Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation / 生物工程学报

Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm2 vs. (1 274.8±327.3) μm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) μm2 vs. (2 610.4±604.4) μm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.

Myostatin coordinating the proliferation and differentiation of adipose and skeletal muscle cells and energy metabolism balance based on gene chip technology / A miostatina na coordenação da proliferação e diferenciação de células musculares adiposas e esqueléticas e no equilíbrio do metabolismo energético com base na tecnologia de chips genéticos / Msrn, basado en la tecnología de chips genéticos, coordina la fertilización y el metabolismo de las células musculares grasas y óseas

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.

RESUMO Os mioblastos se fundem eM fibras musculares multinucleadas para formar e promover o crescimento do músculo esquelético. A fim de analisar o papel da miostatina (MSTN) na gordura corporal, proliferação de células musculares esqueléticas e diferenciação e metabolismo energético, este estudo utilizará a tecnologia anti-RNA de chips genéticos para estudá-la. Os resultados mostraram que o gene MSTN regulava o crescimento e a proliferação de mioblastos e afetava o desenvolvimento do músculo esquelético, afetando a expressão de Cdc42, bnip2, p38 e outros genes; a eliminação ou sobrexpressão do gene MSTN conduziria a uma tendência de os genes adiposos sintetizarem a gordura até sua decomposição; após a eliminação do gene MSTN, os níveis de expressão de cpti-b, PPARG e outros genes nas células mostraram-se correspondentes após a sobrexpressão do gene MSTN, e a expressão relativa do gene PPARG diminuiu. Sugere-se que a eliminação ou sobrexpressão da MSTN possa afetar a acumulação de lipídeos, e o cpti-b e o PPARG podem regular diretamente o nível lipídico. Espera-se que esta experiência possa fornecer uma referência para o estudo do efeito da MSTN sobre a deposição de gordura.

RESUMEN Los mioblastos se funden en fibras musculares multinucleadas para formar y promover el crecimiento del músculo esquelético. A fin de analizar el papel de la miostatina (MSTN) en la grasa corporal, proliferación de células musculares esqueléticas y diferenciación y metabolismo energético, este estudio utilizará la tecnología anti-RNA de chips genéticos para estudiarla. Los resultados mostraron que el gen MSTN regulaba el crecimiento y la proliferación de mioblastos y afectaba el desarrollo del músculo esquelético, afectando la expresión de Cdc42, bnip2, p38 y otros genes; la eliminación o sobreexpresión del gen MSTN conduciría a una tendencia de que los genes adiposos sinteticen la grasa hasta su descomposición; después de la eliminación del gen MSTN, los niveles de expresión de cpti-b, PPARG y otros genes en las células se mostraron correspondientes después de la sobreexpresión del gen MSTN, y la expresión relativa del gen PPARG disminuyó. Se sugiere que la eliminación o sobreexpresión de la MSTN pueda afectar la acumulación de lipídos, y el cpti-b y el PPARG pueden regular directamente el nivel lipídico. Se espera que esta experiencia pueda proveer una referencia para el estudio del efecto de la MSTN sobre el depósito de grasa.

Effect of exogenous miR206 on the development of denervated muscular atrophy by inhibiting the expression of myostatin of rats / 中华显微外科杂志

