Key Laboratory for Poultry Genetics and Breeding of Jiangsu Province,Poultry Institute,Chinese Academy of Agricultural Sciences,Yangzhou 225125,P.R.China

Abstract Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α)played a prominent role in regulating muscle fiber type transition and composition.However,the role of PGC-1α in chicken muscle has seldom been explored.To investigate the effect of PGC-1α on chicken skeletal muscles in this study,the PGC-1α gene was overexpressed or silenced in chicken primary myoblasts by using lentivirus,and then the effects of the PGC-1α gene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation.The results showed that overexpression of PGC-1α from proliferation to differentiation was accompanied by the up-regulated expression of Pax7,MyoD,and CnAα,which was significantly (P<0.01) increased after one day of transfection (1I).The enhancement of MyoG,MEF2c,and MyHC SM expression lagged,which was improved significantly (P<0.01) after four days of transfection (1I3D).Overexpression of PGC-1α decreased (P<0.01) the MyHC FWM expression after four days of transfection (1I3D),and it had no significant impact (P>0.05) on the expression of CnB1,NFATc3,and MyHC FRM during myofiber formation.The effective silence (P<0.01) of PGC-1α by lentivirus mediating short hairpin RNA (shRNA) was detected after four days of transfection (1I3D) in cultures,and the lack of its function in chicken primary myoblasts significantly (P<0.01) down-regulated the expression of Pax7,MyoD, CnAα,MyoG,MEF2c,and MyHC SM,significantly (P<0.01) up-regulated the expression of MyHC FWM,and had no significant impact (P>0.05) on the expression of CnB1, NFATc3,and MyHC FRM.These results indicated that the role of PGC-1α in regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals,which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.

Keywords:PGC-1α gene,chicken,myofiber type specificity,mRNA expression profile

1.lntroduction

Vertebrate skeletal muscles contain a heterogeneous population of syncytial muscle fibers that developviathe fusion of a variable number of myoblasts.Traditionally,skeletal muscle fiber types are classified into types I (slowtwitch,oxidative),IIA (red fast-twitch,oxidative-glycolytic),and IIB (white fast-twitch,glycolytic) according to their contraction properties and metabolic properties (Brooke and Kaiser 1970a,b;Ashmore and Doerr 1971).Each fiber type is defined by the expression of specific myosin heavy chain (MyHC) isoforms.Previous studies have shown that the characteristics of muscle fibers influence meat quality in many aspects,such as the water-holding capacity,tenderness,color,juiciness,and flavor (Meynier and Gandemer 1994;Larzulet al.1997;Kimet al.2013;Wuet al.2015).It is commonly suggested that the proportion of type I fibers is positively correlated with meat quality.However,the mechanism for regulating specific skeletal muscle fiber types is complex,which involves induction of a phenotype during embryonic development independent of neural activity and maintenance or switching of this phenotype during activity-dependent postnatal development(Condonet al.1990;Booth and Thomason 1991;Jarviset al.1996;Hood 2001).To date,the biochemical basis for the regulation of fiber-type determination in skeletal muscle is not well understood.

Peroxisome proliferator-activated receptor gamm coactivator-1α (PGC-1α),as a potent transcription coactivator for nuclear receptors and certain other transcriptions in various signaling pathways,displays a dominant role in regulating mitochondrial biogenesis and oxidative metabolism (Linet al.2002;Villena 2015).Studies on mammal skeletal muscles have implicated that PGC-1α is expressed preferentially in muscle-enriched type I fibers and plays a principal role in regulating the switching and determination of muscle fiber types (Linet al.2002).Overexpression ofPGC-1αin both mice and pigs increases the proportion of oxidative type I fibers (Linet al.2002;Zhanget al.2017),whilePGC-1αknock-out (KO) mice exhibit a shift from oxidative type I and IIA fibers toward type IIX and IIB fibers (Handschinet al.2007).It is proposed that the cooperation betweenPGC-1αand the calcineurin signaling pathway is probably critically important in the switching of muscle fiber types or myofiber transformation to the oxidative muscle (Linet al.2002).However,few studies have reported the role of thePGC-1αgene in the specificity of skeletal muscle fiber in chickens.

