ZHANG Ya-wen, YANG Qian,WANG Feng, ZHANG Li-feng

(Department of Cardiology,Children’s Hospital,Fudan University,Shanghai 201102,China)

【Abstract】 Objective To study the effect of retinaldehyde dehydrogenase 2 (Raldh2) on the differentiation of P19 cell to cardiomyocyte-like cells and explore the potential mechanism. MethodsA miRNA expression plasmid specific to Raldh2 was packaged and constructed by RNA interference (RNAi) method.The P19 stable cell line carried Raldh2 miRNA expression was selected by adding blasticidin and induced to differentiate towards cardiomyocyte-like cells.The mRNA levels of myocardium development-related markers were determined by qPCR at different stages during the differentiation process. Results The miRNA expression plasmid specific to Raldh2 could effectively suppress Raldh2 expression,and the MiRaldh2 group,a P19 stable cell line was established successfully in which the knockdown efficiency on Raldh2 was 91% (t=25.52,P<0.000 1,95% CI:0.81-1.01).When compared with P19 group,the mRNA levels of cardiac transcription factors were generally decreased in the MiRaldh2 group during the whole differentiation process.In detail,on the 7th day,the relatively low expression rates of these cardiac markers including Gata4,Tef-1,N-myc,α-mhc and Ctnt was 0.16±0.01 (t=17.29,P<0.000 1),0.51±0.02 (t=3.564,P=0.023 5),0.23±0.01 (t=13.17,P=0.000 2),0.20±0.02 (t=17.76,P<0.000 1) and 0.59±0.06 (t=3.642,P=0.021 9) in MiRaldh2 group when compared with the P19 group.Conversely,the mRNA levels of Nkx2.5 and Hand2 were dramatically increased in MiRaldh2 group on day 2 to 7 and the expression rates on the 7th day was 2.25±0.35 (t=3.526,P=0.024 3) compared with the P19 group while Hand2 was 3.58±0.20 (t=9.214,P=0.011 6). Conclusions Knockdown of Raldh2 inhibits the P19 cells differentiated into cardiomyocyte-like cells,which suggests that Raldh2 may play a potential role in early development of heart.The low expression of Raldh2 might be an explanation of the cardiac malformations associated with retinoic acid deficiency.

【Key words】 retinaldehyde dehydrogenase (Raldh); retinoic acid; cardiomyocyte-like cell; cell differentiation; P19 cell

Congenital heart disease (CHD) is termed as the most common congenital defects in children.The worldwide incidence of CHD is 8‰-12‰[1],and in China it is approximately 11‰[2].From the perspective of genetics,it has always been the hotspot of cardiology researchers to reveal the pathogenesis of CHD.During cardiac development,endogenous retinoic acid (RA) is necessary to restrict second heart field posteriorly,andinvivotreatment with an inhibitor of RA synthetase could delay the migration of epicardial-derived precursor cells into the myocardium[3].Nutritional and clinical studies on animals and humans have shown that maternal vitamin A insufficiency can result in fetal death,or many types of abnormalities such as heart defects[4].Secreted from the epicardium,RA is required for the proliferation of cardiomyocytes.Notably,retinaldehyde dehydrogenase (Raldh) catalyzes the oxidation of retinaldehyde to RA in the retinoic acid pathway,andRALDH2,also known as aldehyde dehydrogenase 1 family,member A2 (ALDH1A2) gene plays an essential role in this metabolism.It was reported thatRaldh2-/-mice embryos exhibited several cardiac malformations including defects of heart looping,reduced atria and sinus venosus outgrowth[5].Moreover,a severe hypoplasia of the ventricular myocardium was characterized inRaldh2-/-mouse embryo,and the activity of RA was undetectable in epicardium as well as the adjacent myocardium[4,6].However,the role ofRaldh2 in myocardial development and differentiation has not been completely revealed.