Objective:To investigate the effects and mechanism of miR206 in rat model of denervated muscular atrophy.Methods:From September, 2020 to December, 2020, a total of 40 rats were selected for this study. Denervated muscular atrophy model was established on 16 SPF Sprague-Dawley rats, by removing 1 cm in length of sciatic nerve. The rats were classified into 4 groups according to the sampling time: 0 d, 3 d, 7 d and 14 d(4 rats per group). The other 24 rats were also established into denervated skeletal muscle atrophy models and assigned into 3 groups: denervation add miR206 group, denervation add NC transfection reagent group, and sham-operated group( n=8 in each group). After sampling, the area of cross section of the gastrocnemius muscle and gastrocnemius muscle mass were measured to evaluate muscle atrophy. The mRNA and protein expression of myostatin were determined by real-time PCR and Western blot. Combining with luciferase report to explore the underlying mechanism of miR206, the t-test and oneway ANOVA were used for data analysis used in this study. In one-way ANOVA analysis, if the difference between groups was statistically significant, Bonferroni method would be used for further comparing of all pairs. P<0.05 was considered statistically significant. Results:After excision of a part of sciatic nerve of rat models, gastrocnemius muscle mass of denervation plus miR206 group, denervation plus NC transfection reagent group and sham-operated group were: (0.63±0.04), (0.51±0.02) and (1.05±0.02), respectively. The cross section areas of gastrocnemius muscle in each groups were: (761.30±21.79) μm 2, (640.30±30.31) μm 2 and (1066.00±51.65) μm 2, respectively( P<0.05). Myostatin mRNA expression showed lower in miR206 group than in NC group tested by Western blot, which were(0.57±0.04) in miR206 and (0.81±0.04) in NC group tested by qPCR( P<0.05). The protein expression measured by Western blot test revealed same expression pattern as mRNA expression pattern. The different of relative expression between miR206 group and NC group( P<0.05). Finally, in the mmu-miR206 co-transfected with the MSTN 3'UTR-luciferase sensor group, the relative luciferase activity was measured at 0.26±0.07 and it was significant lower than any other groups( P<0.05). Conclusion:The miR206 can counteract denervated skeletomuscular atrophy through down regulating the myostatin expression. Myostatin is a new discovered target gene of miR206.

Association between myostatin and sarcopenia in end-stage liver disease / 临床肝胆病杂志

Patients with end-stage liver disease (ESLD) are often accompanied by various complications such as sarcopenia and cachexia including lipopenia, and it was believed in the past that such status was associated with malnutrition, while recent studies have shown that myostatin (MSTN) is associated with the progression of ESLD. MSTN can lead to sarcopenia and cachexia by affecting the metabolism of glucose, fat, and protein and the number of myocytes, and it can be used as a screening indicator for hepatocellular carcinoma (HCC) and an indicator for disease progression. Intervention via the MSTN pathway might be an effective method for controlling sarcopenia and cachexia in patients with ESLD, and MSTN may be an effective indicator for predicting the progression of liver cirrhosis to HCC.

The evaluation of RS1805086 and RS1805065 polymorphisms in Mstn gene and anthropometric properties of national and amateur arm wrestlers / Evaluación de los polimorfismos RS1805086 y RS1805065 en el gen Mstn y las propiedades antropométricas de los luchadores de brazo nacionales y amateurs

The aim of this study is to investigate rs1805086 and rs1805065 polymorphisms of MSTN gene of national and amateur Turkish arm wrestlers and people leading a sedentary lifestyle, and the anthropometric properties such as hand, wrist, and forearm circumferences of national and amateur Turkish arm wrestlers are aimed to be explored. In this study, a total of 79 volunteers who were 24 national (7 females, 17 males) Turkish arm wrestlers, 21 amateur (7 females, 14 males) Turkish arm wrestlers and 34 sedentary people (12 females, 22 males) participated. To analyse the data, Statistical Package for the Social Sciences, SPSS 22 (SPSS Inc., Chicago, IL, USA) was used. As a result of the study, when data on rs1805086 and rs1805065 polymorphisms of MSTN gene were examined respectively, it was found out that MSTN 153KK genotype was 100.0% dominant in both national (n=24) and amateur (n=21) arm wrestlers, and it was 94.12 % dominant in sedentary people. KR genotype was observed in 5.88 % of the sedentary people. The data from the other rs1805065 polymorphism of MSTN gene showed that all participants (n = 45, 100.0 %) were carriers of normal homozygous genotype. Furthermore, for both female group and male group, there found to be statistically significant difference in terms of anthropometric properties. It can be concluded that though there was no significant difference between national and amateur Turkish arm wrestlers in terms of their MSTN gene characteristics; in terms of anthropometric properties, significant differences were discovered. It was found out that on these athletes, not MSTN gene polymorphisms but anthropometric properties were effective.

