Effects of electroacupuncture on gait and proliferation and differentiation of muscle satellite cell in rats with acute blunt trauma of gastrocnemius muscle / 中国针灸

OBJECTIVE@#To observe the effects of electroacupuncture on threshold of pain, gait, proliferation and differentiation of muscle satellite cell in rats with acute blunt trauma of gastrocnemius muscle, and to explore the possible mechanism of electroacupuncture in promoting the repair of acute injury of skeletal muscle.@*METHODS@#A total of 48 SD rats were randomly divided into a blank group (6 rats), a model group (24 rats) and an electroacupuncture group (18 rats). In the model group and the electroacupuncture group, the model of acute blunt trauma of gastrocnemius muscle was established by self-made impactor. In the electroacupuncture group, electroacupuncture was applied at "Chengshan" (BL 57) and "Yanglingquan" (GB 34) on the right side, with disperse-dense wave, in frequency of 2 Hz/100 Hz, once a day, 30 min each time. Electroacupuncture intervention was performed for 3, 7 and 14 days according to the sampling time. On the 1st, 3rd, 7th and 14th days after modeling, the mechanical withdrawal pain threshold of hindfoot was detected by Von Frey method; the standing time and the maximum contact area of the right hindfoot were recorded by Cat Walk XTTM animal gait analysis instrument; the morphology of the right gastrocnemius muscle and the number of inflammatory cells were observed by HE staining; the positive expression of paired box gene 7 (Pax7) and myogenic differentiation (MyoD) of the right gastrocnemius muscle was detected by immunofluorescence.@*RESULTS@#After modeling, the muscle fiber rupture and massive infiltration of red blood cells and inflammatory cells were observed in the right gastrocnemius muscle; after electroacupuncture intervention, the morphology of muscle fiber was intact and the infiltration of inflammatory cells was improved. Compared with the blank group, in the model group, the differences of mechanical withdrawal pain threshold between the left and right foot were increased (P<0.05), the standing time was shortened and the maximum contact area of the right hindfoot was decreased (P<0.05), the number of inflammatory cells and the positive expression of Pax7 and MyoD of the right gastrocnemius muscle were increased (P<0.05) on the 1st, 3rd, 7th and 14th days after modeling. Compared with the model group, in the electroacupuncture group, the differences of mechanical withdrawal pain threshold were decreased (P<0.05), the standing time was prolonged (P<0.05), the number of inflammatory cells of right gastrocnemius muscle was decreased (P<0.05) on the 7th and 14th days after modeling; the maximum contact area of the right hindfoot was increased (P<0.05), the positive expression of MyoD of the right gastrocnemius muscle was increased (P<0.05) on the 3rd, 7th and 14th days after modeling; the positive expression of Pax7 of the right gastrocnemius muscle was increased (P<0.05) on the 3rd day after modeling.@*CONCLUSION@#Electroacupuncture can effectively improve the pain threshold and gait in rats with acute blunt trauma of gastrocnemius muscle, and promote the repair of skeletal muscle injury, the mechanism may be related to the up-regulation of Pax7 and MyoD, so as to promoting the proliferation and differentiation of muscle satellite cell.

Expression of EGR1 gene and location of EGR1 protein in differentiation of bovine skeletal muscle-derived satellite cells / 中国应用生理学杂志

OBJECTIVE@#To investigate the expression of EGR1 gene and the localization of EGR1 protein in bovine skeletal muscle-derived satellite cells (MDSCs), as well as to investigate the mechanism that EGR1 protein enters the nucleus.@*METHODS@#Bovine MDSCs were cultured in differentiation medium for 1 day, 3 days and 5 days, respectively, and each group was triplicate. The expression of EGR1 gene and the localization of EGR1 protein were studied at different differentiation period in MDSCs by qRT-PC and Western blot. Moreover, the changes on the expression of endogenous EGR1 gene and EGR1 proteins were explored by CRISPRi, site-directed mutagenesis and laser confocal method.@*RESULTS@#The results from the qRT-PCR and Western blot showed that the expressions of EGR1 gene on transcription level and translation level were significantly higher in differentiated cells than those in undifferentiated cells. The highest expression was found on the third day after the differentiation, and then began to decline. Immunofluorescence assays showed that EGR1 proteins were preferentially expressed in differentiated MDSCs, and increased along with the increase of number of myotubes. Confocal observation revealed that some EGR1 proteins were transferred into the nucleus in the differentiation of cells, however, the EGR1 proteins would not be detected in the differentiated MDSCs nuclei if a site directed mutagenesis (serine) on EGR1 protein occurred.@*CONCLUSION@#During the differentiation of bovine skeletal muscle satellite cells, the transcriptional level of EGR1 gene is increased, and some EGR1 proteins are transferred into the nucleus. The serine phosphorylation at position 533 of the C terminal of EGR1 protein is necessary for the nucleus transfer.

