Effects of N-Oleoylglycine and Oleate on Mitochondrial UCP1-independent Thermogenesis / 中国生物化学与分子生物学报

Besides UCP1-dependent thermogenesis pathways, UCP1-independent thermogenesis pathways also could increase heat production in adipose tissue to combat obesity. N-Acyl amino acids (NAAs) have been suggested as novel endogenous uncouplers to induce mitochondria UCP1-independent thermogenesis in adipose tissue. Here, we use mouse skeletal muscle C2C12 cells which lack of UCP1 as UCP1 negative cell models. Comparing with its corresponding common fatty acid—oleate, one of the NAAs—N-Oleoylglycine (NOGly), which is highly expressed in the plasma of HFD mice, is selected to study their effects and mechanisms on mitochondrial thermogenesis. We found that 60 μmol / L oleate could induce mitochondrial oxidative phosphorylation protein levels, as well as increase mitochondria thermogenesis-related genes (COX8b, DIO2, UCP3) expression (P < 0. 05) . However, 60 μmol / L NOGly damaged the production and oxidative phosphorylation of mitochondria, significantly down-regulated expression of thermogenic genes (PGC1a, COX8b, COX2, DIO2, UQCRFS1and UCP3) (P< 0. 01), induced the production of reactive oxygen species (ROS) in the mitochondria, and enhanced the oxidative stress in cells. Our study found that oleate can induce UCP1-independent thermogenesis under 60 μmol / L addition dose, whereas NOGly does not due to the induction of oxidative stress in cells.

Action Mechanism of Main Components from Glycyrrhizae Radix et Rhizoma in Improving Insulin Resistance in L6 Rat Myoblasts / 中国实验方剂学杂志

Objective::To determine whether the main components of Glycyrrhizae Radix et Rhizoma can improve insulin resistance by regulating glycogen synthesis, glycolysis pathway and fatty acid synthesis in myoblasts of L6 rat myoblasts. Method::Insulin resistance (IR) model of L6 rat myoblasts was established through incubation with 0.05 mmol·L-1 palmitic acid (PA) for 9 hours. Normal group, model group, glycyrrhizic acid (GA, 25 μmol·L-1) group, 18β-glycyrrhetinic acid (18β-GA, 25 μmol·L-1) group, isoliquiritigenin (ILG, 25 μmol·L-1) group and isoliquiritin (ILQ, 25 μmol·L-1) group were set up, glucose content in supernatant of cell culture medium was detected by glucose kit, myoblasts glycogen content was determined by glycogen detection kit, protein expression levels of Sterol regulatory element binding protein-1c(SREBP-1c), fatty acid synthetase (FAS) and glycogen synthase kinase3β(GSK3β) were detected by Western blot, and the mRNA expressions of key enzymes in glycolysis were detected by quantitative real-time fluorescence polymerase chain reaction(Real-time PCR). Result::Compared with those in the normal group, the glucose consumption rate was significantly down-regulated in model group (P<0.01), the glycogen content was decreased (P<0.05), the protein expressions of Sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) were decreased (P<0.05, P<0.01), the mRNA expressions of fructose phosphate kinase 1 (PFK1), pyruvate kinase (PK) and hexokinase (HK) were down-regulated (P<0.05), and the protein expression of glycogen synthase kinase 3 (GSK3β) protein was increased (P<0.05). Compared with model group, GA, 18β-GA and ILG could significantly increase glycogen content in myoblasts of IR-L6 rats (P<0.05, P<0.01). GA, 18β-GA and ILQ could significantly increase the expression of SREBP-1c (P<0.05, P<0.01), and GA, 18β-GA, ILG and ILQ could significantly increase the expression of FAS (P<0.05, P<0.01), the mRNA expressions of PFK1, PK and HK (P<0.05, P<0.01), and down-regulate the protein expression of GSK3β (P<0.05). Conclusion::The main components of licorice improve the insulin resistance by promoting glycolysis and glycogen synthesis and regulating fatty acid synthesis.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca²⁺ signaling of differentiated C2C12 myotubes

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca²⁺]i transient and reduced sarcoplasmic reticulum (SR) Ca²⁺ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca²⁺-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca²⁺ signaling by downregulating the expression of DHPR and SERCA proteins.

