Severe muscle injury is hard to heal and always results in a poor prognosis. Recent studies found that extracellular vesicle-based therapy has promising prospects for regeneration medicine, however, whether extracellular vesicles have therapeutic effects on severe muscle injury is still unknown. Herein, we extracted apoptotic extracellular vesicles derived from mesenchymal stem cells (MSCs-ApoEVs) to treat cardiotoxin induced tibialis anterior (TA) injury and found that MSCs-ApoEVs promoted muscles regeneration and increased the proportion of multinucleated cells. Besides that, we also found that apoptosis was synchronized during myoblasts fusion and MSCs-ApoEVs promoted the apoptosis ratio as well as the fusion index of myoblasts. Furthermore, we revealed that MSCs-ApoEVs increased the relative level of creatine during myoblasts fusion, which was released via activated Pannexin 1 channel. Moreover, we also found that activated Pannexin 1 channel was highly expressed on the membrane of myoblasts-derived ApoEVs (Myo-ApoEVs) instead of apoptotic myoblasts, and creatine was the pivotal metabolite involved in myoblasts fusion. Collectively, our findings firstly revealed that MSCs-ApoEVs can promote muscle regeneration and elucidated that the new function of ApoEVs as passing inter-cell messages through releasing metabolites from activated Pannexin 1 channel, which will provide new evidence for extracellular vesicles-based therapy as well as improving the understanding of new functions of extracellular vesicles.
Creatine/metabolism* ; Extracellular Vesicles ; Muscle, Skeletal/metabolism* ; Myoblasts/metabolism* ; Regeneration ; Connexins/metabolism*

Animals ; Cell Differentiation ; physiology ; Cell Hypoxia ; Cell Line ; Mice ; Myoblasts ; cytology ; Transcription Factors ; metabolism

OBJECTIVE: To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. METHODS: Firstly, hyaluronic acid was oxidized with NaIO ₄ and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P<0.05). CONCLUSION The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.
Hydrogels ; Hyaluronic Acid/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Tissue Engineering/methods* ; Cell Differentiation ; Myoblasts/metabolism* ; Cell Proliferation

AIMTo study the effects of PKC activation on apoptosis during ischemia/reperfusion in L-6TG rat skeletal myoblasts.

METHODSCultured L-6TG cells were divided into 3 groups: control group (C), ischemia/reperfusion group (I/R), PMA + ischemia/ reperfusion group (PMA), SOD, XOD and free calcium and mitochondrial respiration in L-6TG cell were evaluated in each group. Apoptosis was detected by flow cytometer with PI staining method and agarose gel electrophoresis, the immunohistochemical method was used to determine the expression of caspase-3.

RESULTSCompared with I/R group, in PMA group, XOD , free calcium in L-6TG cell and apoptotic percentage all decreased significantly, while SOD and mitochondrial respiration in L-6TG cell increased. DNA fragmentation analysis of L-6TG cell showed no laddering pattern. The expression of caspase-3 was down regulated significantly.

CONCLUSIONActivation of PKC can lessen ischemia/reperfusion injury and apoptosis through lessening oxidative injury and mitochondrial injury, adjusting calcium dyshomeostasis and down expression of caspase-3.

Animals ; Apoptosis ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cells, Cultured ; Mitochondria ; metabolism ; Myoblasts, Skeletal ; cytology ; metabolism ; Oxidative Stress ; Protein Kinase C ; metabolism ; Rats ; Reperfusion Injury ; metabolism ; pathology

OBJECTIVETo observe human tumor necrosis factor-alpha (hTNF-alpha) expression and secreting level of human embryo myoblasts transfected by hTNF-alpha gene.

METHODSHuman embryo myoblasts were transfected with shuttle plasmid pSV23SHTNF containing hTNF-alpha gene by cationic liposomes DOSPER. The control group was only given equivalent liposomes except plasmid. After culturing for 24, 48, 72 and 96 hours, hTNF-alpha expression level of human embryo myoblasts was observed with immunocytochemistry staining, and hTNF-alpha secreting of human embryo myoblasts was analyzed by ELISA.