Effect of a combination of astaxanthin supplementation, heat stress, and intermittent reloading on satellite cells during disuse muscle atrophy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We examined the effect of a combination of astaxanthin (AX) supplementation, repeated heat stress, and intermittent reloading (IR) on satellite cells in unloaded rat soleus muscles. Forty-nine male Wistar rats (8-week-old) were divided into control, hind-limb unweighting (HU), IR during HU, IR with AX supplementation, IR with repeated heat stress (41.0-41.5 °C for 30 min), and IR with AX supplementation and repeated heat stress groups. After the experimental period, the antigravitational soleus muscle was analyzed using an immunohistochemical technique. Our results revealed that the combination of dietary AX supplementation and heat stress resulted in protection against disuse muscle atrophy in the soleus muscle. This protective effect may be partially due to a higher satellite cell number in the atrophied soleus muscle in the IR/AX/heat stress group compared with the numbers found in the other groups. We concluded that the combination treatment with dietary AX supplementation and repeated heat stress attenuates soleus muscle atrophy, in part by increasing the number of satellite cells.

Implantation of muscle satellite cells overexpressing myogenin improves denervated muscle atrophy in rats

This study evaluated the effect of muscle satellite cells (MSCs) overexpressing myogenin (MyoG) on denervated muscle atrophy. Rat MSCs were isolated and transfected with the MyoG-EGFP plasmid vector GV143. MyoG-transfected MSCs (MTMs) were transplanted into rat gastrocnemius muscles at 1 week after surgical denervation. Controls included injections of untransfected MSCs or the vehicle only. Muscles were harvested and analyzed at 2, 4, and 24 weeks post-transplantation. Immunofluorescence confirmed MyoG overexpression in MTMs. The muscle wet weight ratio was significantly reduced at 2 weeks after MTM injection (67.17±6.79) compared with muscles injected with MSCs (58.83±5.31) or the vehicle (53.00±7.67; t=2.37, P=0.04 and t=3.39, P=0.007, respectively). The muscle fiber cross-sectional area was also larger at 2 weeks after MTM injection (2.63×103±0.39×103) compared with MSC injection (1.99×103±0.58×103) or the vehicle only (1.57×103±0.47×103; t=2.24, P=0.049 and t=4.22, P=0.002, respectively). At 4 and 24 weeks post-injection, the muscle mass and fiber cross-sectional area were similar across all three experimental groups. Immunohistochemistry showed that the MTM group had larger MyoG-positive fibers. The MTM group (3.18±1.13) also had higher expression of MyoG mRNA than other groups (1.41±0.65 and 1.03±0.19) at 2 weeks after injection (t=2.72, P=0.04). Transplanted MTMs delayed short-term atrophy of denervated muscles. This approach can be optimized as a novel stand-alone therapy or as a bridge to surgical re-innervation of damaged muscles.

Protective effect of ATP on skeletal muscle satellite cells damaged by H₂O₂ / 华中科技大学学报（医学）（英德文版）

This study investigated the protective effect of ATP on skeletal muscle satellite cells damaged by H₂O₂in neonatal rats and the possible mechanism. The skeletal muscle satellite cells were randomly divided into four groups: normal group, model group (cells treated with 0.1 mmol/L H₂O₂for 50 s), protection group (cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h, and then with 0.1 mmol/L H₂O₂for 50 s), proliferation group (cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h). MTT assay, FITC+PI+DAPI fluorescent staining, Giemsa staining and immunofluorescence were performed to examine cell viability and apoptosis, and apoptosis-related proteins. The results showed that the survival rate of skeletal muscle satellite cells was decreased and the apoptosis rate was increased after H₂O₂treatment (P<0.01). Different doses of ATP had different effects on skeletal muscle satellite cells damaged by H₂O₂: the survival rate of muscle satellite cells treated with ATP at 4, 2, or 1 mmol/L was increased. The protective effect was most profound on cells treated with 2 mmol/L ATP. Immunofluorescence showed that ATP could increase the number of Bcl-2-positive cells (P<0.01) and decrease the number of the Bax-positive cells (P<0.01). It was concluded that ATP could protect skeletal muscle satellite cells against H₂O₂damage in neonatal rats, which may be attributed to the up-regulation of the expression of Bcl-2 and down-regulation of Bax, resulting in the suppression of apoptosis.