El objetivo de este estudio fue investigar los polimorfismos rs1805086 y rs1805065 del gen MSTN de luchadores de brazos turcos, nacionales y aficionados, y personas que llevan un estilo de vida sedentario, y las propiedades antropométricas además de las circunferencias de manos, muñecas y antebrazos de los luchadores de brazos turcos nacionales y aficionados. En este estudio, participaron un total de 79 voluntarios: 24 luchadores de brazos turcos nacionales (7 mujeres, 17 hombres), 21 luchadores de brazos turcos aficionados (7 mujeres, 14 hombres) y 34 personas sedentarias (12 mujeres, 22 hombres). Para analizar los datos, se utilizó el Paquete Estadístico para las Ciencias Sociales, SPSS 22 (SPSS Inc., Chicago, IL, EE. UU.). Como resultado del estudio, cuando se examinaron los datos sobre los polimorfismos rs1805086 y rs1805065 del gen MSTN respectivamente, se descubrió que el genotipo MSTN 153KK era 100,0 % dominante en luchadores de brazos nacionales (n = 24) y aficionados (n = 21) , y era 94,12 % dominante en personas sedentarias. El genotipo KR se observó en el 5,88 % de las personas sedentarias. Los datos del otro polimorfismo rs1805065 del gen MSTN mostraron que todos los participantes (n = 45; 100,0 %) eran portadores del genotipo homocigoto normal. Además, tanto para el grupo femenino como para el masculino, se encontró una diferencia estadísticamente significativa en términos de propiedades antropométricas. Se puede concluir que, aunque no hubo una diferencia significativa entre los luchadores de brazos turcos nacionales y aficionados en términos de sus características genéticas MSTN; en términos de propiedades antropométricas, se descubrieron diferencias significativas. Se descubrió que, en estos atletas, no fueron los polimorfismos del gen MSTN sino las propiedades antropométricas las efectivas.

CRISPR/Cas9-mediated MSTN gene editing induced mitochondrial alterations in C2C12 myoblast cells

Background: Myostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn−/+ KO cells, and wondered whether the mitochondria biogenesis are affected. Results: In this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels. Conclusion: These findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.

Serum myostatin levels are associated with abdominal aortic calcification in dialysis patients

BACKGROUND: Serum myostatin levels are increased according to renal function decline and myostatin may be a main mediator of chronic kidney disease–related sarcopenia. A previous study reported that serum myostatin level was negatively associated with abdominal aortic calcification (AAC) in older males. The aim of this study was to assess the association between serum myostatin level and AAC among dialysis patients of both sexes. In addition, we analyzed the relationship between serum myostatin level, muscle mass, and bone mineral density (BMD).METHODS: In this cross-sectional study, we evaluated AAC in the lateral lumbar spine using plain radiography and BMD in 71 patients undergoing dialysis. We classified patients into two groups according to the median value of myostatin as follows: those with high myostatin levels (≥ 5.0 ng/mL) and those with low myostatin levels (< 5.0 ng/mL).RESULTS: The proportion of patients with an AAC score of five points or more was higher among those with low myostatin levels. Myostatin level was negatively associated with AAC scores on plain radiography and had a positive association with skeletal muscle mass and T-scores for BMD measured at the total hip and femur neck. Lower myostatin levels were independently associated with higher AAC scores following adjustment for age, sex, diabetes mellitus, dialysis vintage, dialysis modality, and osteoprotegerin level.CONCLUSION: Lower serum myostatin levels were associated with higher AAC scores, lower muscle mass, and lower BMD in dialysis patients. Further, prospective studies and those with larger cohorts are necessary to validate these findings.

Effects of Electroacupuncture on Skeletal Muscle Atrophy-associated Protein in Hind Limbs of Traumatic Spinal Cord Injury Rats / 中国康复理论与实践