P19 cell is an embryonic carcinoma cell line derived from an embryo-derived teratocarcinoma in mice.At concentration of 0.5%-1% (v/v),DMSO induced P19 cell aggregates to develop into a wide variety of mesodermal and endodermal cell types efficiently[7-8].The cardiac myocytes derived from P19 cells display the biochemical and physiological properties that occur on day 6 to 7 of induction of DMSO[9].Thus,the P19 cell line is known as one of the most suitable models for cardiac differentiation at the molecular and functional levels[10].

In addition,recent studies have also suggested that transcription factors (TFs) such asGata4,Tef-1,N-myc,α-mhc,Ctnt,Nkx2.5 andHand2,all of which are involved with the cardiomyocyte commitment and have been identified to modulate the process of cardiomyocyte differentiation[11].While how those factors involved in RA-Raldh2 signaling pathway on myocardial differentiation is not clear.During July 2015 to Oct.2016,we investigated the effect ofRaldh2 on myocardial differentiation by using P19 cell model and testing the expressions of cardiac markers whenRaldh2 was knocked down.

Materials and Methods

Cell cultureP19 cells were purchased from Invitrogen (Shanghai) Trading Co.,Ltd.,and cultured in 100 mm tissue culture grade bacteriological dish containing 1640 (Invitrogen,USA) supplemented with 10% fetal bovine serum (Invitrogen,USA),100 U/mL penicillin (Invitrogen,USA) and 100 μg/mL streptomycin (Invitrogen,USA) at 37 ℃ in 5% CO2(Thermo,USA).

P19 cell differentiation experimentsProtocol of inducing cardiomyocyte-like cells differentiation:day 0,cells were transferred into 10 cm bacterial dishes containing a thin layer of 5 g/L agar medium to prevent adhesion,in a concentration of 1*106cells/mL in a volume of 9 mL culture medium in the presence of 1% DMSO (Sigma,USA).After 1 day,7 mL of fresh medium was added and replaced by fresh medium on day 2.Day 4,aggregates (embryoid bodies) were digested and transferred into 6 cm tissue culture dishes at a density of 1*105cells/mL and allowed to adhere.Medium was refreshed every two days.Cells were harvested and digested by 0.25% trypsin (Invitrogen,USA) and 0.02% ethylenediamine tetraacetic acid (EDTA;Invitrogen,USA) for induced subculture on day 0,2,4,7 and 14 of differentiation.

Construction ofRaldh2 miRNA interference expression plasmids and determination of knockdown efficiencyThe sequences of four pairs of miRNA oligo (Tab 1) were designed and synthesized based onRaldh2 sequences.The oligos were annealed into double chains and inserted into miRNA expression vectors pcDNATM6.2-GW/EmGFPsiR respectively using BLOCK-iTTMPol Ⅱ miR RNAi Expression Vector Kit with EmGFP (Invitrogen,USA).These four miRNA interference plasmids were constructed using HQ High Purity Plasmid Extraction Kit (Invitrogen,USA) and transformed into receptive cell DH5α (Invitrogen,USA).PCR and sequencing were performed to ensure the positive clone was constructed successfully.The primer sequences of pcDNATM6.2-GW/EmGFPmiR were synthesized by Invitrogen.Using Lipofectamine 2 000 reagent (Invitrogen,USA) according to the manufacturer’s instructions,3 μg of interference plasmids and negative control plasmids MR_neg (C-) were transfected into 293T cells (Invitrogen,Shanghai) transiently.After 48 h transfection,the knockdown efficiency of fourRaldh2 miRNA interference expression plasmids were detected by qPCR carried out using Power SYBR Green Kit and Applied Biosystems (Thermo,USA).The transcripts for GAPDH were used for internal normalization and the sample transfected with negative interference plasmid was used as control.