In China,consumers often prefer native yellow feathered broiler chickens rather than fast-growing breeds,as they have high oxidative metabolism leading to desirable traits in muscle,such as a higher red muscle ratio and satisfactory meat color,marbling,and flavor (Shuet al.2014).Our previous studies on the differences in gene expression profiles between oxidative and glycolytic skeletal muscles in chicken suggested that thePGC-1αgene might play an important role in myofiber composition (Shuet al.2017).We hypothesized that thePGC-1αgene might regulate the specificity of skeletal muscle fiber in chickens.To verify this hypothesis,we herein overexpressed or silenced thePGC-1αgene in chicken primary myoblasts by using lentivirus,and then investigated its effects on the expression profile of genes related to the myofiber type specificity over a developmental time-course of fiber formation.

2.Materials and methods

2.1.Experimental design and treatments

A completely randomized design was adopted in this study.The isolated primary chicken myoblasts seeded in 24-well plates were infected with recombinant lentivirus,and then the cells were collected on one day in PM (1I),one day in PM and then three days of differentiation (1I3D),one day in PM and then eight days of differentiation (1I8D),respectively.At each detected time point,there were three treatment groups:the experimental group,the GFP group,and the cell control group.Each group of each detected time point was performed in at least six replicates.Three replicates were used for RNA extraction and quantitative real-time PCR,and the rest were used for protein extraction and Western blotting.

2.2.Recombinant lentiviral vector construction

The coding sequence (CDS) of chickenPGC-1αgene(GenBank accession no.NM_001006457.1) was generated by using PCR-mediated methodology and inserted intoNotI andNsiI restriction sites of pGLV5-CMV-GFP vector plasmid (GenePharma,China),then lentiviral expression vector (pGLV5-PGC-1α-GFP) carrying thePGC-1αgene,2 388-bp cDNA,was constructed.The set primers for PCR were forward 5´-ATTTGCGGCCGCGCCACCATGG-3´and reverse 5´-CCAATGCATTACCTGCGCAGGC-3´.Based on our previous research results,the sequences of shRNA (5´-GCAGGGATCCCAAGGTAATAA-3´)designed for chickenPGC-1αgene and NC shRNA(5´-TTCTCCGAACGTGTCACGTTTC-3´) designed as the control were synthesized and inserted respectively intoBamHI andEcoRI restriction sites of pGLV3-CMV-GFP vector plasmid supplied by Suzhou GenePharma Co.,China (Shuet al.2017).

Lentiviral vector particles were produced following the manufacturer’s instructions (GenePharma,China).The recombinant plasmid vector pGLV5-PGC-1α-GFP,pGLV3-PGC-1α-shRNA,pGLV3-NC-shRNA,and pGLV5-CMV-GFP empty-vector plasmid were co-transfected respectively into 293T cells with the third-generation packaging plasmids pGag/Pol and pRev and the envelope plasmid pVSV-G by using RNAi-Mate Reagent (GenePharma,China).Viral supernatants were harvested at 48 and 72 h posttransfection,centrifuged for 10 min at 3 000 r min–1under 4°C to remove cell debris,filtered through 0.45-μm cellulose acetate filters and concentrated using ultracentrifugation.The viral pellet was resuspended in serum-free Dulbecco’s Modified Eagle’s Medium (DMEM),and the infectious titer was determined by counting GFP-positive cells after gradient dilution.The produced viral titers were approximately 3×108TU (transduction units) mL–1.

2.3.lsolation and culture of chicken primary myoblasts

Chicken primary myoblasts isolation and culture were prepared according to the previous method (Luoet al.2014)with few modifications.Leg muscle samples were isolated from the embryos of Chinese Qingyuan Partridge chickens on the 11th day of hatching.The muscle tissue was washed twice with DMEM and minced into small pieces of 1 mm2with sharp scissors in proliferation medium (PM) consisting of DMEM/F12 medium (Gibco,USA),20% fetal bovine serum(FBS) and 1% penicillin/streptomycin.To obtain separate cells,the suspension was shaken by vortexing and filtrated with 100-,200-,and 400-mesh filters to remove large debris,and then the cells were collected by centrifugation at 1 000 r min–1for 5 min and resuspended in PM.Serial plating was performed to enrich myoblasts and eliminate fibroblasts.Differentiation of the myoblasts was induced in the differentiation medium (DM) by replacing 20% FBS with 2% horse serum (Hyclone,USA).