Objective:To explore the effects and mechanism of electroacupuncture (EA) on expression of myostatin (MSTN), muscle-specific ring finger protein 1 (MuRF1/Trim63), F-box only protein 32 (Atrogin-1/ Fbxo32), myogenic differentiation antigen (Myod) and myogenin (Myog) in traumatic spinal cord injury (TSCI) rats. Methods:A total of 45 adult female Sprague-Dawley rats were randomly divided into sham operation group (n = 12) and operation group (n = 33). The TSCI model was established with the modified Allen's method. After modeling, there were 24 survival rats and they were randomly divided into model group (n = 12) and EA group (n = 12). EA group was electroacupunctured at Dazhui (DU 14), Mingmen (DU 4) and bilateral Zusanli (ST 36) for 10 minutes, once a day, six times a week for 28 days. Basso-Beattie-Bresnahan (BBB) score was tested before modeling, and three days, seven days, 14 days, 21 days and 28 days after modeling. The rats were measured their body mass before and 28 days after modeling. The ratio of gastrocnemius wet mass was calculated; the cross-sectional area (CSA) and fiber diameter were measured by HE staining; the expression of MSTN, Trim63, Fbxo32, Myod and Myog mRNA were tested with real-time quantitative polymerase chain reaction (qPCR). Results:Three days, seven days, 14 days, 21 days, and 28 days after modeling, the score of BBB was lower in the model group than in the sham operation group (P < 0.01); seven days, 14 days, 21 days, and 28 days after modeling, the score of BBB was higher in EA group than in the model group (P < 0.01). Compared with the sham operation group, the mass of rats, the gastrocnemius wet mass, the CSA and the diameter of the muscle fiber were smaller in the model group (P < 0.05), while the expression of MSTN, Trim63, Fbxo32, Myod and Myog mRNA were higher (P < 0.05). Compared with the model group, the mass of rats, the gastrocnemius wet mass, the CSA, the expression of Myod and Myog mRNA were higher (P < 0.05) in EA group, while the expression of MSTN, Trim63 and Fbxo32 mRNA were lower (P < 0.05). Conclusion:EA might delay the gastrocnemius atrophy in TSCI rats by down-regulating the expression of MSTN, Trim63, Fbxo32 mRNA and up-regulating the expression of Myod and Myog mRNA via controlling the differentiation of the muscle satellite cells and the degradation of protein in skeletal muscle cells.

Progress of myostatin on duchenne muscular dystrophy / 国际儿科学杂志

Duchenne muscular dystrophy (DMD) is a severe muscular genetic disorder,which is caused by a mutation in the DMD gene encoding the dystrophin protein.The clinical manifestation of DMD is the progressive muscular dystrophy.Myostatin is a negative regulator of skeletal muscle growth and development.In recent years,it has received extensive attention in the treatment of muscle diseases.This review focuses on the structural and activity of myostatin,its regulation of skeletal muscle growth and noval approaches to Myostatin inhibitor in the treatment of DMD.

Rheumatoid arthritis-increased gene expressions in muscle atrophy are restored back to control as a response to acute resistance exercise / Estímulo agudo de musculação promove regulação de genes alterados pela atrofi a muscular

This study had as objective to analyze the acute eff ects of resistance exercise (RE) on the mRNA levels of the following genes (MyoD, myogenin, IGF-1, atrogin-1, MuRF-1, and myostatin) in rheumatoid arthritis (experimental arthritis). Therefore, 26 females rats were randomly allocated into four groups, control (CT, n=7), exercise (Ex, n=6), rheumatoid arthritis (RA, n=6) and RA with exercise (RAEx, n=7). Met-BSA was injected into the tibiotarsal joint in the RA and RAEx groups. After 15 days from injection, the animals were submitted to an acute bout of RE and six hours post protocol the animals were euthanized. We evaluated the joint thickness, infl ammation score, cross-sectional area (CSA) of gastrocnemius muscle fi bers and mRNA expression of the IGF-1, MyoD, myogenin, myostatin, MuRF-1, atrogin-1 and GAPDH. It was observed that the joint thickness and score strongly increased in arthritic rats (p <0.001) while the CSA decreased (p ≤ 0.05). Increased mRNA levels of IGF-1 (2.0 fold), myostatin (4.5 fold), atrogin-1 (2.5 fold), MyoD (3.7-fold) and myogenin (5 fold) were observed in muscle of arthritic rats. The mRNA expression of myostatin, atrogin-1, MyoD and myogenin decreased in the RAEx group. In this way, we can conclude that experimental arthritis-increased gene expressions in muscle atrophy myostatin, atrogin-1, MyoD and myogenin) are restored back to control as a response to acute RE....(AU)