Recent Advances in Skeletal Muscle Stem Cells for Duchenne Muscular Dystrophy Treatment / 한양의대학술지

Muscle stem cells, which are known as satellite cells have heterogeneous components of committed myogenic progenitors, non-committed satellite cells, and mesenchymal stem cells. This distinguishing organization of self-renewal and differentiation capacities encourages the remarkable regenerative ability of skeletal muscles. Lately it has been proved that the satellite cell is the derivation of muscle regeneration and with the self-renew function, it roles as a true muscle stem cell. Therefore, stem cell therapy using satellite cells is considered to be ideal therapy for muscular dystrophies, which is deficient in specific muscle protein and causes muscle degeneration. Especially, Duchenne Muscular Dystrophy (DMD), which is caused by mutations at the dystrophin gene, has been targeted by much research. In this article the satellite cell characteristics, regulation of cell function, and stem cell therapy for DMD and the present progressive clinical trials will be reviewed.

Ultrassom terapêutico na atividade mitocondrial e diferenciação de células musculares C2C12 / Therapeutic ultrasound in the mitochondrial activity and differentiation of muscle cells C2C12

Introdução: O ultrassom terapêutico (US) é muito utilizado na prática clínica, mas há poucos estudos sobre seu efeito na regeneração muscular. Objetivo: Avaliar os efeitos do US sobre a atividade mitocondrial e diferenciação de células musculares C2C12, quando aplicado concomitantemente à indução do processo de diferenciação. Métodos: As células musculares foram submetidas ao processo de diferenciação pela adição de meio de cultura DMEM, suplementado com 2% de soro de cavalo, e receberam simultaneamente tratamento com US (pulsado a 20%, 3 MHz, 0,2 e 0,5 W/cm², 5 minutos). A atividade mitocondrial foi avaliada após 24h, 48h e 96h pelo método MTT, e a diferenciação celular após um e três dias pela atividade de creatina quinase (CK). Resultados: Não houve alteração da atividade mitocondrial e de CK nos grupos que receberam tratamento com US nos diferentes períodos avaliados. Conclusão: O US, nos parâmetros avaliados, não foi capaz de alterar a atividade mitocondrial e a diferenciação de células musculares C2C12.

Introduction: The therapeutic ultrasound (US) has been widely used in clinical practice, but there are few studies on its effect on muscle regeneration. Objective: To evaluate the effects of the US on mitochondrial activity and differentiation of muscle cells C2C12 when applied concomitantly the induction of the differentiation process. Methods: Muscle cells were subjected to differentiation process by addition of DMEM culture medium supplemented with 2% horse serum and received concomitant treatment with US (pulsed at 20%, 3 MHz, 0.2 and 0.5 W/cm², 5 minutes). The mitochondrial activity was assessed after 24, 48 and 96 hours by MTT assay and cell differentiation after one and three days for the activity of creatine kinase (CK). Results: There was no change in mitochondrial activity and CK in the groups receiving US treatment in different periods. Conclusion: In the evaluated parameters, the US was not able to change mitochondrial activity and differentiation of muscle cells C2C12.

Accelerated regeneration of the skeletal muscle in RNF13-knockout mice is mediated by macrophage-secreted IL-4/IL-6

RING finger protein 13 (RNF13) is a newly identified E3 ligase reported to be functionally significant in the regulation of cancer development, muscle cell growth, and neuronal development. In this study, the function of RNF13 in cardiotoxin-induced skeletal muscle regeneration was investigated using RNF13-knockout mice. RNF13(-/-) mice exhibited enhanced muscle regeneration-characterized by accelerated satellite cell proliferation-compared with wild-type mice. The expression of RNF13 was remarkably induced in macrophages rather than in the satellite cells of wild-type mice at the very early stage of muscle damage. This result indicated that inflammatory cells are important in RNF13-mediated satellite cell functions. The cytokine levels in skeletal muscles were further analyzed and showed that RNF13(-/-) mice produced greater amounts of various cytokines than wild-type mice. Among these, IL-4 and IL-6 levels significantly increased in RNF13(-/-) mice. The accelerated muscle regeneration phenotype was abrogated by inhibiting IL-4/IL-6 action in RNF13(-/-) mice with blocking antibodies. These results indicate that RNF13 deficiency promotes skeletal muscle regeneration via the effects on satellite cell niche mediated by IL-4 and IL-6.