RESULTSAfter transfected by hTNF-alpha gene for 24, 48, 72 and 96 hours, the human embryo myoblasts displayed significant secretion of hTNF-alpha in the cultural supernatant (P < 0.05), and overexpression in cytoplasma and cell membrane.

CONCLUSIONTransfection of hTNF-alpha gene to human myoblasts made myoblasts secrete high concentration of hTNF-alpha, implying it is feasible that transfecting muscle cells surrounding tongue carcinoma lesion with hTNF-alpha gene can prevent tongue carcinoma from intruding into deeper muscle tissue.

Animals ; Genetic Therapy ; Humans ; Myoblasts ; Neoplasm Invasiveness ; prevention & control ; Plasmids ; Tongue Neoplasms ; therapy ; Transfection ; Tumor Necrosis Factor-alpha ; metabolism

AIMTo investigate the effects of pravastatin on endothelin(ET) expression induced by aldosterone in cultured neonatal rat cardiac fibroblasts.

METHODSET concentration in conditioned medium was measured by radioimmunoassay, intracellular ET-1 level was evaluated by flow cytometry, and the expression of preproendothelin-1 (ppET-1) was detected and quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) method.

RESULTSThe cardiac fibroblasts, treated with aldosterone at 107 mol/L, significantly up-regulated ppET-1 mRNA expression, as well as ET-1 synthesis and release. Pravastatin (10(-5), 10(-4), 10(-3) mol/L) dose-dependently blocked these effects. In contrast, pravastatin-induced inhibitory effects were reversed in the presence of mevalonate.

CONCLUSIONPravastatin down-regulated ppET-1 mRNA expression, as well as ET-1 synthesis and release induced by aldosterone in a process specifically related to mevalonate in cardiac fibroblasts.

Aldosterone ; metabolism ; Animals ; Cells, Cultured ; Endothelins ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Myoblasts, Cardiac ; drug effects ; metabolism ; Pravastatin ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the effect of oxidative stress on the accumulation of heat shock protein 70 (HSP70) within C2C12 myogenic cells. METHODS: Heat shock response (42 degrees C for 1 h and recovery for 12 h at 37 degrees C) was used to induce the expression of heat shock protein 70. We constructed a recombinant plasmid of HSP70 with enhanced green fluorescent protein (EGFP). After being transfected transiently into C2C12 cells, immunoblotting was used to detect the expression of HSP70 induced by heat shock response and transfection. Immunocytochemistry, fluorescent microscopy and immunoblotting were used to detect the translocation of HSP70. RESULTS: Immunoblotting showed that the overexpression of HSP70 was induced by heat shock response and transient transfenction. HSP70 localized within the cytoplasm of the normal cells, but HSP70 translocated from the cytoplasm to the nucleus and the nucleolus at 1 h after the treatment of oxidative stress (0.5 mmol/L H2O2) by using immunocytochemistry, fluorescent microscopy and immunoblotting for cellular partial proteins. CONCLUSION Oxidative stress may induce the accumulation of heat shock protein 70 within the nucleolus.
Cell Nucleolus ; metabolism ; Cells, Cultured ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Myoblasts ; cytology ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Oxidative Stress ; physiology

AIMTo study the effect of thyroid hormone on protein kinase C activity and isoprotein expressions in cardiac myocytes and fibroblasts of rats in vitro.

METHODSCardiac myocytes and fibroblasts were cultured according to the method of Simpson. Cells were pretreated with 1% newborn calf serum (NCS) or Angiotensin II (Ang II) for 24 hours, then Triiodothyronine (T3) was added to the culture medium and the culture was kept for another 48 hours. The protein kinase C activation were measured by PepTaga non-radioactive PKC assay, and the expressions of PKC alpha and PKC epsilon were detected by Western blot method.

RESULTSAt the condition of 1% NCS culture medium, T3 could inhibit PKC activity and PKC epsilon expression in cardiac myocytes significantly, but the expression of PKC alpha in cardiac myocytes was not influenced by T3. In cardiac fibroblasts, neither PKC activity nor PKC alpha and PKC epsilon expressions was influenced by T3. When cells were pretreated with Ang II for 24 hours, PKC activities in cardiac myocytes and fibroblasts were increased significantly, and PKC epsilon expressions in cardiac myocytes were also markedly increased. Following a T3 treatment, PKC activity and PKC epsilon expression in cardiac myocytes were markedly decreased, but PKC activity in cardiac fibroblasts was not changed.

