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*

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

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

OBJECTIVE: To determine the improvements of post-infarction heart function after transplantation of autologous skeletal myoblasts transfected with VEGF165 in rabbits. METHODS: Myocardium infarction was induced in rabbits by left anterior descending coronary artery ligation. At 2 weeks, 1.75×10(7) autologous skeletal myoblasts transfected with pcDNA3.1-VEGF165 were infused into the region of MI via direct intramuscular injection; pcDNA3.1 served as a control. RESULTS: The DAPI-labeled and Desmin-positive immunostained skeletal myofibers were found throughout the infracted areas and border zones, and the density of blood capillary in the MI region transplanted by myoblasts with VEGF165 was increased (measured 4 weeks later and compared with controls). Heart function was examined by the Buxco system and demonstrated that maximum dp/dt [(1607.23±102.67) mmHg/s vs (1217.77±89.91) mmHg/s] and minimum dp/dt [(-1535.09 ± 81.34) mmHg/s vs (1174.58 ± 91.5) mmHg/s] were improved in the heart transplanted with the transfected myoblasts(P<0.05). CONCLUSION Autologous skeletal myoblasts transfected with VEGF165 could ameliorate the blood supply in the MI region, and aid recovery of heart function more quickly in post-infarction hearts. This suggests an effective treatment for myocardium infarction.
Animals ; Female ; Myoblasts, Skeletal ; metabolism ; transplantation ; Myocardial Infarction ; physiopathology ; therapy ; Rabbits ; Recovery of Function ; Transfection ; Transplantation, Autologous ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Ventricular Function, Left ; physiology

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.
Animals ; Cells, Cultured ; Gene Expression Regulation ; MEF2 Transcription Factors ; Mice ; Muscle, Skeletal ; metabolism ; Myoblasts ; cytology ; Myogenic Regulatory Factors ; genetics ; physiology ; Myostatin ; genetics ; physiology ; Promoter Regions, Genetic ; Swine

OBJECTIVETo explore the role of hypoxia in skeletal myoblasts proliferation and related mechanism.

METHODSThe numbers and proliferous indexes of skeletal myoblasts were detected by flow cytometer under 20%, 3%, and 10% oxygen concentration. Hypoxia inducing factor 1alpha (HIF-1alpha) mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). HIF-1alpha proteinum in endochylema and intranucelus were respectively detected by Western blot.

RESULTSThe numbers and proliferous indexes were higher (P < 0.05) in hypoxia group than those of control group. The expression HIF-1alpha mRNA had no difference in hypoxia and in normal groups. The level of HIF-1alpha proteinum in endochylema under normoxia was more than that in intranucelus and it was opposite under hypoxia.

CONCLUSIONHypoxia can promote the proliferation of skeletal myoblasts. The possible mechanism of hypoxia promoting the proliferation of skeletal myoblasts might be that low oxygen concentration regulates HIF-la nuclear translocation.

Animals ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Male ; Myoblasts, Skeletal ; cytology ; metabolism ; Oxygen ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo investigate changes in proliferative activity of myoblasts in skeletal muscle and potential role of phosphorylated Akt on it, so that a better understanding in mechanisms of skeletal muscle atrophy after burn injury will be got.

METHODSOne hundred and twenty Wistar rats were randomly divided into 2 groups: control and severe thermal injury group. Rats in severe thermal injury group were subjected to a 40% total body surface area full-thickness scald injury, and Tibialis Anterior (TA) muscles were collected on 0, 1, 4, 7, 10, 14 days post-injury. After muscle mass determined, immunohistochemical double staining was used for detection of Proliferative Cell Nuclear Antigen (PCNA) of myoblasts. Protein expression of total Akt and phosphorylated Akt was determined by Western Blot.

RESULTSBurn injury induced significant reduction of TA muscle mass and maximal reduction of it appeared by 4 days after injury (P < 0.01). Proliferative activity of myoblasts decreased significantly from the first day post-injury (P < 0.01) and increased slowly to basal level of controls after 7 days post-injury. The phosphorylated Akt was undetectable in both of controls and injured samples before 4 days but increased significantly after 7 days post-injury (P < 0.01), though total Akt expression had no significant alteration at any time points (P > 0.05).

CONCLUSIONSDecrease in proliferative activity of myoblasts may be one of the contributors of significant atrophy of skeletal muscle after burn injury. Effect of phosphorylated Akt on proliferation attenuated in early stage and increased significantly in later stage after burn injury may partly explain the changes in proliferative activity of myoblasts.