Molecular regulation of skeletal satellite cell's self-renewal / 生物医学工程学杂志

Skeletal muscle possesses a remarkable ability for its regeneration and injured tissue repair. This ability depends on the activity and contributions of muscle satellite cells. Proliferating satellite cells, termed myogenic precursor cells or myoblasts, are activated and driven out of their quiescent state upon muscle injury. In this summary, we present a review to summarize the molecular regulation in skeletal satellite cells to light on the satellite cells' self-renewal mechanism.

Relation between microcurrent therapy and satellite cells in the regeneration of induced skeletal muscle injury in rat

Severe injuries in skeletal muscle result in muscle weakness, which delays recovery and contributes to progressive decline in muscle function. Microcurrent therapy is a novel treatment method used in soft-tissue injury and tissue regeneration therapy. The regenerative capacity of skeletal muscle tissue resides in satellite cells, the quiescent adult stem cells. The present work aimed at investigating the possible relation between microcurrent therapy and satellite cells in regeneration of induced skeletal muscle injury in albino rats. Twenty-four male albino rats were divided into 2 groups: Control group and experimental group [II]: rats were subjected to gastrocnemius-soleus muscle injury [subgroup IIa], they were subdivided into subgroups IIa1 and IIa2 sacrificed 1 and 3 weeks after injury respectively. Subgroup IIb: Rats were subjected to muscle injury and micro-current electric stimulator, was applied for 20 minutes for three sessions per week. The animals were subdivided into subgroups IIb1 and IIb2 sacrificed 1 and 3 weeks following the day of injury. Muscle sections were stained with hematoxylin and eosin, alpha smooth muscle actin [alpha-SMA] and CD34 immunostaining. Morphometric studies and statistical analysis were performed. Atypical fibers were widely separated by connective tissue cells and revealed partial loss of striations in subgroup IIa. Some fibers recruited strong acidophilic sarcoplasm with focal vacuolations in subgroup IIa1. In subgroup IIb1, some typical fibers, some centrally located nuclei, and a few deeply acidophilic fibers were found. Striations were found in some areas of the sarcoplasm. In subgroup IIb2 striations were found in most areas of the sarcoplasm. A significant decrease in the mean area of atypical fibers, a significant increase in the mean area% of alpha-smooth muscle actin-positive cells, and a significant increase in the mean area% of CD34-positive cells were found in subgroup IIb compared with subgroup IIa. A definite therapeutic effect of the microcurrent was found on induced skeletal muscle injury, which was time dependent. This effect was proved to be related to satellite cell activation

Correlation between Innervation of Skeletal Muscles and Myoblast Stem Cells / Correlación entre la Inervación de los Músculos Esqueléticos y las Células Precursoras Mioblásticas

In order to explore the change rule of myoblast stem cells (satellite cells, SCs) in the denervated and re-innervated muscle and to investigate the cellular mechanism of the morphological and functional changes of the muscle, denervated muscle atrophy and nerve regeneration models were established in one-month-old rats. Postoperative indexes such as muscle wet weight, cell section areas, content of collagen fibers and DNA, electrophysiology, numbers of SCs in the triceps muscle of calf were dynamically tested. After denervation, the muscle wet weight and cell area reduced rapidly, and the collagen fiber content increased slowly. The number of SCs increased at first, and then declined suddenly two months later. From 4 to 5 weeks after re-neuralization, muscle action potentials could be evoked, but the best innervation effect was found in the groups, which received re-neuralization at 2 months and 3 months after denervation. Denervation causes a progressive progress of muscle atrophy. SCs proliferate within 3 months after denervation, and then atrophy becomes irreversible from 4 months. At 4 or 5 weeks after re-neuralization, muscle action potentials can be evoked. Re-neuralization at 2 months and 3 months after denervation can achieve a good effect on the functional recovery of the atrophic muscle.