Mitochondrial dysfunction reduces the activity of KIR2.1 K⁺ channel in myoblasts via impaired oxidative phosphorylation

Myoblast fusion depends on mitochondrial integrity and intracellular Ca²⁺ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with [Ca²⁺]i regulation in normal and mitochondrial DNA-depleted (ρ0) L6 myoblasts. The ρ0 myoblasts showed impaired myotube formation. The inwardly rectifying K⁺ current (I(Kir)) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated Ca²⁺ channel and Ca²⁺-activated K⁺ channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the I(Kir). The ρ0 myoblasts showed depolarized resting membrane potential and higher basal [Ca²⁺]ᵢ. Our results demonstrated the specific downregulation of I(Kir) by dysfunctional mitochondria. The resultant depolarization and altered Ca²⁺ signaling might be associated with impaired myoblast fusion in ρ0 myoblasts.

Molecular cloning and expression pattern of duck Six1 and its preliminary functional analysis in myoblasts transfected with eukaryotic expression vector.

Skeletal muscle development is regulated by Six1, an important myogenic transcription factor. However, the functional analysis of duck Six1 has not been reported. Here, we cloned the coding domain sequence (CDS) region of the duck Six1 gene using RT-PCR and RACE methods. Bioinformatics analysis revealed that duck Six1 CDS region comprised of 849 bp and encoded 282 amino acids and had a high degree of homology with other species, suggesting that the functions of duck Six1 gene are conserved among other animals. Real-time PCR used to determine the mRNA expression profiles of duck Six1 in different tissues and different developmental stages showed that Six1 was highly expressed in skeletal muscle and the embryonic stage. Furthermore, the eukaryotic expression vector pEGFP-duSix1 was constructed and transfected into the duck myoblasts; the MTT assay revealed an obvious increase of cell proliferation after transfection. The expression profiles of Six1, Myf5 and MyoD showed that their expression levels were significantly increased. These results together suggested that pEGFP-duSix1 vector was constructed successfully and overexpression of duck Six1 in the myoblasts could promote cell proliferation activity and significant up-regulate expression of Myf5 and MyoD.

Chemical Composition, Antioxidant Activities and Protective Effects of Sideritis italica Extract on C2C12 Oxidative Stress.

Aims: Sideritis italica is a medicinal plant used for medical purposes mainly based on experiences rather than scientific evidence. Biological properties, composition of primary and secondary metabolites as well as the antioxidant capacity were investigated on samples from wild plant. Methodology: The ultrastructure of aerial parts and quantitative distribution of pigments, including chlorophylls and amino acids, as well as the main class of secondary metabolites (phenols, flavonoids, flavonols and proanthocyanidins) were investigated. The extracts were tested by radical scavenging assays (DPPH, ABTS) and pharmacological assays (antiproliferative activity, effects on ROS production and protective effects against DNA damage induced by hydrogen peroxide) for their effects on C2C12 cell line. Results: Scanning electron microphotography confirms the presence of pharmacognostic characteristics, such as glandular and non-glandular trichomes on aerial parts. The chemical analysis indicates that the leaves are the most important part of the plant, and ethanol/water 70/30 is the preferable extraction solvent. The highest concentration of all metabolites was found in 70% ethanol extract of leaves. The antiradical assays and the in vitro tests on mouse myoblast cells C2C12 confirm the biological activities of the extract. C2C12 culture medium supplemented with extract, at doses (5-200μg/ml) not interfering with cell viability, was seen to modulate the ROS production and balance the increased oxidative stress induced by hydrogen peroxide. The treatment of C2C12 cells with 200 μg/ml of extract results in a percentage reduction of ROS of -60% and -71%, compared to untreated and H2O2 treated groups, respectively, P<.05. The quantitative reduction of 8- hydroxy-2’-deoxyguanosine (8-OH-dG), which is a biomarker of free radical DNA damage, confirms the protective effect of S. italica extract on oxidative stress at basal condition as well as in presence of exogenous stimuli (-11 and -7%, at 20μg/ml, respectively versus untreated and H2O2 groups, P<.05). Conclusion: The results obtained in the present study support the rational base for the medicinal use of plant and extracts in modulating the free radical metabolism and balancing the oxidative stress.