O presente estudo teve como objetivo analisar o efeito agudo do Exercício com pesos sobre os níves de mRNA de genes envolvidos no anabolismo ou catabolismo muscular em um modelo experimental de Artrite Reumatóide. Para tanto, 26 ratas fêmeas foram randomicamente alocadas em quatro grupos, controle (CT, n=7), Exercício (Ex, n=6), Artrite Reumatóide (AR, n=6) e Artrite Reumatóide com exercício (AREx, n=7). Uma substância contendo Albumina bovina metilada foi injetada na articulação tíbio-tarsal nos grupos AR e AREx para indução da Artrite Reumatóide. Após 15 dias da injeção, os animais foram submetidos a um estímulo agudo de treinamento com pesos e 6 horas após o exercício os animais foram eutanasiados. Nós avaliamos a espessura da articulação, escore de infl amação, a área de secção transversa (AST) das fi bras do músculo Gastrocnêmio e a mRNA de IGF-1, MyoD, Myogenina (genes envolvidos no anabolismo muscular), e MuRF-1, atrogina-1 (genes envolvidos no catabolismo muscular), além do gene controle , GAPDH. Foi observado que a espessura articular e o escore de infl amação aumentaram fortemente nas ratas induzidas a Artrite Reumatóide (p <0,001), enquanto a AST reduziu (p ≤ 0,05). Um aumento nos níveis de mRNA de IGF-1 (2,0 vezes), miostatina (4,5 vezes), atrogina-1 (2,5 vezes), MyoD (3,7 vezes) e miogenina (5 vezes) foi observado no músculo das ratas induzidas a Artrite Reumatóide. mRNA de miostatina, atrogina-1, MyoD e miogenina reduziu no grupo RAEx. Desta forma, podemos concluir, que o modelo experimental de Artrite Reumatóide induziu um aumento da expressão de genes durante a atrofi a muscular (myostatin, atrogin-1, MyoD and myogenin) e que estas alterações foram reguladas pelo Exercício com peso....(AU)

Co-administration of myostatin-targeting siRNA and ActRIIB-Fc fusion protein increases masseter muscle mass and fiber size / Шинэ санаа Шинэ нээлт

@#Myostatin, a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle cell growth and differentiation, and binds with high affinity to the activin type IIB receptor (ActRIIB). The soluble ligand-binding domain of ActRIIB fused to the Fc domain of IgG (ActRIIB-Fc) potently binds and inhibits TGF-beta family members in muscle, leading to rapid and marked muscle growth. The present study was designed to assess the effectiveness of the co-delivery of myostatin-targeting siRNA (Mstn-siRNA) and ActRIIB-Fc into skeletal muscle as a potential treatment of atrophic myopathies. Eleven-week-old, male C57BL/6 mice were injected with atelocollagen (ATCOL)-mediated Mstn-siRNA with/ without ActRIIB-Fc locally into the masseter muscle twice a week. Inhibition of myostatin function by the combination of Mstn-siRNA and ActRIIB-Fc increased muscle weight and myofibril size in murine masseter muscle. Real-time RT-PCR analysis revealed significant downregulation of myostatin mRNA expression in both the Mstn-siRNA-treated and the combination treatment group. Furthermore, myogenin mRNA expression was upregulated in the combination treatment group, while MuRF-1 and Atrogin-1 mRNA expression was downregulated compared to administration of each compound alone. These findings suggest that double inhibition of myostatin is a potentially useful treatment strategy to increase muscle mass and fiber size and could be a useful treatment of patients with various muscle atrophies, including muscular dystrophy.

Effects of co-transfection with myostatin-targeting siRNA and ActRIIB-Fc fusion protein on skeletal muscle growth / Шинэ санаа Шинэ нээлт

@#Myostatin (Mstn) is a secreted TGF- β family member that controls skeletal muscle growth, and binds with high affinity to the activin type IIB receptor (ActRIIB). The soluble ligand-binding domain of ActRIIB fused to the Fc domain of IgG (ActRIIB-Fc) potently binds and inhibits TGF-β family members in muscle, leading to rapid and marked muscle growth. The present study was designed to assess the combinative effects of myostatin-targeting siRNA (Mstn-siRNA) and ActRIIB-Fc on murine myoblast in vitro and in vivo. C2C12 cells were treated by Mstn-siRNA with or without ActRIIB-Fc at 0 and 48 h after differentiation. Myotube size was measured, and gene expression of Mstn, MuRF-1, MyoD and myogenin were analyzed. Furthermore, 11-week-old, male C57BL/6 mice were injected with atelocollagen (ATCOL)-mediated Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc locally into the masseter muscle twice a week. Histological and biochemical analyses were performed using the dissected muscles. Transfection of Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc resulted in significant increases in the myotube diameter of the C2C12 cells compared with untreated control. Also, treatment with Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc could lead to an upregulation of MyoD and myogenin gene expression and downregulation of Mstn and MuRF-1. In vivo, muscle fibril hypertrophy was observed in both Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc treated groups. Moreover, western blotting analysis showed that the p-Smad2/3 expression level was decreased by treatment of Mstn-siRNA/ActRIIB-Fc. In contrast, MyoD and myogenin protein levels were increased by combined treatment, compared with the other groups. These suggest that double inhibition of myostain is potentially useful for myogenesis and muscle growth promotion. This may be a good as new treatment remedy for patients with various muscle atrophies, including muscular dystrophy.