2.4.Transfection and expression assays of chicken myoblasts

The concentration of chicken myoblasts was adjusted to 5×106cells mL–1with PM,and then seeded in 24-well plates and cultured in a 5% CO2incubator at 37°C.When the cells had grown to approximately 80% confluence,myoblasts were infected with recombinant lentivirus pGLV5-PGC-1α-GFP,pGLV5-CMV-GFP,pGLV3-PGC-1α-shRNA,and pGLV3-NC-shRNA respectively,at a multiplicity of infection(MOI) of 10 in PM.On the next day,the transfection medium was replaced with DM.The cells were harvested after one day,four days,and nine days of infection,respectively.

2.5.RNA isolation,reverse transcription,and quantitative real-time PCR

Total RNA was extracted from cells with RNA Isolation Kit(TIANGEN,Beijing,China) according to the manufacturer’s instructions.The RNA integrity and concentration were assessed by denatured gel electrophoresis and NanoDrop 2000c (Thermo,Waltham,MA,USA).cDNA synthesis for mRNA was carried out using QuantScript RT Kit (TIANGEN,Beijing,China) before quantitative real-time PCR (qPCR).qPCR was conducted in the Stratagene Mx3000P qPCR System (Agilent Technologies Inc.,USA) using SuperReal PreMix Plus (SYBR Green) (TIANGEN,Beijing,China),and quantification was done as previously described (Taoet al.2015).Primer sequences ofPGC-1α,Pax7,MyoD,MyoG,CnAα,CnB1,MEF2c,NFATc3,SM,FWM,andFRMgenes were shown in Table 1.To calculate the mRNA expression of the target genes,ΔCt values were used for the detection of mRNA related to internal controlACTBexpression using the 2–ΔΔCtmethod.

Table 1 Primers for quantitative real-time PCR

2.6.Protein isolation and Western blotting

Total protein was extracted from cells using radio immune precipitation assay (RIPA) buffer (Beyotime,China) with phenylmethane sulfonyl fluoride (PMSF) protease inhibitor(Beyotime,China).After incubation on ice for 10 min,the supernatant was collected by centrifugation at 10 000 r min–1for 5 min at 4°C.The protein concentration was assessed by Coomassie blue staining.Western blotting was performed according to the standard procedures,and the antibodies used in this study were as follows:anti-PGC-1α antibody(ab 106814;Abcam,USA),anti-GAPDH antibody (A00191-40;GenScript,China) and peroxidase-donkey anti-goat IgG antibody (A00178;GenScript,China).

2.7.Statistical analysis

Data were analyzed by ANOVA using SAS 8.2 package(SAS Institute Inc.,Cary,NC).The obtained results were presented as the mean±SE.Statistical significance of differences among means was analyzed by the least significant difference (LSD) with a significant difference level ofP-value less than 0.05 and a highly significant difference level ofP-value less than 0.01.Each replicate served as the experimental unit for all statistical analyses.

3.Results

When the cells had grown to approximately 80% confluence,chicken myoblasts were infected respectively with recombinant lentivirus pGLV5-PGC-1α-GFP,pGLV5-CMVGFP,pGLV3-PGC-1α-shRNA,and pGLV3-NC-shRNA and cultured in PM for one day (1I),then allowed to differentiate for eight days in DM (1I8D) on which muscle differentiation reached an advanced stage (Fig.1).Cells collected from the uninfected control group and infected groups over a three-point time-courses (1I,1I3D,and 1I8D) were subjected to quantitative analysis of targeted genes by qPCR and Western blotting.The chicken myoblasts transfected with recombinant lentivirus for one day in PM and then three days in DM (1I3D) were observed using fluorescence microscopy,and the positive cells were showed as green (Fig.2).