Con el fin de explorar la regla de cambio de las células precursoras mioblásticas (células satélite, CSs) en el músculo denervado y re-inervado e investigar el mecanismo celular de los cambios morfológicos y funcionales del músculo, se establecieron, en ratas de un mes de edad, modelos de atrofia del músculo denervado y regeneración del nervio. Fueron examinados de manera dinámica índices postoperatorios tales como, el peso húmedo del músculo, áreas celulares de la sección, contenido de fibras de colágeno y ADN, electrofisiología, número de CSs en el músculo tríceps de las crías. Luego de la denervación, el peso del músculo húmedo y el área celular se redujeron rápidamente, mientras que el contenido de fibras colágenas aumentó lentamente. El número de CSs aumentó al inicio, pero más tarde, a los dos meses, disminuyó repentinamente. Entre las 4 a 5 semanas después de la reneuralización, los potenciales de acción muscular pudieron ser evocados, pero el mejor efecto de inervación se encontró en los grupos que recibieron reneuralización a los 2 y 3 meses después de la denervación. La denervación causó un avance progresivo de la atrofia muscular. Las CSs proliferaron dentro de los primeros 3 meses post-denervación, y luego de los 4 meses la atrofia fue irreversible. A las 4 o 5 semanas después de la reneuralizacón, los potenciales de acción muscular pueden ser evocados. La reneuralización a los 2 y 3 meses después de la denervación puede lograr un buen efecto en la recuperación funcional del músculo atrófico.

Effects of angelica polysaccharides on the proliferation of mouse skeletal muscle satellite cells and the expression of stem cell factor receptor protein / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effects of angelica polysaccharides (APS) on the proliferation of mouse skeletal muscle satellite cells (MSCs) and c-kit expression in different in vitro hematopoietic microenvironments.</p><p><b>METHODS</b>MSCs were primarily cultured. The desmin protein was examined by immunohistochemical assay five days later. The MSCs were randomly divided into 8 groups, i. e., the control group, the supernatant from cultured bone marrow stroma cells group, 100, 200, 300 microg/mL APS added in the DMEM/F12 medium experimental groups, and 100, 200, 300 microg/mL APS intervened medium groups. The effects of the proliferation activities of MSCs were detected using MTT method. The c-kit protein of the MSCs was stained by immunohistochemistry.</p><p><b>RESULTS</b>The desmin protein was positive in the isolated cultured MSCs. Results of MTT method showed the proliferation of MSCs in APS intervened medium groups was significant. The strong positive c-kit immunoreactivity existed in APS intervened medium groups. The strong positive c-kit immunoreactivity was present in the cytoplasmic of the MSCs in the DMEM/F12 medium experimental groups and the APS intervened medium groups.</p><p><b>CONCLUSIONS</b>The APS intervened MSC medium could effectively change the growth properties of MSCs, obviously promote the proliferation of MSCs and c-kit expression. The c-kit protein might play some regulative roles in the proliferation of the MSCs.</p>

Effect of danggui buxue decoction on the hemopoiesis reconstruction of mouse transplanted by the muscle satellite cell receptor / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effects of Danggui Buxue Decoction on the hematopoietic reconstruction of mice transplanted by muscle satellite cells ( MSCs).</p><p><b>METHODS</b>MSCs were procured from newly born male mice of the homologous series. The female Kunming receptor mice irradiated with 8Gy137Cs-gamma ray were randomly divided into six groups, i. e., the blank control group, the transplanted MSC group, four groups intervened by different doses of Danggui Buxue Decoction after transplanted MSC (gastrogavage by one, three, five, and ten times of clinically equivalent dose for seven days, as the DGBX 1 group, the DGBX 2 group, the DGBX 3 group, and the DGBX 4 group). After transplantation changes of splenic colony forming unit spleen (CFU-S), white blood cells (WBC), hemoglobin (Hb), and platelet (PLT) in the peripheral blood were observed in pos-transplanted 1-, 2-, and 3-week receptor mice. The 3-week survival rate was calculated. The source of hematopoietic reconstruction was identified using PCR.</p><p><b>RESULTS</b>The desmin protein was positive in the cultured MSCs. WBC of each transplanted MSC group obviously increased at the 2nd week (P<0.05). Hb obviously increased in the DGBX 2 group, the DGBX 3 group, and the DGBX 4 group (P<0.05). WBC and Hb obviously increased in the DGBX 3 group and the DGBX 4 group when compared with the transplanted MSC group at the 3rd week (P<0.05). The recovery of PLT was significant in the DGBX 4 group (P<0.05). Compared with the blank control group at the 2nd week, CFU-S obviously increased in the DGBX 3 group and the DGBX 4 group (P<0.05). PCR results of Y chromosome in the survived transplant mice indicated that the hematopoietic cells of reconstruction female receptor mice were originated from male donors.</p><p><b>CONCLUSION</b>The hematopoiesis of mice transplanted by muscle satellite cells could be constructed after intervention of Danggui Buxue Decoction.</p>