Relationship of myostatin with the nutritional status and outcome of gastric cancer patients / 中华临床营养杂志

Objective To investigate the expressions of myostatin (MSTN) in the gastric cancer patients' rectus abdominis and serum,and to discuss its relation with the nutritional status and outcome of the gastric cancer patients.Methods We recruited 102 patients with gastric cancer and another 53 patients with benign abdominal disease who were admitted to Zhejiang Province People's Hospital and Tongde Hospital of Zhejiang Province from February 2008 to February 2011.We tested MSTN expression in the malignant patients' rectus abdominis and serum using reverse transcription polymerase chain reaction,Western blot,and enzymelinked immunosorbent assay.Relationship between MSTN expression and nutrition status of gastric cancer patients was explored.Results The relative expression of MSTN mRNA of rectus abdominis muscle in gastric cancer group and control group was 3.43 ± 1.11 and 1.07 ± 0.31 (t =19.406,P ＜ 0.01),the relative expression level of MSTN protein in gastric cancer group and control group was 0.115±0.696 and 0.085±0.499 (t=2.856,P＜0.05) and its serum expression 8.79±4.32 and 1.21±0.55 in these two group (t =16.701,P＜0.05),showing significant difference.The high (defined as higher than median) expression of MSTN in rectus abdominis muscle of gastric cancer patients was significantly correlated with age (x2 =22.039,P =0.000),percentage of more than 10％ decline in body weight in half a year (x2 =14.365,P=0.000),body mass index (BMI) ＜22 kg/m2 (x2 =6.800,P=0.009),serum albumin (x2 =31.018,P=0.000),hemoglobin (x2=10.079,P =0.001),prognostic nutritional index (x2 =10.074,P =0.002),higher Nutritional Risk Screening 2002 Score (Z=2.628,P=-0.009),tumor stage of TNM (Z=2.550,P=0.011).The expression of the rectus abdominis muscle in patients with gastric cancer was significantly correlated with the survival rate of the patients (x2 =12.200,P＜0.01).Conclusions The expression of MSTN increases in the rectus abdominis and serum of gastric cancer patients with cachexia.The increased MSTN expression is closely related with malnutrition and TNM stage in these patients,suggesting MSTN may also play an important role in the muscle consumption of the gastric cancer patients.

A research progress on metabolism-associated myokines / 复旦学报（医学版）

Cytokines and peptides produced and released by muscle cells and exert either autocrine,paracrine or endocrine effects are defined as myokines.Myokines are capable of exerting specific endocrine effects on organs such as adipose tissue,liver,pancreas and bone,thereby impacting the structure and function of numerous organs and tissues,as well as playing an important role in the maintenance of biological homeostasis.So far,researches have confirmed hundreds of different myokines,including irisin,myonectin,myostatin and brain-derived neurotrophic factor (BDNF),which can affect glucose and lipid metabolism,as well as energy equilibrium.The generation、physiology、mechanism of action and potential clinical value of these metabolism-associated myokines will be reviewed in this article.

Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents

Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

Identification of polymorphisms in the myostatin and leptin genes of santa inês breed and crossbreed sheep and association with carcass traits / Identificação de polimorfismo nos genes da miostatina e leptina em ovinos santa inês e cruzados e associação com características de carcaça

The objective of this study was to identify polymorphisms in the myostatin and leptin genes in Santa Inês (SI) and crossbreed (SI x Dorper) sheep, to verify the effect of these polymorphisms on carcass traits. We evaluated seventy sheep of 8-month-old at the Federal Institute of Espírito Santo, Brazil. Data collected were slaughter weight (SW), hot carcass weight (HCW), cold carcass weight (CCW), loin weight, tenderloin weight and fat thickness (FT). The hot carcass yield (HCY) was calculated by the formula (HCW/SW) x 100. We collected hairs from each animal for DNA extraction by the alkaline protocol. The animals were genotyped for the G>A mutation in nucleotide 9827 of the myostatin gene and for three polymorphisms in exon 3 of the leptin gene, by the PCR-RFLP technique. The amplicons the myostatin and leptin gene were cleaved with restriction enzyme for allelic discrimination. The alleles were recorded for each animal and analysis of variance was performed to check the influence of the mutations on the carcass traits. The mutant allele of the myostatin gene showed association with increased measures of CCW, FT and with reduced HCY. Among the three alleles of the leptin gene, only one showed an effect (increased CCW). The other alleles were not associated with any traits.

