Enhanced hyperplasia in muscles of transgenic zebrafish expressing Follistatin1

Myostatin is a member of the transforming growth factor-β(TGF-β) super-family and functions as a negative regulator of muscle growth.Binding of the specific receptor,Activin receptor IIB(Act RIIB),with myostatin or other related TGF-β members,could be inhibited by the activin-binding protein follistatin(Fst) in mammals.Overexpressing Fst in mouse skeletal muscle leads to muscle hypertrophy and hyperplasia.To determine if Fst has similar roles in fish,we generated transgenic zebrafish expressing high levels of zebrafish Fst1 using the promoter of the zebrafish skeletal muscle-specific gene,myosin,light polypeptide 2,skeletal muscle(Mylz2).Independent transgenic zebrafish lines exhibited elevated expression levels of myogenic regulatory genes MyoD and Pax7 in muscle cells.Adult Fst1 overexpressing transgenic zebrafish exhibited a slight body weight increase.The high level of Fst1 expression dramatically increased myofiber numbers in skeletal muscle,without significantly changing the fiber size.Our findings suggest that Fst1 overexpression can promote zebrafish muscle growth by enhancing myofiber hyperplasia.

Dynamic resistance exercise increases skeletal muscle-derived FSTL1 inducing cardiac angiogenesis via DIP2A-Smad2/3 in rats following myocardial infarction

Purpose:The aim of this study was to investigate the potential of dynamic resistance exercise to generate skeletal muscle-derived follistatin like-1(FSTL1),which may induce cardioprotection in rats following myocardial infarction(MI)by inducing angiogenesis.Methods:Male,adult Sprague-Dawley rats were randomly divided into 5 groups(n=12 in each group):sham group(S),sedentary MI group(MI),MI+resistance exercise group(MR),MI+adeno-associated virus(AAV)-FSTL1 injection group(MA),and MI+AAV-FSTL1 injection+resistance exercise group(MAR).The AAV-FSTL1 vector was prepared by molecular biology methods and injected into the anterior tibialis muscle.The MI model was established by ligation of the left anterior descending coronary artery.Rats in the MR and MAR groups underwent 4 weeks of dynamic resistance exercise training using a weighted climbing-up ladder.Heart function was evaluated by hemodynamic measures.Collagen volume fraction of myocardium was observed and analyzed by Masson’s staining.Human umbilical vein vessel endothelial cells culture and recombinant human FSTL1 protein or transforming growth factor-b receptor 1(TGFbR1)inhibitor treatment were used to elucidate the molecular signaling mechanism of FSTL1.Angiogenesis,cell proliferation,and disco interacting protein 2 homolog A(DIP2A)location were observed by immunofluorescence staining.The expression of FSTL1,DIP2A,and the activation of signaling pathways were detected by Western blotting.Angiogenesis of endothelial cells was observed by tubule experiment.One-way analysis of variance and Student’s t test were used for statistical analysis.Results:Resistance exercise stimulated the secretion of skeletal muscle FSTL1,which promoted myocardial angiogenesis,inhibited pathological remodeling,and protected cardiac function in MI rats.Exercise facilitated skeletal muscle FSTL1 to play a role in protecting the heart.Exogenous FSTL1 promoted the human umbilical vein vessel endothelial cells proliferation and up-regulated the expression of DIP2A,while TGFbR1 inhibitor intervention down-regulated the phosphorylation level of Smad2/3 and the expression of vascular endothelial growth factor-A,which was not conducive to angiogenesis.FSTL1 bound to the receptor,DIP2A,to regulate angiogenesis mainly through the Smad2/3 signaling pathway.FSTL1-DIP2A directly activated Smad2/3 and was not affected by TGFbR1.Conclusion:Dynamic resistance exercise stimulates the expression of skeletal muscle-derived FSTL1,which could supplement the insufficiency of cardiac FSTL1 and promote cardiac rehabilitation through the DIP2A-Smad2/3 signaling pathway in MI rats.

Establishment of a Technique to PCR Detection about Follistatin Transgenic Sheep by Injection of Lentivirus

[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead lambs were collected and used to extract DNA for PCR with primers designed for N＋D1 fragment of follistatin gene.At the same time,we detected the CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector.The body tissues of dead lambs and muscle tissues of transgenic lambs in vivo were used to extract RNA for RT-PCR.[Result]The results showed that the DNA Extraction Kit was faster and more efficient than conventional method in extracting DNA and the DNA extracted with kit was easier to be amplified than that with conventional method.In order to avoid false positive caused by the interference of endogenous gene,the primers were designed for amplifying the combination of upstream of vector gene and downstream of target gene,increasing the specificity of detection.Tail tissue of newborn transgenic lambs could be used for detection and the detected results were reliable and accurate.The detection of CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector provided a data support for the biological safety of transgenic animals and verify the detected results of target gene of transgenic lambs.The transcription products of RNA extracted from three of the lambs were not detected.[Conclusion] The PCR method established in our research for detecting transgenic sheep was efficient,fast and accurate.It would provide experimental basis for further detection at protein level,lay a foundation for the establishment of multi-level and systematic detection method of transgenic sheep and provide a stable technology platform for safety monitoring of transgenic sheep.