Quantitative comparison of the expression of myogenic regulatory factors in flounder(Paralichthys olivaceus) embryos and adult tissues

MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, MyJ5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions ofMyf5, MyoD, and myogenin at the early stages have a common characteristic： expression gradually increased to a peak level, and then gradually decreased to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages, the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage.

Characterization of flounder (Paralichthys olivaceus) FoxD5 and its function in regulating myogenic regulatory factor

As one member of winged helix domain transcription factors, FoxD5 was reported to be a trunk organizer. Recent study showed that zebrafish foxd5 is expressed in the somites. To further understand the function of FoxD5 in fish muscle development, the FoxD5 gene was isolated from flounder. Its expression pattern was analyzed by in situ hybridization, while its function in regulating myogenic regulatory factor, MyoD, was analyzed by ectopic expression. It showed that flounder FoxD5 was firstly expressed in the tailbud, adaxial cells, and neural plate of the head. In flounder embryo, FoxD5 is expressed not only in forebrain but also in somite cells that will form muscle in the future. When flounder FoxD5 was over-expressed in zebrafish by microinjection, the expression of zebrafish MyoD in the somites was reduced, suggesting that FoxD5 is involved in myogenesis by regulating the expression of MyoD.

Molecular Cloning,Characterization and Expression Profile of Myf5 and Myf6 During Growth and Development in the Seriola lalandi

Myogenic Regulatory Factors(MRFs)is involved in the muscle growth and differentiation.In this study,the cDNA sequence of yellowtail kingfish MRFs genes were cloned by rapid amplification of cDNA ends(RACE)method;then,the character-istics of these genes and the predicted protein sequences were analyzed by bioinformatics methods,the tissue and embryonic stages differential expression pattern were detected by the quantitative real-time PCR.Our results showed that the yellowtail kingfish(YTK)Myf5 cDNA has a full length of 951 bp,encoding 266 amino acids.The yellowtail kingfish Myf6 cDNA has a full length of 1105bp,encoding 250 amino acids.The proteins containα-helix,β-strand,and loops.The Neighbour-joining tree revealed that YTK Myf5 and Myf6 are closely related to Seriola dumerili.The yellowtail kingfish Myf5 and Myf6 gene expressed significantly higher in mus-cle than in other tissues(P<0.05).In addition,Myf5 and Myf6 in muscle was significantly expressed in 400g and 500 g fish but not in 50 g,suggesting that myogenic regulatory factors expression had a great relationship with the fish size.Our results also indicated that Myf5 and Myf6 have different functions during embryonic development,because Myf5 showed highest expression level at the neuroembryo period,but Myf6 had the highest expression level at embryo coverage yolk 70%stage.Myf5 gene showed highest ex-pression at 30 d of age,suggesting it played key roles in myogenic period.However,the Myf6 gene was significantly highly ex-pressed at 60 d,revealing this gene functioned in the later muscle formation period.

Effect of constant compressive stress induced by imitating tuina stimulation with various durations on the cell cycle,cellular secretion,apoptosis,and expression of myogenic differentiation and myogenic factor 5 of rat skeletal muscle cells in vitro

OBJECTIVE:To investigate the effect of constant compressive stress induced by imitating Tuina stimulation with various durations on the cell cycle,cellular secretion,apoptosis,and expression of myogenic regulatory factors(MRFs),myogenic factor 5(Myf5)and myogenic differentiation(MyoD)of rat skeletal muscle cells(RSkMCs)in vitro.METHODS:Third passage RSkMCs were subjected to constant compressive stresses with various durations at 2000μstrain for 15,30,60,90,and 120 min via a four-point bending system.The control group(CG)was cultured in the absence of mechanical loading.Alterations of the cell cycle and apoptosis rate were detected by flow cytometry(FCM).The concentrations of interleukin 6(IL-6)/prostaglandin E2(PGE2)and nitric oxide(NO)in supernatants were determined by enzyme-linked immunosorbent assays and the nitrate reductase method,respectively.Expression of Myf5 and MyoD was detected by immunohistochemistry.RESULTS:Compared with the CG,a significant alteration was observed in the synthesis phase fraction(SPF)(P<0.01).The SPF and proliferation index(PI)were reduced from 15 to 90 min,but reached levels similar to those at 120 min.Apoptosis was increased significantly at 30 min(P<0.05)and especially at 90 and 120 min(P<0.01).Expression of MyoD and Myf5 was increased significantly at 15,30,and 90 min(P<0.01).Compared with 15 and 30 min,MyoD and Myf5 expression at 60 and 120 min was decreased significantly(P<0.01).Compared with 60 min,M yoD expression at 90 min was increased significantly(P<0.05),whereas MyoD and Myf5 expression at 120 min was significantly lower(P<0.05).The IL-6 concentration was increased at 60 min compared with the CG and 15 min(P<0.05),whereas the concentrations of PGE2 and NO were the highest at 15 and 30 min,respectively,compared with the CG and other time points(P<0.05).CONCLUSION:The cell cycle,secretion,apoptosis,and Myf5 and MyoD expression of RSkMCs were regulated by compressive stress in a time-dependent manner.SPF and PI were inhibited at short durations(<90 min),but NO and PGE2 secretion was the highest at shorter durations(<30 min).With the prolongation of stimulation time,SPF,PI,and apoptosis were increased,but Myf5 and MyoD expression was decreased gradually at 15-30 min.

Gyejibokryeong-Hwan improves recovery of injured muscles in mouse model of muscle contusion

OBJECTIVE: To investigate the effects of Gyejibokryeong-Hwan(Guizhifuling-wan, GBH) on muscle injury in a mouse model of muscle contusion.METHODS: C57/BL6 mouse biceps femoris muscles were injured using the drop-mass method and injured animals were treated orally with GBH(50,100, or 500 mg/kg) once a day for 7 d. Open field and treadmill running tests were performed to assess functional recovery from muscle injury. The production of pro-inflammatory cytokines was examined by enzyme-linked immunosorbent assay and Western blotting analysis. Expression of the muscle regeneration biomarkers, myoblast determination(Myo D), myogenic factor 5(Myf5), and smooth muscle actin(α-SMA), in the biceps femoris muscle was investigated at the protein and m RNA level by Western blotting and real time-PCR, respectively. Histological analysis was performed using hematoxylin and eosin staining. Finally, myosin heavy chain production was investigated in differentiated C2C12 myoblasts in the presence of GBH.RESULTS: GBH treatment markedly improved locomotion and running behavior. GBH significantly inhibited the secretion of monocyte chemoattractant protein-1 into the bloodstream in muscle-contused animals. The levels of Myo D, Myf5, and α-SMA protein and m RNA were significantly up-regulated by GBH in injured muscle tissue. Histological studies suggested that GBH facilitated recovery from muscle damage. However, GBH did not induce the production of myosin heavy chain in vitro.CONCLUSION: Overall, the present study suggested that GBH improves the recovery of the injured muscles in the mouse model of muscle contusion.