3.1.Physiological overexpression or inhibition of the PGC-1α gene in chicken myoblasts

Throughout the experimental period from proliferation to differentiation,the expression trends ofPGC-1αmRNA and protein in three groups were coincident,and there was no significant difference (P>0.05) between the GFP group and cell control group at each detected time (Fig.3).Compared with the GFP group and cell control group,the expression level ofPGC-1αmRNA in thePGC-1α-overexpressed group was the highest over a three-point time-course which resulted in 9309,137 and 97.6% increase (P<0.01) relative to that of the control group on each detected time from proliferation to differentiation (Fig.3-A).The expression level of PGC-1α protein in thePGC-1α-overexpressed group was also the highest of three groups from proliferation to differentiation and significantly up-regulated relative to that of the control group on 1I3D (P<0.05) and 1I8D (P<0.01)(Fig.3-B).Conversely,compared with the GFP group and cell control group,the expression level ofPGC-1αmRNA in thePGC-1α-shRNA group was decreased on 1I3D,which resulted in 63.7% decrease (P<0.01) on 1I3D and 59.1%decrease (P<0.01) on 1I8D respectively relative to that of the control group (Fig.3-C).Corresponding to the expression level of mRNA,the expression level of PGC-1α protein in thePGC-1α-shRNA group was significantly down-regulated(P<0.01) relative to that of the control group on 1I3D and 1I8D (Fig.3-D).

Fig.1 Microscopic images of chicken primary myoblasts during fiber formation.A,chicken primary myoblasts were isolated from leg muscle of chicken embryos on the 11th day of hatching and cultured in proliferation medium (PM) for one day.B,the myoblasts were cultured in PM for one day (1I),and a few myotubes appeared.C,the myoblasts were cultured in differentiation medium(DM) for eight days (1I8D).Muscle differentiation reached an advanced stage.Lots of myotubes appeared (black arrows),and became longer and thicker.

Fig.2 Chicken myoblasts infected with recombinant lentivirus for one day,allowed to differentiate for three days,and viewed under fluorescence microscopy for GFP.A,proliferating chicken myoblasts were infected with recombinant lentivirus pGLV5-PGC-1α-GFP and empty-vector lentivirus pGLV5-CMV-GFP under fluorescent light and normal light.B,proliferating chicken myoblasts were infected with recombinant lentivirus pGLV3-PGC-1α-shRNA and empty-vector lentivirus pGLV3-NC-shRNA under fluorescent light and normal light.Scar bar:100 μm.

3.2.Regulatory mRNA expression profile of several key genes in chicken skeletal myocyte differentiation

During embryonic development,myogenic regulatory factors(MRFs) in concert with co-factors directing the expression of muscle-specific genesviatheir DNA-binding elements,E-boxes,control the formation of muscle fiber (Armandet al.2008;Daouet al.2013).MyoDandMyoGare two main members of the MRF family and have been recognized to play a crucial role in myocyte specification and differentiation for three decades (Zammit 2017).The muscle satellite cell marker genePax7as an upstream regulator of myogenesis plays an important role in satellite cell transition from proliferation/self-renewal to differentiation (Chenet al.2010;Buckingham and Relaix 2015).

Since the molecular pathway regulating fiber type specificity is superimposed on the myocyte differentiation program,here we examined the time-course effect ofPGC-1αoverexpression or inhibition in chicken myoblasts on the mRNA expression of several key genes includingPax7,MyoD,andMyoGduring the process of skeletal myocyte differentiation.Quantitative analysis by real-time PCR revealed that overexpression ofPGC-1αin chicken myoblasts significantly increased (P<0.01) thePax7andMyoDexpression throughout the experimental period,while the expression enhancement ofMyoGlagged,which was significantly improved (P<0.01) on 1I3D,as shown in Fig.4-A.In contrast,the inhibition ofPGC-1αin chicken myoblasts significantly down-regulated (P<0.01) the expression ofPax7,MyoD,andMyoGon 1I3D and 1I8D,as shown in Fig.4-B.Throughout the experimental period from proliferation to differentiation,the expression trends ofPax7andMyoDmRNA in all cell groups were declined,whereas that ofMyoGmRNA in all cell groups declined on 1I3D (Fig.4).

Fig.3 Physiological overexpression or inhibition of the PGC-1α gene in chicken myoblasts.A,the relative mRNA expression level of PGC-1α gene from PGC-1α-overexpressed chicken myoblasts.B,the relative protein level of PGC-1α gene from PGC-1αoverexpressed chicken myoblasts.C,the relative mRNA expression level of PGC-1α gene from PGC-1α-silenced chicken myoblasts.D,the relative protein level of PGC-1α gene from PGC-1α-silenced chicken myoblasts.1I,one day in proliferation medium (PM);1I3D,one day in PM and then three days of differentiation (DM);1I8D,one day in PM and then eight days of differentiation.The results were expressed as the mean±SE of three replicates within the same experiment.Statistical significance of differences among means was analyzed by the least significant difference (LSD) (＊,P<0.05;＊＊,P<0.01).