Tab 1 miRNA oligo sequences (including negative control sequence)

Four pairs of miRNA oligo were designed and synthesized based onRaldh2.miRaldh2-1:pcDNATM6.2-GW/EmGFPmiRNA_1;miRaldh2-2:pcDNATM6.2-GW/EmGFPmiRNA_2;miRaldh2-3:pcDNATM6.2-GW/EmGFPmiRNA_3;miRaldh2-4:pcDNATM6.2-GW/EmGFPmiRNA_4;F:Forward;R:Reverse.

Tab 2 Primer sequences used for qPCR amplify Raldh2 and cardiomyocyte-specific markers in P19 cell lines

Construction of lentivirus expression vector with highestRaldh2 interference efficiency and multiplicity of infection (MOI) testThe fragment ofRaldh2 miRNA expression plasmid with the highest interference efficiency was inserted into the lentivirus expression vectors pLenti6.3/V5-DEST (Invitrogen,USA).BP and LR recombination systems were provided by Invitrogen.DNA sequencing was carried out to verify the sequence information of inserted fragments in recombinant clones.The interference plasmid and the packaging plasmid (Invitrogen,USA) were transfected into 293T cells.After 48 h transfection,the biological titer of lentivirus was determined by collecting supernatant concentrated by ultracentrifugation.293T cells were plated in 96-well plates at 8 000 cells per well and transfected with virus liquid using gradient dilution method contain 8 μg/mL polybrene (Sigma,USA) and 2% FBS at a fusion density of 30%-50%.Transfection efficiencies were determined by counting the green fluorescent protein (GFP) with fluorescence microscope OLYMPLS IX71 (Olympls,Japan) and the titer of the virus was the number of GFP multiplied by the corresponding dilution multiple.After calculating the virus titer,the virus solution was added to the cultured P19 cells (2*104cells per well in 96-well plates) at MOI=1,5,10,30,60,120,respectively.Cells were observed every 24 h and photos were taken by fluorescence microscope in the same field of vision.Cells were harvested on day 0,2,4,7 and 14 of differentiation.

Establishment of the MiRaldh2 group,a P19 stable cell line carried both the blasticidin-resistent gene andRaldh2 miRNAP19 cells were plated in 6-well plates at a concentration of 5*105cells per well.Lentivirus liquid ofRaldh2 miRNA interference expression vector as well as control miRNA vectors were added into P19 cells.Then 24 h later,the antibiotic blasticidin (Invitrogen,USA) was added to remove untransduced cells.StableRaldh2 silenced P19 cell lines were isolated and amplified after 14 days of selection in blasticidin-resistent condition.

Kill curve testP19 cells in logarithmic growth were plated in 24-well plates at a density ratio of 1∶10.Twenty-four hours later,blasticidin were added into cells with different concentration gradients,and three complex holes were set for each gradient.Medium supplemented with blasticidin was refreshed every 3 days.

Total RNA isolation and real-time polymerase chain reaction (qPCR)Using reverse transcription kit (Invitrogen,USA) according to the manufacturer’s instructions,total RNA from each group was extracted with Trizol reagent(Invitrogen,USA) and 1 μg was used to synthesize cDNA.qPCR reactions were performed using the SYBR Green qPCR Kit (Toyobo,Japan) on an Applied Biosystems 7500 Sequence Detection System (Thermo,USA).Samples were subject to the following cycles:holding at 95 ℃ for 2 min;40 repeat cycles at 95 ℃ for 10 s,60 ℃ for 30 s,and 70 ℃ for 45 s;melt at 70-95 ℃.GAPDH was used as a reference to obtain the relative expression for target genes using the comparative cycle threshold (Ct) method calculated by the formula:2-(ΔΔCt)(ΔΔCt=Ct(target gene)-Ct(reference gene)-Asame value).

Statistical analysisEach experiment was performed at least three times and all data are presented as mean ± standard error of the mean (SEM).Statistical analysis was performed by two-tailed unpaired Student’sttest with Graphpad Prism 6.0 software.Differences were considered statistically significant atP<0.05.