O objetivo deste estudo foi identificar os polimorfismos nos genes da miostatina e leptina em Santa Inês (SI) e mestiças (SI x Dorper) ovelhas, para verificar o efeito desses polimorfismos sobre características de carcaça. Foram avaliadas setenta ovelhas com oito meses de idade, do Instituto Federal do Espírito Santo, Brasil. Os dados coletados foram o peso de abate (PA), peso de carcaça quente (PCQ), peso de carcaça fria (PCF), peso de lombo, peso lombinho e espessura de gordura de cobertura (ECG). O rendimento de carcaça quente (RCQ) foi calculado pela fórmula (PCQ/PA) x 100. Foram coletados pelos de cada animal para a extração de DNA pelo protocolo alcalino. Os animais foram genotipados para a mutação G>A no nucleotídeo 9827 do gene da miostatina e para três polimorfismos no exon 3 do gene da leptina, através da técnica PCR-RFLP. Os produtos de amplificação do gene da miostatina e da leptina foram clivados com a enzima de restrição para diferenciação alélica. Os alelos encontrados foram registrados para cada indivíduo e então foi realizada a análise de variância para verificar os efeitos das mutações sobre as características de carcaça pelo procedimento GLM do SAS. O alelo mutante do gene da miostatina mostrou associação com o aumento das médias PCF e ECG e com a redução do RCQ. Entre os três alelos do gene da leptina, apenas um apresentou efeito com aumento do PCF. Os demais alelos e as demais características não apresentaram associação.

Effect of follistatin on skeletal muscle wasting of cancer cachexia mice and its mechanism / 吉林大学学报(医学版)

Objective: To observe the effects of follistatin (FST)on the skeletal muscle wasting of cancer cachexia mice and the expressions of Mstn, LncRNA-MALAT1 and Caspase-3, and to elucidate its associated molecular mechanisms.Methods:Thirty-two BALB/c mices were randomly assigned into:healthy control (HC) group,FST prevention (FP)group,FST treatment (FT)group and cancer cachexia (CC)group.The murine colon adenocarcinoma CT26 cells were inoculated subcutaneously into the mices in FP, FT and CC groups to establish the cancer cachexia models. The body weight, spontaneous activity and tumor growth were daily monitored.The mice in FP and FT groups were administrated with FST intraperitoneally on day 6 and 12 after inoculation.The samples were collected on day 20.The tumor and gastrocnemius weights of the mice were detected. The biochemical metabolism indexes and myofiber cross-sectional area of gastrocnemius tissue were detected.The mRNA expression levels of Mstn,Caspase-3 and LncRNA-MALAT1 were examined by Real-time PCR.The protein expression levels of Mstn and Caspase-3 were measured by Western blotting method. Results:Compared with CC group,the body weights,spontaneous activities,gastrocnemius weights and myofiber cross-sectional areas were increased (P <0.05);the serum levels of glucose,total protein and albumin of the mice in FP and FT groups were increased (P <0.05).The protein and mRNA expression levels of Mstn and Caspase-3 in gastrocnemius of the mice in CC group were significantly higher and the expression level of LncRNA-MALAT1 was significantly lower than those in HC group (P < 0.05).The mRNA and protein expression levels of Mstn and Caspase-3 in FP and FT groups were reduced and the expression level of LncRNA-MALAT1 was increased compared with CC group (P < 0.05).The prevention effect in FP group is better than FT group (P < 0.05). Conclusion:FST may alleviate the muscle wasting of the mice with cancer cachexia by inhibiting the expression of Mstn,thus upregulating the expression of LncRNA-MALAT1 which in turn to suppress the expression of Caspase-3.