Autologous myoblasts transplantation improves heart function after myocardiac infarction / 中南大学学报(医学版)

Objective To determine whether autologous skeletal myoblasts implantation improves the cardiac function after myocardial infarction and the possible mechanism. Methods Myocardial infarction was induced by ligation of the left anterior descending coronary artery in rabbits. At 2 weeks, 1. 34 × 107 to 1.75 × 107 autologous skeletal myoblasts were infused into the lesion via direct intramuscular injection. In the control group, the postinfarction hearts were infused with medium alone. Buxco invasive cardiac function testing and histopathological examination were utilized to evaluate the functional and structural changes in the myocardium 4 weeks later. Results Both maximum rising rate of the left intraventricular pressure [+dp/dtmax,( 1 217.77 +89.91 )mmHg/s vs. (897.83 ±70.04) mmHg/s] and maximum falling rate of the left intraventricular pressure [- dp/dtmax,( -1174.58 ± 91.5 ) mmHg/s vs. ( - 753.67 ± 69.66 ) mmHg/s] were improved in the myoblast transplanted group compared with medium infusion group. The positive desmin immunostaining skeletal myofibers in the myocardium were found throughout the infracted areas and the border zone. Conclusion Autologous skeletal myoblasts can establish muscle tissue when transplanted into postinfarction hearts, and this mucle can treat myocardiac infarction effectively.

Muscle Regeneration: Research for the Treatment of Fecal Incontinence

Fecal incontinence remains a socially isolating condition, which can have a profound impact on all aspects of quality of life. It affects 2% to 17% of people living in the community and is an iatrogenic disease that develops after a restorative proctectomy for rectal cancer. Conservative management, such as biofeedback and medication, or surgical therapy may be ineffective, the symptomatic benefit being disappointing. In a few recent reports, autologous myoblasts injected into the urinary or anal sphincter were used successfully for the treatment of incontinence, and these cells improved the muscle function. These autologous cell therapies can avoid adverse events, such as tumor formation, compared to the use of embryonic stem cells. However, the limited regenerative capacity of cell therapy has prompted the development of replacing dysfunctional muscle tissue. Regenerative medicine for functioning muscles may be a therapeutic tool for fecal incontinence in the future. Now, many challenges remain to be overcome prior to reaching the ultimate goal of a fully functional 3-D vascularized engineered muscle: These include development of highly organized 3-D scaffolds, development of scaffolds that specifically direct cellular differentiation, development of co-culture systems of multiple cell types on smart surfaces, development of vascularized constructs, reduction of serum dependence, and innervation into constructed muscle. The successful generation of functional muscle tissues requires an in-depth knowledge of both muscle tissue physiology and advanced engineering practices. The recent advances in tissue engineering technique and cell biology suggest that artificially-derived muscle constructs may be used in clinical settings in the near future.

The possible role of Myosin light chain in myoblast proliferation

Skeletal muscles have the potential to regenerate by activation of quiescent satellite cells, however, the molecular signature that governs satellite cells during muscle regeneration is not well defined. Myosin light chains (Myls) are sarcomere-related proteins as traditional regulator of muscle contraction. In this report, we studied the possible role of Myl in the proliferation of skeletal muscle-derived myoblasts. Compared to diaphragm-derived myoblasts, the extraocular muscle-derived myoblasts with lower levels of Myl proliferated faster, maintained a longer proliferation phase, and formed more final myotubes. It was found that blockading Myl with anti-Myl antibody or knockdown of Myll by siRNA targeted against Myll could enhance the myoblast proliferation and delay the differentiation of myoblasts. Our results suggested that Myl, likely Myll, can negatively affect myoblast proliferation by facilitating myoblast withdrawal from cell cycle and differentiation.

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces alfa2beta1 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.