Results

Identification of the most suitableRaldh2 miRNA interference plasmidsFirst,we detected the mRNA level ofRaldh2 in P19 cells by qPCR.Then we constructed four interference plasmids specific toRaldh2.The consistency between the sequence of inserted fragment in recombinant clone and the designed oligos demonstrated that miRNA interference plasmids were successfully constructed.To determine the most suitable interference plasmid,we evaluated silencing efficiency ofRaldh2 by qPCR.Raldh2 mRNA expression levels were significantly reduced in 293T cells transfected withRaldh2 miRNA interference plasmids (miRaldh2-1:pcDNATM6.2-GW/EmGFPmiRNA_1;miRaldh2-2:pcDNATM6.2-GW/EmGFPmiRNA_2;miRaldh2-3:pcDNATM6.2-GW/EmGFPmiRNA_3;miRaldh2-4:pcDNATM6.2-GW/EmGFPmiRNA_4) when compared with the P19 group cells (Fig 1A).The results demonstrated that silence efficiency were 44% (t=3.834,P=0.018 6,95%CI:0.12-0.76) in miRaldh2-1,67% (t=6.145,P=0.003 6,95%CI:0.37-0.97) in miRaldh2-2,13% (t=1.056,P=0.350 6,95%CI:-0.21-0.47) in miRaldh2-3 and 31% (t=2.505,P=0.066 4,95%CI:-0.03-0.64) in miRaldh2-4 respectively,suggesting that P19 miRaldh2-2 group achieved the best efficiency ofRaldh2 mRNA suppression compared with the P19 group.Then we constructed lentivirus pLenti6.3-miRaldh2-2 using BP and LR recombination systems,and DNA sequencing analysis confirmed the correct construction.GFP signal in 293T cells transfected with theRaldh2 miRNA expression plasmid and the packaging plasmid suggested the successful packaging of lentivirus Lenti6.3-miRaldh2-2 (Fig 1B).

A:Raldh2 mRNA levels in 293T cells transfected withRaldh2 miRNAs.Expression ofRaldh2 mRNA normalized to GAPDH in 293T cells at 48 h after transfection.Data were shown as mean±SEM.vs. the control group(1)P<0.05,(2)P< 0.01,n=3.B:Green fluorescent protein (GFP) signal in 293T cells transfected withRaldh2 miRNA (5*).

Fig 1 The evaluation ofRaldh2 expression knocked down by the interference vectors

Establishment of MiRaldh2 group,the P19 stable cell line harboringRaldh2 miRNAIn pLenti6.3-miRaldh2-2 infected 293T cells,the number of expressing GFP cells were 16 and 14 in 10-6dilution holes respectively.The titer of the lentivirus is the numbers of cells with GFP signal multiply by the corresponding multiple of dilution.The biological titer of Lenti6.3-miRaldh2-2 lentivirus was 1.5*108TU/mL.After 96 hours Lenti6.3-miRaldh2-2 lentivirus infection,Lenti6.3-miRaldh2-2 was expressed in a large percentage of cells at a MOI of 120 (Fig 2).To select a P19 stable cell line carried both the blasticidin-resistent gene andRaldh2 miRNA,named as MiRNA group,we used blasticidin.The best screening dose of antibiotics is the antibiotics concentration capable to kill a number of cells within 3 days and kill all the cells within 2 weeks.After continuous observation and recording of cell growth consequently,it was showed that the best screening dose of blasticidin for P19 cells was 4 μg/mL.We then successfully established the MiRaldh2 group (Fig 3A) and detected the expression ofRaldh2 in MiRaldh2 group comparing with P19 group (Fig 3B).Our qPCR results showed that the silencing efficiency ofRaldh2 miRNA was approximately 91% (t=25.52,P<0.0001,95% CI:0.81-1.01).