3.3.Regulatory mRNA expression profile of key component genes in the calcineurin signaling pathway

Calcineurin signaling pathway is involved in multiple aspects of skeletal muscle development,and it demonstrates a major role in both specifying activity-dependent fiber types in adult skeletal muscle (Semsarianet al.1999;Armandet al.2008;Hudsonet al.2014) and establishing the early muscle fiber types in immature myofibers during embryogenesis(Daouet al.2013).Calcineurin is an enzyme complex that comprises a calcineurin A catalytic subunit (CnA) and a calcium-binding calcineurin B regulatory subunit (CnB).There are three major gene isoforms ofCnA(α,β,and γ)and two gene isoforms ofCnB(1 and 2).OnlyCnAα,CnAβ(Aαis more abundant thanAβ) andCnB1are expressed in skeletal muscles.TheNFATfamily andMEF2family are two known effectors of calcineurin signaling on gene expression in skeletal muscles (Costaet al.2007).In C2C12 myoblasts,NFATc3is the onlyNFATfamily member that translocates to the nucleus at the onset of myoblast differentiation in response to calcineurin,enhancing myogenesis (Dellinget al.2000).During mouse and chicken embryogenesis,MEF2cis the firstMEF2family member to be expressed(Edmondsonet al.1994).In cultured C2C12,PGC-1αwas showed to activate transcription of fiber-type specific promoters in cooperation with MEF2 proteins and served as a target for calcineurin signaling,which has been implicated in slow fiber gene expression (Linet al.2002).

Fig.4 Regulatory mRNA expression profile of several key genes in chicken skeletal myocyte differentiation.Quantitative real-time PCR was performed by the ΔCt method to quantify the expression levels of Pax7,MyoD,and MyoG mRNAs in cells collected from control and infected groups on one day,four days and nine days of transfection (1I,1I3D and 1I8D),respectively.A,the relative mRNA expression level of Pax7,MyoD and MyoG genes from PGC-1α-overexpressed chicken myoblasts.B,the relative mRNA expression level of Pax7,MyoD and MyoG genes from PGC-1α-silenced chicken myoblasts.The results were expressed as the mean±SE of three replicates within the same experiment.Statistical significance of differences among means was analyzed by the least significant difference (LSD) (＊＊,P<0.01).

To investigate whether thePGC-1αgene cooperates with the calcineurin signaling pathway in chickens,we examined the time-course effect ofPGC-1αoverexpression or inhibition in chicken myoblasts on the mRNA expression ofCnAα,CnB1,NFATc3,andMEF2c.The real-time PCR results showed that overexpression ofPGC-1αin chicken myoblasts significantly increased (P<0.01) the mRNA expression level ofCnAαthroughout the experimental period and significantly increased (P<0.01) the mRNA expression level ofMEF2con 1I3D (Fig.5-A).In contrast,the inhibition ofPGC-1αin chicken myoblasts significantly down-regulated(P<0.01) the expression ofCnAαandMEF2con 1I3D and 1I8D (Fig.5-B).Both overexpression and inhibition ofPGC-1αin chicken myoblasts did not affect(P>0.05) the expression ofCnB1andNFATc3,as shown in Fig.5-A and B.In the GFP group and cell control group,the expression trends ofCnAα,CnB1,andNFATc3mRNA were rising while the expression trend ofMEF2cmRNA was declined during the entire experimental period from proliferation to differentiation (Fig.5).