Animals ; Burns ; metabolism ; pathology ; Cell Proliferation ; Disease Models, Animal ; Male ; Muscle, Skeletal ; metabolism ; pathology ; Myoblasts ; metabolism ; pathology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.
Animals ; Calcium/metabolism ; Calcium Channels/metabolism ; Calcium Channels, L-Type/genetics/metabolism ; Cations/metabolism ; *Cell Differentiation ; *Cell Proliferation ; Cells, Cultured ; Excitation Contraction Coupling ; Gene Knockdown Techniques ; Membrane Potentials ; Mice ; Muscle Fibers, Skeletal/*metabolism ; Muscle Proteins/metabolism ; Myoblasts, Skeletal/*metabolism ; Ryanodine Receptor Calcium Release Channel/metabolism ; Sarcoplasmic Reticulum/*physiology ; Synaptophysin/metabolism ; TRPC Cation Channels/genetics/*metabolism ; Transient Receptor Potential Channels/metabolism

OBJECTIVETo study the modulatory effect of insulin on apoptosis of skeletal myoblast (L6 cells) by serum of scalded rat and its mechanism.

METHODSL6 cells cultured with DMEM medium containing 10% FBS were divided into control (C, added with 20% normal rat serum), serum from rat with scald injury (S, added with 20% serum from scalded rat), insulin (I, added with 20% normal rat serum and 100 nmol/L insulin), and serum of scalded rat + insulin (SI, added with 20% serum of scalded rat + 100 nmol/L insulin) groups according to the random number table. After being cultured for 48 hours, apoptosis was observed with Hoechst 33258 staining and its number counted, annexin V -FITC/PI double-labeling method was used to assess apoptosis rate, the protein levels of phosphorylated (p-) Akt, p-PI3K, Bax, Bcl-2, and active caspase-3 were determined by Western blotting. Data were processed with grouped or paired t test.

RESULTS(1) The amount of apoptosis with typical morphological change in S group [(59.6 +/- 3.9) per visual field] was more than that in C, I, and SI groups [(4.9 +/- 2.6), (5.5 +/- 2.1), (19.7 +/- 2.3) per visual field, with t value respectively 28.53, 29.86, 21.53, P values all below 0.01]. (2) Apoptotic rate in S group was (18.5 +/- 1.8)%, which was markedly higher than that in C, I, and SI groups [(1.1 +/- 0.6)%, (1.5 +/- 0.3)%, (7.8 +/- 0.6)%, with t value respectively 22.41, 22.83, 13.92, P values all below 0.01]. (3) Compared with those in C group, the protein levels of Bax and active caspase-3 in S group were up-regulated (1.12 +/- 0.63 vs. 0.16 +/- 0.03, 2.15 +/- 0.51 vs. 0.21 +/- 0.03, with t value respectively 3.80, 10.69, P values all below 0.01), the protein level of p-Akt was lowered (0.20 +/- 0.03 vs. 0.42 +/- 0.07, t = -8.46, P < 0.01), and the protein levels of p-PI3K and Bcl-2 showed no statistical difference (0.19 +/- 0.03 vs. 0.26 +/- 0.09, 0.17 +/- 0.03 vs. 0.28 +/- 0.07, with t value respectively -2.73, - 1.14, P values all above 0.05). The protein levels of Bax (0.40 +/- 0.14) and active caspase-3 (0.83 +/- 0.18) in SI group were lowered (t = -3.23, P < 0.05; t = 6.66, P < 0.01) and the protein levels of p-Akt, Bcl-2, and p-PI3K in SI group were elevated (0.39 +/- 0.10, 0.78 +/- 0.03, 0.47 +/- 0.12, with t value respectively 4.07, 18.71, 5.05, P < 0.05 or P < 0.01) as compared with those in S group.

CONCLUSIONSSerum from scalded rat can induce apoptosis in skeletal myoblast, and the effect can be inhibited by insulin through PI3K/Akt signal pathway.

Animals ; Apoptosis ; drug effects ; Burns ; blood ; metabolism ; pathology ; Caspase 3 ; metabolism ; Cell Line ; Insulin ; pharmacology ; Male ; Myoblasts, Skeletal ; cytology ; drug effects ; pathology ; Rats ; Rats, Wistar ; Serum ; immunology ; Signal Transduction ; bcl-2-Associated X Protein ; metabolism

Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. This study investigated the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 microgram/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group. A histological examination at the postoperative 7th day revealed that the rhEGF treatment increased the number of proliferating nuclear antigen immunoreactive cells in the epidermis layer. In addition, the immunoreactive area of alpha-smooth muscle actin and the expression of prolyl 4-hydroxylase were significantly higher than those of the control group. Overall, a topical treatment of rhEGF ointment promotes wound healing by increasing the rate of epidermal proliferation and accelerating the level of wound contraction related to myofibroblast proliferation and collagen deposition.
Actins/genetics/metabolism ; Administration, Topical ; Animals ; Cell Proliferation/drug effects ; Collagen/*biosynthesis ; Epidermal Growth Factor/*administration&dosage/*pharmacology ; Gene Expression Regulation ; Male ; Myoblasts, Skeletal/*drug effects ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Wound Healing/*drug effects