Transfection of rat myoblasts with leuflvirus carrying autocrine motility factor gene / 中华创伤杂志

Objective To explore a safe and high efficiency way of gene transfection of autocrine motility factor(AMF) in order to provide experimental basis for transplantation of myoblasts carrying AMF gone. Methods Sprague Dawley rat myoblasts were cultured, purified, proliferated and immunohisto-chemically verified. Then, the myoblasts were transfected with AMF and eGFP (enhanced green fluores-cent protein) gene by FIV (feline immunodeficiency virus). Fluorescence microscope and laser scanning confocal microscope were employed to detect eGFP so as to verify positive transfection rate. Expression of AMF was detected by immunohistochemical method. Results Myoblasts with 98% purity could he ob-tained after two weeks of primary culture and purification. Positive transfection rate reached 90.4% when MOI (multiplicity of infection) was 100 (P <0.01). The transfected AMF gene could express normally. Conclusions Explant culture is a proper way in rat myoblast culture. Meanwhile, AMF gene can he effectively transfected into rat myoblast and well expressed via FIV.

Dysferlin Expression in Skeletal muscles of Patients with Myopathy and Cultured Human Myoblast/Myotube

BACKGROUND: Dysferlin is a 230 kDa protein of the sarcolemma. This encoding gene is mutated in patients with dysferlinopathy (limb-girdle muscular dystrophy 2B and Miyoshi myopathy), which is characterized byan active muscle degeneration and regeneration process. Dysferlin is known to play an essential role in muscle signaling and muscle fiber repair. We studied the gene to define its functional role in muscle repair and differentiation in human skeletal muscle of the patients with myopathies and cultured human myoblast. METHODS: An immunohistochemical analysis of dysferlin and N-CAM in biopsied muscle tissue obtained from eleven patients with myopathies [six patients with Duchenne muscular dystrophy (DMD), two patients with dermatomyositis (DM), two patients with polymyositis (PM), and one patient with dysferlinopathy (MM)] and eight normal controls. Cultured human myoblast obtained from normal muscle tissue was also analyzed by the expression of dysferlin through immunocytochemical staining and western blot. RESULTS: The immunoreactivity of dysferlin was strongly expressed in regenerative muscle fibers of myopathies except dysferlinpathy, which was co-localization with N-CAM by double immunohistochemistry. By western blot analysis, the expression level of dysferlin was variable in myopathies compared to normal controls, but no expression in dyferlinopathy. The expression of dysferlin in myotubes was significantly increased compared to that in myoblast by immunostaining and western blot analysis. CONCLUSIONS: These results indicated that the expression of dysferlin increased in regenerative and degenerative muscle fibers and also increased in myoblast differentiation. Our study supports that dysferlin not only has a role in skeletal muscle development but also in regeneration/repair process.

Myoblast transplantation for heart repair: A review of the state of the field / 老年心脏病学杂志（英文版）

Over 200 humans have been treated with myoblast transplantation for heart muscle repair since June 2000. Bioheart sponsored percutaneous delivery studies began in May 2001 in Europe. Approximately one third of the patients have exhibited substantial improvement in left ventricular ejection fraction (LVEF) of over 30% and two heart failure class improvements. Over 80% of the patients have exhibited one heart failure class improvement with moderate improvement of LVEF. Clinical trials seem to demonstrate a marked reduction in emergency hospitalizations in myoblast treated patients. Many years of careful studies have lead to randomized controlled studies that are enrolling patients now at numerous centers worldwide. A firm conclusion on the safety and efficacy of myoblast transplantation cannot be determined until these randomized studies are completed. Final results from randomized controlled studies should be available soon. (J Geriatr Cardiol 2006;3:165-7.)

Primary culture of adipose-derived stem cells and differentiation induction into myoblasts / 上海第二医科大学学报

Objective To explore the method of culture of rat adipose-derived stem cells(ADSCs) and differentiation induction into myoblasts. Methods Adipose tissues were obtained from SD rats,and were isolated by enzyme digestion and cultured into ADSCs.The expression of surface antigen CD90,CD105 and CD34 was detected by immunofluorescence and flow cytometry.ADSCs of the second passage with logarithmic growth were obtained,and culture media containing 5-azacytidine(5-aza) and basic culture media were employed for cells in induction group and control group,respectively.The induction lasted for 7 d,14 d,21 d,28 d and 35 d,respectively.Cell growth and cell morphology were observed by inverted phase contrast microscope,and immunofluorescence and flow cytometry were utilized to detect the expression of myoblast specific antigens desmin and myosin. Results ADSCs were successfully isolated and cultured,and were identified to be stem cells.On the 28th day of induction,cells in induction group displayed "swirl" morpholgy,and multinucleation was observed.It was revealed by immunofluorescence and flow cytometry that the highest expression rates of desmin and myosin were 52.57% and 50.04%,respectively on the 28th day of induction,while there was no expression before induction and in control group. Conclusion ADSCs can be isolated and cultured from rat adipose tissues,and can further differentiate into myoblasts after induction by culture media with 5-aza.The expression of myoblast specific antigen is the highest on the 28th day of induction.