3.4.Regulatory mRNA expression profile of myosin heavy chain genes

Myofiber type is defined by the isoform identity of the skeletal myosin heavy chain (MyHC).Here mRNA expression of threeMyHCgenes,including slow-twitch myofiber(SM),red fast-twitch myofiber(FRM),andwhite fast-twitch myofiber(FWM) during fiber formation of chicken myoblasts over a three-point time-course was detected.The quantitative real-time PCR results showed that overexpression ofPGC-1αin chicken myoblasts significantly increased(P<0.01) the expression ofMyHC SMand significantly decreased (P<0.01) the expression ofMyHC FWMon 1I3D,respectively (Fig.6-A).In contrast,the inhibition ofPGC-1αin chicken myoblasts significantly down-regulated(P<0.01) the expression ofMyHC SMand significantly increased (P<0.01) the expression ofMyHC FWMon 1I3D,respectively (Fig.6-B).Both overexpression and inhibition ofPGC-1αin chicken myoblasts did not affect (P>0.05) on the expression ofMyHC FRM,as shown in Fig.6.During the experimental period from proliferation to differentiation,the expression trends of fastMyHC(FWMandFRM) mRNA were similar in all cell groups,which were the highest on 1I,declined on 1I3D and then raised on 1I8D,while the expression trends ofMyHC SMmRNA in all cell groups began to decline on 1I3D (Fig.6).

Fig.5 Regulatory mRNA expression of key component genes in the calcineurin signaling pathway.Quantitative real-time PCR was performed by the ΔCt method to quantify the expression levels of CnAα,CnB1,NFATc3,and MEF2c mRNAs in cells collected from the control and infected groups on one day,four days and nine days of transfection (1I,1I3D and 1I8D),respectively.A,the relative mRNA expression level of CnAα,CnB1, NFATc3,and MEF2c genes from PGC-1α-overexpressed chicken myoblasts.B,the relative mRNA expression level of CnAα,CnB1, NFATc3, and MEF2c genes from PGC-1α-silenced chicken myoblasts.The results were expressed as the mean±SE of three replicates within the same experiment.Statistical significance of differences among means was analyzed by the least significant difference (LSD) (＊＊, P<0.01).

4.Discussion

The hypothesis that thePGC-1αgene might regulate the specificity of skeletal muscle fiber in chickens was supported by the results of the present study.The present study has demonstrated that thePGC-1αgene and shRNA directed to thePGC-1αgene were transduced efficiently into primary myoblasts by lentivirus vectors,and thePGC-1αgene could induce the expression of myofiber specificity marker genes and key genes of calcineurin signaling which were implicated in slow fiber gene expression.Overexpression ofPGC-1αgene eventually up-regulated the expression ofMyHC SMas well asPax7,MyoD,MyoG,CnAα,andMEF2c,and meanwhile down-regulated the expression ofMyHC FWMin chicken skeletal muscle cells over the development period of fiber formation.Conversely,inhibition of thePGC-1αgene significantly down-regulated the expression ofMyHC SM,MyoD,MyoG,CnAα,andMEF2c,and upregulated the expression ofMyHC FWM.Therefore,the increasedPGC-1αgene expression in chicken skeletal muscle could facilitate more oxidative myofiber formation.This result might ultimately help improve the control of muscle fiber types,which would be beneficial to meat quality improvement in broiler breeding.

Skeletal muscle consists of various types of myofibers,and a higher proportion of oxidative (slow) myofibers is hypothesized to be beneficial for meat quality (Karlssonet al.1999;Lefaucheur 2010).The relative percentage of a specific myofiber type can be estimated by quantification of theMyHCgene expression level in skeletal muscles(Tidymanet al.1997).As the expression pattern of eachMyHCisoform is different in time and myofiber nature,the signaling pathways and transcriptional regulators of eachMyHCisoform are probably different (Daouet al.2013).In this study,the simultaneous response ofMyHC SMandMyHC FWMtoPGC-1αoverexpression or inhibition in chicken myoblasts after four days of transfection (1I3D)indicated that both processes (elevation and repression)were under active regulation during fiber formation.

Fig.6 Regulatory mRNA expression profile of myosin heavy (MyHC) genes.Quantitative real-time PCR was performed by the ΔCt method to quantify the expression levels of MyHC SM,MyHC FWM,and MyHC FRM mRNAs in cells collected from the control and infected groups on one day,four days and nine days of transfection (1I,1I3D and 1I8D),respectively.A,the relative mRNA expression level of MyHC SM,MyHC FWM,and MyHC FRM genes from PGC-1α-overexpressed chicken myoblasts.B,the relative mRNA expression level of MyHC SM,MyHC FWM,and MyHC FRM genes from PGC-1α-silenced chicken myoblasts.The results are expressed as the mean±SE of three replicates within the same experiment.Statistical significance of differences among means was analyzed by least significant difference (LSD) (＊＊,P<0.01).