Isolation, culture and growth characteristics of human muscle stem cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To establish the methods for purification, culture, and identification of adult human skeletal muscle stem cells in vitro and to explore the biological properties of the cells.</p><p><b>METHODS</b>Muscle stem cells were obtained by reformed enzymatic digestion of muscle tissue from the consenting donors and cultured in serum-free medium. The morphology was inspected by an inverted phase contrast microscope. Phenotypic characteristics of the cells and expression of cell-specific markers were determined using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The growth of single cells in suspension culture was observed and recorded continuously. The cells were analyzed for their multi-lineage differentiation potential into osteoblastic, adipocyte, and smooth muscle cell lineages.</p><p><b>RESULTS</b>Primary cultured human skeletal muscle stem cells proliferated and formed the big spheres when cultured with serum free medium. Immunofluorescence staining displayed Pax7 and ALDH1 positive expression in the cell spheres. Furthermore, Myod and Desmin showed positive expression in the monolayer cells derived from the spheres. The gene expressions of Oct3/4, Nanog, Sox2 and Pax7 in the cells were determined by RT-PCR. The cell clones formed from single cells grew well. In addition, they were capable of spontaneous differentiation into myotubes in differentiation medium and into other mesodermal cell lineages in induction medium.</p><p><b>CONCLUSIONS</b>Human muscle stem cells with properties of self-renewal capacity and multi-differentiation could be successfully isolated and expanded in vitro.</p>

Bex1 Participates in Muscle Regeneration by Regulating Myogenic Satellite Cell Differentiation / 한국실험동물학회지

Bex1 protein is upregulated in regenerating muscle and interacts with calmodulin, a Ca2+-binding protein involved in cell cycle regulation. Following cardiotoxin-induced injury the regenerating muscle of Bex1 knock-out mice exhibits prolonged cell proliferation and delayed cell differentiation compared to wild-type mice. To gain insight into this process, we compared the regenerating myogenic morphologies of Bex1 knock-out and wild-type mice at several time points. Bex1-positive cells were identified by double immunofluorescence staining. These studies demonstrated that a population of cells that are Bex1-positive after injury are c-Met/basal lamina-positive and Mac-1-negative indicating that they are derived from at least a subset of myogenic progenitor/satellite cells but not invading immune cells. In addition, in regenerating muscle, Bex1 co-localizes with calmodulin in the cytoplasm of the late myoblast or early myotube stage of myogenesis. These results suggest that Bex1 participates in muscle regeneration through the regulation of satellite cell proliferation and differentiation by its interaction with calmodulin. Current studies of Bex1 may provide a new molecular tool for the identification of activated satellite cell and open the way to new or improved therapeutic regimens against progressive muscular atrophy.

Bex1 Participates in Muscle Regeneration by Regulating Myogenic Satellite Cell Differentiation / 한국실험동물학회지

Bex1 protein is upregulated in regenerating muscle and interacts with calmodulin, a Ca2+-binding protein involved in cell cycle regulation. Following cardiotoxin-induced injury the regenerating muscle of Bex1 knock-out mice exhibits prolonged cell proliferation and delayed cell differentiation compared to wild-type mice. To gain insight into this process, we compared the regenerating myogenic morphologies of Bex1 knock-out and wild-type mice at several time points. Bex1-positive cells were identified by double immunofluorescence staining. These studies demonstrated that a population of cells that are Bex1-positive after injury are c-Met/basal lamina-positive and Mac-1-negative indicating that they are derived from at least a subset of myogenic progenitor/satellite cells but not invading immune cells. In addition, in regenerating muscle, Bex1 co-localizes with calmodulin in the cytoplasm of the late myoblast or early myotube stage of myogenesis. These results suggest that Bex1 participates in muscle regeneration through the regulation of satellite cell proliferation and differentiation by its interaction with calmodulin. Current studies of Bex1 may provide a new molecular tool for the identification of activated satellite cell and open the way to new or improved therapeutic regimens against progressive muscular atrophy.