A Comparative Study of Magnetic-Activated Cell Sorting, Cytotoxicity and Preplating for the Purification of Human Myoblasts

Although cultured myoblast transplantation has been extensively studied as a gene complementation approach to muscular dystrophy treatment, clinical success has still been limited. The inability to adequately isolate and purify myoblasts presents a major limitation to the production of sufficient myoblasts for engrafting purposes. This study attempted to purify myoblasts from primary culture by magnetic-activated cell sorting (MACS), complement-mediated cytotoxicity, and a preplating technique. As a result of positive myoblasts selection by MACS, the average percentage of myoblasts in mixed culture was increased from 30.0% to 41.7%. We observed both myoblast lysis and fibroblast lysis after complement-mediated cytotoxicity. Enrichment of myoblasts in mixed culture was found to increase to 83.1% by using the preplating technique. In addition, higher purification (92.8%) was achieved by following the preplating technique with MACS. Thus, preplating in combination with magnetic-activated cell sorting allows for a rapid and effective isolation of myoblasts from human muscle tissue.

Studies on Differentiation of Cardiac Myoblast Induced by Co-culture with Isolated Neonatal Rat Cardiac Myocytes / 대한해부학회지

Cellular cardiomyoplasty has recently emerged as a potential new treatment of ischemic heart disease. Combining cellular cardiomyoplasty with gene therapy using myogenic transcription factor might facilitate myocardial regeneration. In this study, we engineered H9c2, L6 using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. Each cell overexpressing MEF2 and Nkx2.5 was generated by gene transfection and clonal selection. CO-culture was performed that each cell line added over cultured cardiomyocyte. H9c2-MEF2c and H9c2-Nkx2.5 became long, spindle shape like cardiomyocyte. Troponin T, cardiac specific marker, was found spot-like pattern in H9c2-Nkx2.5. However, co-culture with cardiomyocyte did not induce differentiation all kinds of cells into cardiomyocyte. Connexin43, which is gap junction marker was increased in H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c and L6-Nkx2.5. Especially, co-culture with cardiomyocyte resulted in elevation of connexin43 levels more than monoculture. Ultrastructurally, formations of gap junction and desmosome were found apparently in L6-Nkx2.5. Long-standing, strong, regular and more frequent contraction were observed in cardiomyocyte co-cultured with H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c, L6-Nkx2.5, respectively. Neverthless, any cell did not have active contraction itself, but passive movement except cardiomyocyte. H9c2-MEF2c, L6-MEF2c and L6-Nkx2.5 had resistance to hypoxia compared with other groups. These results suggested that co-culture and overexpressions of MEF2c and Nkx2.5 induced differentiation into cardiomyocyte and played an important role on intercellular junction formation and hypoxic resistance. This would be a promising source of cellular cardiomyoplasty. Therefore, much more research would be essential for clinical application of cellular cardiomyoplasty and this study would be a basic source for further study of MEF2c and Nkx2.5 in cellular cardiomyoplasty.

A Possible Role of Kainate Receptors in C2C12 Skeletal Myogenic Cells

Ca2+ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca2+ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in Ca2+ mobilization of muscle, and that Ca2+ influx is mediated by NMDA receptors in C2C12 myoblasts. Here, we report that other types of ionotropic glutamate receptors, non-NMDA receptors (AMPA and KA receptors), are also involved in Ca2+ influx in myoblasts. To explore which subtypes of non-NMDA receptors are expressed in C2C12 myogenic cells, RT-PCR was performed, and the results revealed that KA receptor subunits were expressed in both myoblasts and myotubes. However, AMPA receptor was not detected in myoblasts but expressed in myotubes. Using a Ca2+ imaging system, Ca2+ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular Ca2+ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular Ca2+ increase was completely suppressed by treatment of nifedifine, a L-type Ca2+ channel blocker. Furthermore, KA stimulated myoblast fusion in a dose-dependent manner. CNQX inhibited not only KA-induced myoblast fusion but also spontaneous myoblast fusion. Therefore, these results suggest that KA receptors are involved in intracellular Ca2+ increase in myoblasts and then may play an important role in myoblast fusion.