CONCLUSIONWhether at the condition of 1% NCS medium or in a pretreatment with Ang II, thyroid hormone could inhibit the PKC activity and PKC epsilon expression in cardiac myocytes. The influence of thyroid hormone on the PKC signal pathway in cardiac myocyte may be involved in many pathophysiological progress of myocardium.

Animals ; Cells, Cultured ; Myoblasts, Cardiac ; drug effects ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Thyroid Hormones ; pharmacology

OBJECTIVE: To apply hypoxia of different oxygen concentration on C2C12 cells to study the changes of Nrf2 antioxidant system under HO. METHODS: The perfect simulative effect time and concentration of HO were chosen. Cell vitality was tested after C2C12 cells cultured in 0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 0.75 mmol/L, 1 mmol/L and 2 mmol/L HO for 1 or 2 h respectively. The C2C12 cells were divided into different oxygen concentration group: 21%O, 12%O, 8%O, 5%O respectively. And then cells were treated with HO for 1 h, and collected for determination. Immunofluorescence of Nrf2 and the protein expression of Nrf2 were detected. The expressions of antioxidant enzymes superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase(CAT), NADPH quinine oxidoreductase-1 (NQO-1), glutathione peroxidase-1 (GPX-1), Heme oxygenase-1 (HO-1) mRNA and cellular ROS levels were tested by high quality fluorescence assay. RESULTS: 0.5 mmol/L HO for 1 h was selected as the conditions of HOstimulation. Compared with 21% O group, the expressions of Nrf2 mRNA and protein, antioxidant enzymes SOD1, SOD2, CAT, HO-1, NQO-1, GPX-1 mRNA were increased significantly (P＜0.05 or P＜0.01), and ROS level was lower (P＜0.01) in 12%O group cells; only the expression of GPX-1 mRNA was increased (P＜0.05) in 8%O group; the expressions of Nrf2 mRNA and protein expression, antioxidant enzymes SOD1, SOD2, NQO-1, GPX-1 mRNA were decreased significantly(P＜0.05 or P＜0.01), and ROS level was higher (P＜0.01) in 5%O group. CONCLUSION Hypoxia can affect the Nrf2 antioxidant system, and the different oxygen concentrations have different impact. In addition, 12% O for 12 h could promote the Nrf2 antioxidant system, and 5% extremely low oxygen may inhibit it.
Animals ; Antioxidants ; metabolism ; Cell Line ; Cell Survival ; Hydrogen Peroxide ; Mice ; Myoblasts ; enzymology ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; Oxygen ; Reactive Oxygen Species ; metabolism

The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism and cell differentiation. However, its role in myoblast differentiation is unclear. To study the biological function of FoxO1 during differentiation in porcine primary myoblast, we constructed stably FoxO1 over-expressed porcine myoblast mediated by liposome and adopted morphological observation, quantitative real-time RT-PCR and Western blotting methods to analyze FoxO1 and early and late myogenic regulation factors MyoD and myogenin expression. During differentiation the mRNA level of FoxO1 was significantly increased. However, the total protein did not change but the phosphorylation of FoxO1 was upregulated. Furthermore, overexpression of FoxO1 in porcine myoblast decreased MyoD and myogenin mRNA, whereas MyoD protein changed little and myogenin was significantly suppressed (P < 0.05). These results indicated that FoxO1 delays and negatively regulates the porcine myoblast differentiation. Moreover, FoxO1 may play a critical role in muscle fiber-type specification through the inhibition of myogenic regulation factors.
Animals ; Animals, Newborn ; Cell Differentiation ; genetics ; Cells, Cultured ; Forkhead Transcription Factors ; biosynthesis ; genetics ; Muscle, Skeletal ; cytology ; metabolism ; Myoblasts ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Swine