Fig 2 P19 cells were infected with Lenti6.3-miRaldh2-2 at different MOIs for 96 (*5)

A:P19 cells infected with Lenti6.3-miRaldh2-2 were successfully screened by adding blasticidin.The left fluorescent image showed positive cells expressing GFP signal and the right figure is the corresponding visible light image (5*).B:Expression ofRaldh2 mRNA normalized to GAPDH in P19 cell lines at 48 h after infection.Data was shown as mean±SEM.vs.the P19 group,(1)P<0.001,n=3.

Fig 3 Silencing efficiency and establishment ofRaldh2miRNA stable P19 cell line (MiRaldh2 group)

Relative expression of cardiac markers inRaldh2 silenced P19 cellsThe qPCR analysis was carried out to evaluate whether or not repression ofRaldh2 could be correlated with different levels of expression of cardiac specific TFs (Fig 4).Currentinvitrodifferentiation protocols generally rely on time of differentiation to distinguish these populations,resulting in a developmentally diverse mixture of cells at any given timepoint[12].Some TFs have been identified as causative,multiple TFs likeGata4 andNkx2.5 can regulate cardiac chamber morphogenesis and might be involved in the formation of cardiomyocytes[13-14].Tef-1,N-myc,α-mhc,CtntandHand2 are also known as regulators of heart-specific gene expression as their essential roles in cardiomyocyte proliferation[14-16].

Considering with the fact that induction of P19 cells into cardiac muscle by day 6 to 7 of differentiation,we focused more on the differentiation from the 4thto the 7thday in this study.The qPCR analysis revealed that they all expressed in both P19 and MiRaldh2 P19 cells.Generally,compared with P19 group,the mRNA levels ofGata4,Tef-1,N-myc,α-mhcandCtntin siRaldh2 group were remarkably decreased whileNkx2.5 andHand2 were significantly increased during the differentiation.

qPCR analysis of mRNA levels ofGata4,Tef-1,N-myc,α-mhc,Ctnt,Nkx2.5 andHand2.Gene expression levels were normalized to GAPDH expression.Data are shown as mean±SEM.vs.the P19 group.(1)P<0.05,(2)P<0.01,(3)P<0.001,n=3.

Fig 4 Repression ofRaldh2 restrains cardiomyocyte differentiation of P19 cells

The mRNA levels ofGata4 were expressed in both MiRaldh2 group and P19 group,and the expression ofGata4 from 0 to the 14thday was consistently lower in MiRaldh2 group than P19 group (Fig 4).In detail,relatively low ratio ofGata4 mRNA expression were detected in MiRaldh2 group (5%) in contrast with P19 group on day 0.Moreover,in the early stage of cell differentiation,theGata4 mRNA level was lower in MiRaldh2 group,and it was remarkably decreased from the 2ndto 14thday with the relatively low expression ratios which were 0.34±0.04 (t=3.341,P=0.028 8),0.18±0.02 (t=14.98,P=0.000 1),0.16±0.01 (t=17.29,P<0.000 1) and 0.19±0.01 (t=28.13,P<0.000 1) on the 2nd,4th,7thand 14thday respectively when compared with that of P19 group at the same time points.Notably,theGata4 mRNA level in MiRaldh2 group on the 7thday was the lowest during the progress of cell differentiation.

In contrast with P19 group,the expression ofTef-1 mRNA in MiRaldh2 group presented an increased trend on day 0 without statistical difference (P>0.05).However,it decreased constantly during the differentiation period and reached bottom level on the 7thday when the mRNA level was down-regulated by half compared with that of P19 group (t=3.564,P=0.023 5,95%CI:0.11-0.90).Except day 4,theN-mycmRNA was generally decreased in MiRaldh2 group when compared with P19 group during the progress of differentiation (Fig 4).In addition,on day 7,the expression ofN-mycwas 50% of that on day 4 in MiRaldh2 group (t=5.161,P=0.0141).As forα-mhc,the mRNA levels were also reduced in MiRaldh2 group.Indeed,compared with P19 group,α-mhcwas remarkably down-regulated in MiRaldh2 group on day 4,7 and 14,and the minimal level was only 0.20±0.02 on the 7thday (t=17.76,P<0.000 1) compared with P19 group.And the expression ofCtntin MiRaldh2 group was also lower than that of P19 group from the 4thto the 7thday (Fig 4).