Estudo comparativo da ação da toxina botulínica tipo A e da crotoxina sobre as células satélites da musculatura extrínseca ocular em modelo animal / A comparative study of the effects of type A botulinum toxin and crotoxin on satellite cells of extraocular muscles in rabbits

OBJETIVOS: Avaliar o efeito da toxina botulínica do tipo A e da crotoxina na ativação de células satélites das fibras musculares de retos superiores de coelhos. MÉTODOS: Os músculos retos superiores do olho direito de 29 coelhos machos albinos neozelandeses foram inoculados com toxina botulínica do tipo A ou com crotoxina, em diferentes doses. Os músculos retos superiores contralaterais de cada animal foram inoculados com solução salina em volume igual ao das toxinas. Os animais foram sacrificados 12, 18 ou 25 dias após as aplicações. Os olhos foram enucleados e cada músculo foi preparado para análise imunoistoquímica, com marcadores de células satélites. Foi realizada contagem dos núcleos corados pelos marcadores a cada cem miofibras. RESULTADOS: A aplicação de toxina botulínica e de crotoxina provocou um aumento no número de células satélites ativadas e em proliferação nos músculos retos superiores. Uma maior ativação foi observada após a aplicação de crotoxina, embora, estatisticamente, a diferença do efeito de ativação entre os grupos botoxina e crotoxina não tenha sido significativa. Nos grupos botoxina e crotoxina, não houve correlação estatisticamente significativa entre a dose e o volume aplicados e o aumento na ativação das células. O tempo de vida após a aplicação contribuiu para o aumento das células ativadas nos grupos. CONCLUSÃO: A observação de maior desorganização na estrutura muscular e de sinais de regeneração mais evidentes no grupo crotoxina parece estar correlacionada ao aumento de células satélites ativadas.

PURPOSE: To evaluate the effect of botulinum toxin A and crotoxin on satellite cell activation in the muscle fibers of superior rectus muscles of rabbits. METHODS: The superior rectus muscles in the right eyes of 29 male, albino, New Zealand rabbits were inoculated with different doses of botulinum toxin A or crotoxin. The contra-lateral superior rectus muscles in each rabbit were inoculated with the same volume of saline solution only. The animals were sacrificed either 12, 18 or 25 days after the inoculation. The eyes were enucleated and subsequently, each muscle was prepared for immunohistochemical analysis, using satellite cell markers. The positive nuclei, revealed by the markers in each 100 myofibers, were counted. RESULTS: The application of the botulinum toxin A and crotoxina triggered a more significant increase satellite cell activation and proliferation in right superior rectus muscles in rabbits when compared with a saline solution inoculation in the contralateral muscles. Greater cell activation was observed after crotoxin application, although, statistically, the difference in the effects of this activation between the botoxin and crotoxin groups was not significant. There was no statistically significant correlation between the dose and the volume applied and resulting cell activation in the botoxin and crotoxin groups. Post-application survival time contributed to the increase in activated satellite cells in all groups. CONCLUSION: The observed increase in disorganization in the muscle structure, together with more obvious signs of regeneration in the crotoxina group, suggests a correlation with the increase in satellite cell activation.

Atuação dos esteróides anabolizantes na regeneração músculo-esquelética / Performance of the anabolic steroids in the skeletal muscle regeneration

A capacidade regenerativa das fibras musculares depende da presença de células satélites, ocorrendo por mecanismos fisiológicos que compõem sua cito-arquitetura. Os esteróides anabolizantes apresentam efeitos de manutenção protéica, anticatabolismo e modulação do cálcio, e associados à regeneração muscular, proporcionam um auxílio na velocidade de recuperação de fibras lesadas, além do aumento do diâmetro da fibra. Assim sendo, o presente trabalho tem por objetivo atualizar informações e reunir descobertas sobre regeneração músculo-esquelética associada aos esteróides anabolizantes por levantamento bibliográfico em banco de dados Medline, Lilacs e Scielo, bem como a utilização de livros, monografias e dissertações da área. Para busca deste material foram utilizados os seguintes descritores: músculo esquelético, células satélite, regeneração muscular, esteróides anabolizantes, consultados em 2006. Analisando as informações obtidas, sugere-se que os esteróides anabolizantes atuam na preservação da integridade da fibra muscular diante do processo regenerativo, sendo necessários fundamentos mais precisos em modelos experimentais humanos.

