myogenic marker.GO analysis indicated that the differentiation and migration factors SVIL,ENSCHIG00000026624(AQP1)and SERPINE1 enhanced the transition from SSCs to myoblasts,accompanied by changes in the apoptotic balance.In the myoblast vs.myotube group,candidates related to cell adhesion and signal transduction were highly expressed in the myotubes.Additionally,CCN2,TGFB1,MYL2 and MYL4 were identified as hub-candidates in this group.These data enhance our existing understanding of myotube formation during early development and repair.

Genetic Cell Therapy in Anti-Aging Regenerative Cosmetology

As post-WWII baby boomer approaching age 80, Anti-Aging Regenerative Cosmetology (AARC) has been developed and patented for beautifying and strengthening the human body using live cells;to enhance the appearance and function of various bodily parts to provide health and aestheticism of human being throughout life. It is a combined cosmetic and preventive medicine to intervene with and to correct the undesirable phenotypic expression of aging. The intrinsic properties of myoblasts and foreskin fibroblasts in development and regeneration are harnessed to formulate a genetic cell therapy program which is safe and efficacious as previously been tested in FDA Phase III clinical trials. Myoblasts are selected for strength development and foreskin fibroblasts for tenacity and smooth-to-the-touch. Both cell types are highly mitotic and non-carcinogenic. In additional to providing large quantities of nuclei as regenerative gene medicine, and of mitochondria as energy generators, myoblasts secret tumor necrosis factor alpha (TNF-α) for skin whitening and melanoma prevention. Myoblasts, because of their small size, spindle shape, and resilience, grow readily on collagen and laminin within wrinkles of skin surfaces, thus enhancing the color, luster, and texture of the skin “plated” with them. Alternatively, they can be injected subcutaneously as cell filler to reduce wrinkles. Intramuscular injection of myoblasts can augment the size, shape, consistency, tone, and strength of muscle groups, improving the lines, contours, and vitality to sculpt a youthful appearance. By improving cell genetics and organ functions, the program holds promise to sustain the human subject in good health and appearance, with a good quality of life and life prolongation.

Benefit of stem cells and skeletal myoblast cells in dilated cardiomyopathies

Although some authors suggest that there is mitotic division in the heart,most cardiomyocytes do not have the capacity to regenerate after myocardial infarction and when this occurs there is a deterioration of contractile function,and if the area of infarction is extensive ventricular remodeling may occur,leading to the development of heart failure.Cell transplantation into the myocardium with the goal of recovery of cardiac function has been extensively studied in recent years. The effects of cell therapy are based directly on the cell type used and the type of cardiac pathology.For myocardial ischemia in the hibernating myocardium, bone marrow cells have functional benefits,however these results in transmural fibrosis are not evident. In these cases there is a benefit of implantation with skeletal myoblasts,for treating the underlying cause of disease,the loss of cell contractility.

Skeletal myoblast based delivery of angiogenic growth factors:a comparison between angiopoietin-1 and VEGF gene delivery for therapeutic angiogenesis in the heart

Objectives This study investigated the efficacy of human skeletal myoblasts (SkM) mediated either human vascular endothelial growth factor-165 (hVEGF165) or angiopoietin-1 (Ang-1) on vascular development and myocardial regional perfusion. Methods A porcine heart model of chronic infarction was created in 28 female swine by coronary artery ligation. The animals were randomized into: (1) group-1, DMEM injected (n=6), (2) group-2, Ad-null transduced SkM transplanted (n=6), (3) group-3, Ad-hVEGF165 transduced SkM transplanted (n=8), and (4) group-4, Ad-Ang-1 transduced SkM (n=8). Three weeks later, 5 ml DMEM containing 3×108 SkM carrying exogenous genes were intramyocardially injected into 20 sites in left ventricle in groups-2, -3 and -4. Animals in group-1 were injected 5 ml DMEM without cells. Animals were kept on 5 mg/kg cyclosporine per day for 6 weeks. Regional blood flow was measured using fluorescent microspheres. The heart was explanted at 2, 6 and 12 weeks after transplantation for histological studies. Results Histological examination showed survival of lac-z expressing myoblasts in host tissue. Capillary density based on Von Willebrand factor-VIII (vWF-VIII) at low power field (×100) was 57.13±11.85 in group-3 at 6 weeks and declined to 32.1±5.21 at 12 weeks, while it was 39.9±10.26 at 6 weeks and increased to 45.14±6.54 at 12 weeks in group-4. The mature blood vessel index was highest in group- 4 at 6 and 12 weeks after transplantation. The regional blood flow in the center and peri-infarct area was significantly increased in animals of groups-3 and -4. Conclusions SkM carrying either hVEGF165 or Ang-1 induced neovascularization with increased blood flow. Ang-1 overexpression resulted in mature and stable blood vessel formation and may be a more potent arteriogenic inducer for neovascularization.