The Effect of Myoblast Carrying Human IGF-1 Injection on the Endogenous mIGF-1 mRNA Expression in Mice Skeletal Muscle following Injury / 中国运动医学杂志

Objective To observe the effect of the injection of myoblast carrying human insulin-like growth factor-1(hIGF-1) on the expression of endogenous IGF-1 mRNA and IGF-1 level in mice skeletal muscle following injury.Methods Seventy two male C3H mice(20~30g,7~11w)were randomly divided into three groups(24 mice in each group) with four mice normal controls.Applied a falling hit from certain height at the medial calf of right lower limbs in three groups,the injured skeletal muscle model was successfully simulated.Three days following injury,the mice in group A and B were injected with 1?106 myoblasts either carried with or without hIGF-1 gene respectively and the mice in group C were injected with 100?l saline at the injured muscle.Three mice in each group were sacrificed randomly at day 2,5,10,15,20,30 after contusion.The expression level of mIGF-1 was assessed by immunohistochemical staining and real time PCR.Results mIGF-1 mRNA expression and mIGF-1 factor secretion were observed in all three groups;the amount of mIGF-1 mRNA expression and mIGF-1 secretion in group A were significant higher than that in group B and C.Conclusion Myoblast carrying hIGF-1 transplantation could promote endogenous IGF-1 secretion in injured skeletal muscle.

Expression of Caveolin-3 in the Muscle Cell and Tissue

OBJECTIVE: Caveolae are the microdomain of the plasma membrane that have been implicated in signal transduction and caveolin is a principal component of the caveolae. Caveolin-3, a family of caveolin related protein, is expressed only in muscle tissue. Here we examined the expression of caveolin-3 in the course of myobalst differentiation and within the muscle tissue. METHOD: L6 cell, rat skeletal myoblast, was cultured in the low mitogen medium and caveolin-3 expression was observed both by immunocytochemistry and western blot analysis. Localization of caveolin-3 within the muscle tissue was investigated and compared to that of dystrophin. RESULTS: While caveolin-3 was not expressed in the proliferating myolast, caveolin-3 was expressed in the differentiated myoblast. Caveolin-3 and dystrophin were co-expressed in the membrane of muscle tissue and integrated density of caveolin-3 was elevated in the area of muscle injury. In the Duchenne muscular dystrophy, caveolin-3 was expressed in the membrane of muscle tissue, but dystrophin was not. CONCLUSION: Caveolin-3 was induced during the myobalst differentiation and its expression was increased during the muscle regeneration. Caveolin-3 was physically associated with dystrophin as a complex, but not absolutely required for the biogenesis of dystrophin complex.

The Expression of Cytokines and Chemokine mRNA by Human Skeletal Muscle Cell Line (SKM14)

BACKGROUND: Muscle is a target of immunological injury in several muscle diseases, such as idiopathic inflammatory myopathy. However, it is also a target for gene therapy. Therefore, it is important to understand the immunological capabilities of muscle cells. To assess as to whether muscle cells are actively involved in the inflamed muscle tissue, a human skeletal muscle cell line was tested for the expression of several cytokines and chemokine at the mRNA level. METHODS: A human skeletal muscle cell line (SKM14) had been developed by a retroviral vector encoding v-myc transfection into a 12-week-old human fetal skeletal muscle tissue characterized by the immunostaining of several musclespecific markers. Human skeletal myoblasts of this cell line were tested for their capacity to express different cytokines (IL-1beta, -6, -10, -12, -15, and TNF-alpha) and chemokine (IL-8) mRNA levels at the basal state and in the presence of TNF-alpha(10 ng/ml). RESULTS: The SKM14 cell line was confirmed to be able to express various cytokines constitutively (IL-6, -8, -12, -15, and TNF-alpha) and in the presence of TNF-alpha(IL-1beta, -6, -8, -10, -12, -15, and TNF-alpha). CONCLUSIONS: Our results suggest that muscle cells may play a role as immunocompetent cells.