The regeneration capacity of skeletal muscle fibers is largely mediated by satellite cells and occurs by means of physiological mechanisms involving their cytoarchitecture. The anabolic steroids are effective in increasing protein synthesis and promoting anti-catabolism and calcium modeling in skeletal muscles. As a result, the use of these drugs led to an increased fiber diameter as well as to an enhanced rate of regeneration of injured muscle fibers. The current study aimed to update information and collect findings about musculoskeletal regeneration associated with the use of anabolic steroids. To do so, a bibliographic survey was carried out in the database of Medline, Lilacs and Scielo, as well as in scientific books, monographs, dissertations and theses. The descriptors used in the survey were: musculoskeletal, satellite cells, muscle regeneration and anabolic steroids. The analysis of the collected information suggests that the anabolic steroids play a rolein the maintenance of the integrity of muscle fibers during the regenerative process. However, further foundations should be built for human experimental models.

Effect of thyroid hormone on contractility and sarco/endoplasmic Reticulum Ca[2+] ATPase -1 [SERCA1] expression in adult rats; role in thermogenesis

Thyroid hormone is one of the main factors that determine skeletal muscle contractile properties and the slow-twitch vs. fast twitch muscle fiber phenotype. Sarcoplasmic reticulum Ca[2+] ATPase is one of the principal regulators of Ca[2+] homeostasis in the skeletal muscle cells. It has been previously shown that modification of thyroid hormone levels has a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. The present study aimed to investigate the effects of thyroid hormone treatment for 4 weeks on slow twitch soleus muscle of rats via recording twitch tension, time to peak tension, and half relaxation time and determination of soleus muscle SERCA1 expression. 24 male rats of local strain were randomly divided into 2 groups [n=12], control euthyroid group and hyperthyroid group in which hyperthyroidism was induced by intra peritoneal [i.p] injection of L-troxin 200microg/kg B.W./day, for 4 weeks. The in-situ isometric measures were done for soleus muscle and twitch tension [TT], [expressed as screen units], time to peak tension [TPT], and half relaxation time, 1/2 Rt [expressed in milliseconds] were recorded. SERCA1 protein expression in rat soleus muscle was measured. Thyroid hormone treatment significantly reduced time to peak tension and half relaxation time and increased twitch tension. These contractile changes were accompanied with significant increase in expression of SERCA1 in soleus muscle of rats. Thyroid hormone stimulates the expression of SERCA1 in slow soleus muscle in rats, thereby speeding-up the contraction-relaxation cycle and this increases energy expenditure and these effects of thyroid hormone participate at least in part to thermogenic action of thyroid hormone

Isolation, identification, culture and bionomics of skeletal muscles satellite cells from green fluorescent protein transgenic mouse in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the green fluorescent protein (GFP) expression and the bionomics of skeletal muscles satellite cells (SMSCs) in vitro in GFP transgenic mouse.</p><p><b>METHODS</b>The newborn transgenic mice were acquired to separate skeletal muscles satellite cells with enzyme digestion method. Cells were cultured and subcultured in vitro. Morphological observation, growth curve were investigated to evaluate the proliferation and differentiation characteristics of skeletal muscles satellite cells, fluorescence microscope was used to observe the GFP expression. The cells were identified by immunocytochemical stain. In the basis of identification of anti-sarcometric actin anti-body, the combination of anti-desmin antibody and DAPI (4, 6-diamidino-2-phenylindole) were used to detect the purification of skeletal muscles satellite cells.</p><p><b>RESULTS</b>Immunocytofluorescence suggested the good retain of GFP fluorescence in skeletal muscles satellite cells. The cells showed strong proliferative ability and they were positive with immunocytochemical stain of anti-sarcometric actin antibody and anti-desmin antibody. The combination of anti-desmin and DAPI stain can be used to determine the purification of SMSCs.</p><p><b>CONCLUSION</b>Skeletal muscles satellite cells cultured in vitro showed strong proliferation and differentiation ability. They are fit to construct the cell bank of tissure engineering and to be a useful tool to explore cells fate after transplantation since these cells retain the expression of GFP.</p>