UnlikeGata4,Tef-1,N-myc,α-mhcandCtnt,the results showed that the expressions ofNkx2.5 andHand2 in MiRaldh2 group were dramatically higher than those of P19 group.Nkx2.5 is known as one of the earliest cardiac progenitor cell markers.WithNkx2.5 haploinsufficiency,mice would exhibit atrial septal defects accompanied with decreased cardiomyocyte proliferation[17].In our study,on day 0,the expression ofNkx2.5 in MiRaldh2 group was 0.12±0.01 (t=4.320,P=0.012 4) of that in P19 group.However,from day 2 to 7,the expressions ofNkx2.5 mRNA were increased in MiRaldh2 group and they were remarkably higher than those of P19 group,the relative multiples were 3.41±0.15 (t=12.83,P=0.000 2),3.11±0.05 (t=8.629,P=0.003 3) and 2.25±0.35 (t=3.526,P=0.024 3) respectively (Fig 4).Hand2 is also known as a cardio-transdifferentiating factor,and overexpression ofHand2 might be related to CHDs[18].Our study showed that during the whole 14 days,the relative mRNA levels ofHand2 in MiRaldh2 group were remarkably higher than that of P19 group,especially on day 4 and 7 with the relative expression were 64.24±1.336 (t=47.15,P=0.000 4) and 3.58±0.20 (t=9.214,P=0.011 6) (Fig 4).

In this study,the changes of mRNA level of each cardiomyocyte-specific markers exhibited time-dependent manner in P19 cells when suppressing Raldh2 expression.Indeed,the period from the 4thto the 7this specific for the induction of P19 cells into cardiomyocyte-like cells as reported before,the expression changes of cardiomyocyte-specific markers became more obviously in contrast with the other time points.

Discussion

CHD is currently the leading noninfectious cause of infant mortality,it occurs in 10% of aborted fetuses and more than 29% of newborns who died of birth defects have cardiovascular developmental abnormalities[19-20].The genetic etiology of CHD is currently discernible in 20% of cases with CHD but only 2% with isolated CHD[21].Genetic and epigenetic information will be used increasingly frequently to individualize the care of CHD patients,to identify family members at risk,and to counsel with regard to risk of heritable disease[22].It is well known that P19 cells can differentiate into cardiomyocyte-like cells induced by low concentration of DMSO,which means the P19 cell line is a good model for cardiac developmentinvitro,also has great value for studying the pathogenesis of cardiovascular system[10,23].RA is required during the early development of heart.Because the conversion of retinaldehyde to RA catalyzed byRaldh2 is a critical step in retinoic acid metabolism,we investigated the expression changes of cardiac markers in myocardial differentiation when silencingRaldh2 on P19 cell model.

We successfully constructed a miRNA interference vector to suppress endogenousRaldh2 expression in P19 cells in this study.To explore the effect ofRaldh2 on the differentiation of P19 cells into cardiomyocyte-like cells,the expression patterns of cardiac TFs were observed on P19 cells treated with DMSO for 14 days.Our results showed that silencing ofRaldh2 would generally downregulate the expression ofGata4,Tef-1,N-myc,α-mhcandCtnt,but upregulate the expression ofNkx2.5 andHand2,suggesting that repression of Raldh2 might bring negative effects on the cardiomyocyte-like cells differentiation of P19 cells by interfering cardiac TFs.