Effects of Electroacupuncture on Insulin-like Growth Factor-1, Myostatin and Satellite-cell Proliferation in Rats with Denervated Skeletal Muscle Atrophy / 中国康复理论与实践

@#Objective To observe effects and mechanism of electroacupuncture (EA) on denervated skeletal muscle atrophy. Methods Forty-nine male Sprague-Dawley rats were randomly divided into normal group (group A, n=7), natural recovery group (group B, n=21) and EA group (group C, n=21). The groups B and C, established the model of denervated skeletal muscle atrophy by transecting the sciatic nerve of rats, were divided into subgroups of 7 days, 14 days, 21 days postoperation, seven in each subgroup. Electroacupuncture was given to the group C at Zusanli (ST36) and Chengshan (BL57) once a day since 24 hours after modeling. The muscle wet weight ratio of the affected gastrocnemius was determined. Cross-sectional area and fiber diameter of the gastrocnemius were measured with HE staining. The expression of insulin-like growth factor-1 (IGF-1), Myostatin and proliferating cell nuclear antigen (PCNA) protein and gene in the gastrocnemius were detected with Western blotting and RT-PCR. Results The wet weight ratio, cross-sectional area and fiber diameter were less in the groups B and C than in the group A (P<0.001), and they were more in the group C than in the group B (P<0.001). Compared with the group B, the protein and gene of IGF-1, PCNA increased in the group C (P<0.05), while the Myostatin decreased (P<0.05). Conclusion Electroacupuncture can increase the expression of IGF-1 and decrease the expression of Myostatin, to promote the proliferation of satellite cell, which may relate with the prevention of denervated skeletal muscle atrophy.

Inhibitory effect of RNA interference of MSTN gene expression on the downstream genes in Schizopygopsis pylzovi / 中国实验动物学报

Objective To investigate the silencing effect of RNA interference on MSTN gene ( myostatin, MSTN) expression, and detect the effects on the downstream genes in Schizopygopsis pylzovi. Methods To construct the recombi?nant adenovirus vector 1P3 (DSP MSTN 273+250+1737) and 1P2 (DSP MSTN 195+ 1670) for RNA interference of the MSTN gene in Schizopygopsis pylzovi, and to conduct the RNA interference in vivo experiment by injecting the vector in?to the muscle tissue of Schizopygopsis pylzovi. Real?time PCR and Western blotting were used to evaluate the silencing effects on MSTN gene expression, and to detect the regulatory function of M?CK at gene transcription level after RNA inter?ference of the MSTN gene. Results The result of real?time PCR showed that compared with the HK team ( Virus general negative control group) and N team (blank control group), the 1P3 had significant interference effect on the MSTN gene transcription in Schizopygopsis pylzovi (P<0?05), with an inhibition rate of 53?5%, but the 1P2 had no significant inter?ference effect on the MSTN gene transcription. The result of Western blotting was consistent with the results of real?time PCR. At the same time, after the 1P3 interference, the level of MSTN gene transcription was declined, and the level of M? CK gene expression was significantly increased. Conclusions Our results demonstrate that the expression of MSTN gene can be effectively suppressed, and the expression of M?CK gene can be up?regulated through the RNA interference. There?fore, it proves that MSTN gene can inhibit the transcription of M?CK gene in Schizopygopsis pylzovi, and reveals the regula?tory role of MSTN gene in the muscle growth and development in the plateau fish Schizopygopsis pylzovi.

The potential role of myostatin and neurotransmission genes in elite sport performances.

Elite athletes are those who represent their sport at such major competition as the Olympic Games or World contests. The most outstanding athletes appear to emerge as a result of endogenous biologic characteristics interacting with exogenous influences of the environment, often described as a ‘Nature and Nurture’ struggle. In this work, we assessed the contribution given by 4 genes involved in muscles development (MSTN) and behavioural insights (5HTT, DAT and MAOA) to athletic performances. As for neurotransmission, 5HTT, DAT and MAOA genes have been considered as directly involved in the management of aggressiveness and anxiety. Genotypes and allelic frequencies of 5HTTLPR, MAOA-u VNTR, DAT VNTR and MSTN K153R were determined in 50 elite athletes and compared with 100 control athletes. In this work we found a significant correlation between the dopamine transporter genotype 9/9 and allele 9 and elite sport performances. On the contrary, no association was found between muscle development regulation or serotonin pathway and elite performances. Our data, for the first time, suggest a strong role of dopamine neurotransmitter in determining sport success, highlighting the role of emotional control and psycological management to reach high-level performances.