Calcineurin signaling is a chief regulatory pathway of gene expression selected by slow-twitch fiber (Olson and Williams 2000).Several reports have shown thatPGC-1αserves as a target for calcineurin signaling,playing a prominent role in regulating muscle fiber type determination(Linet al.2002;Roberts-Wilsonet al.2010).In mice,thePGC-1αpromoter was found regulated by theCnAactivity.Moreover,PGC-1αwas observed to regulate its promoter through interactions with the components in the calcineurin signaling pathway.These results showed that there existed an autoregulatory loop controllingPGC-1αexpression in mice muscles (Handschinet al.2003).At present,there is fewer study on the effect ofPGC-1αon calcineurin.Here we found that overexpression ofPGC-1αin chicken myoblasts could increase the expression ofCnAα,while inhibition ofPGC-1αin chicken myoblasts could decrease the expression ofCnAα.This result suggested that in chicken myoblasts,PGC-1αcould also retro-regulate the expression ofCnsince CnA acted as an active subunit of Cn.It might be explained that there was complex gene network regulation in organisms,and there existed a reciprocal regulationbetweenPGC-1αand key genes related to myofiber type specificity.

TheNFATfamily andMEF2family are two known effectors of calcineurin signaling on gene expression of type I fibers in skeletal muscle.MEF2cwas up-regulated byPGC-1αin this study.However,the response ofNFATc3toPGC-1αwas not detected throughout our experiment.The result was in agreement with the previous study by Linet al.(2002) in mice,whereinPGC-1αwas shown to increase the expression of type I fibrillar proteins by co-activating MEF2 transcription factors and no functional interaction between PGC-1α and NFAT on troponin I (slow) promoter was observed.

The molecular pathway regulating the fiber type specificity is superimposed on the myocyte differentiation program.Until now,the role ofPGC-1αin myogenic differentiation has seldom been elucidated.Several reports have suggested the existence of a positive regulatory loop to control the expression of bothMyoDandPGC-1α.MyoDcould activate thePGC-1αgene expression by targeting its promoter in C3H10T1/2 cells (Changet al.2006).Guerfaliet al.(2007) found that thePGC-1αgene electrotransfer transactivatedMyoDpromoter in ratTibialis anteriormuscle.In this study,we determined that the expression trend ofPGC-1αin chicken skeletal muscle cells was declined from proliferation to differentiation,which was similar to that ofPax7andMyoD.Together with the results thatPGC-1αoverexpression increased the expression of these two genes andPGC-1αinhibition decreased the expression of these two genes during fiber formation,we speculated thatPGC-1αwas associated with chicken muscle differentiation,as the role ofPGC-1αreported in murine C2C12 (Linet al.2014).GivenPax7 and MyoD are up-regulators of MyoG (Collinset al.2009),and MEFsare the substrates of calcineurin (Costaet al.2007),it is not surprising that the up-regulated expression ofMyoGandMEF2cwas later than that ofPax7,MyoD,andCnAαwhen thePGC-1αgene was overexpressed in chicken myoblasts.

5.Conclusion

In this study,we successfully studied the function of thePGC-1αgenein vitrousing recombinant lentivirus.The responses of key genes related to calcineurin signaling pathway and myocyte differentiation toPGC-1αoverexpression or inhibition during myofiber formation in chicken may explain,at least in part,howPGC-1αregulates the specificity of chicken skeletal muscles.These results might ultimately be useful in improving meat quality traits in broiler breeding.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31301967),the Natural Science Foundation of Jiangsu Province,China (BK20161322),the projects of Key Laboratory for Poultry Genetics and Breeding of Jiangsu Province (JQLAB-ZZ-201703),the Major Breeding Programs in Jiangsu Province,China(PZCZ201728),the earmarked fund for China Agriculture Research System (CARS-41),the Independent Scientific Foundation of Public Welfare Scientific Institutes in Jiangsu Province,China (BM2018026) and the Open Projects of Key Laboratory of Chicken Genetics and Breeding,Ministry of Agriculture and Rural Affairs of China (CGB-201704).