Knockdown ofRaldh2 may inhibit cardiomyocyte-like cells developmentCardiomyocyte-specific TFs includingGata4,Tef-1,N-myc,α-mhc,Ctnt,Nkx2.5 andHand2 are essential to heart development,and the abnormal expressions of these TFs are associated with a variety of heart deformities.The GATA zinc finger TFs are a group of DNA-binding proteins characterized by preferential binding to the consensus DNA sequence GATA of target gene promoters.Gata4 was the most extensively explored cardiac GATA TFs,and manyGata4 mutations have been identified in patients with a wide variety of congenital heart diseases including atrial septal defect,tetralogy of Fallot,and patent ductus arteriosus[24].One cause of these congenital heart abnormalities might be the defects in cardiomyocyte proliferation during embryogenesis[25].Interestingly,studies on zebrafish have showed that overexpressingGata4 might inhibitRaldh2 expression whereas depletingGata4 would increasesRaldh2 expression[26].Moreover,it was reported that knockdown ofRaldh2 was sufficient to inhibit myocardial proliferation and decrease the numbers ofGata4-positive cardiomyocytes after ventricular resection in zebrafish,which suggested the potential interactions betweenRaldh2 andGata4[27].In this study,after repressing the expression ofRaldh2 in P19 cells,theGata4 mRNA levels were dramatically decreased and lower than control group (P<0.001),suggesting that the downregulation ofRaldh2 might break the balance betweenRaldh2 andGata4 and consequently inhibit the differentiation of P19 cells into cardiomyocyte-like cells.

We also found that cardiac markers includingTef-1,N-myc,α-mhcandCtntwere downregulated by means of knockdown ofRaldh2.It was reported thatTef-1 knockout could result in defective heart development and embryonic lethal phenotypes in mice,histological examination revealed that the ventricular wall in the heart ofTef-1 knockout mouse embryos was abnormally thin with a reduced number of trabeculae[28].Our study showed thatTef-1 andα-mhcmRNA were downregulated inRaldh2 silenced P19 cells.Interestingly,N-mycis considered to be a marker of compact myocardium and participates in convergent obligate pathways of cardiomyocyte maturation.Moreover,cell culture studies have demonstrated thatN-mycpromotes cell cycle progression at the G1/S-phase,andN-mycmutant mice have complex cardiac abnormalities including a lack of endocardial cushions and hypoplasia of the compact myocardium[29].In addition,researches also showed that deficiency ofα-mhcwas related with atrial septal defect,hypertrophic cardiomyopathy and dilated cardiomyopathy[30-31].Consequently,the significantly low expression ofN-mycin our study demonstrated that cardiomyocyte-like cells proliferation was repressed inRaldh2 silenced P19 cells.Indeed,as an important indicator of myocardial function,Ctntwas downregulated inRaldh2 silenced P19 cells in the early and middle stages of differentiation,which also reflected the restraint of myocardial development to some extent.

As the earliest expressed TF in vertebrate heart,Nkx2.5 was also a repressor of premature cardiac differentiation.It was reported thatNkx2.5 plays a central role in progenitor proliferation and outflow tract morphogenesis interaction with Smad1,which suggested that abnormal expression ofNkx2.5 may cause several cardiac malformations[32].Our data showed that silencingRaldh2 could upregulateNkx2.5 expression,which demonstrated that silencingRaldh2 could inhibit the cardiomyocyte-like cells differentiation at an early stage.In addition,genetic studies have revealed that precise dosage ofHand2 is essential for normal cardiomyocyte development and morphogenesis.Moreover,over dosage ofHand2 could cause hypertrophy of the myocardium in the adult mouse,and trisomy of theHand2 gene directly causes congenital heart disease[18,33].These studies show that overexpression ofHand2 may cause heart defects.Different from the general downregulations ofGata4,Tef-1,N-myc,α-mhcandCtnt,the expressions ofHand2 andNkx2.5 mRNA in MiRaldh2 group were significantly upregulated,demonstrating that silencing ofRaldh2 might have a potential negative effect on cardiomyocyte-like cells development.

Gata4 might play an essential role in the regulation of other cardiac markers byRaldh2Based on the evidence that abnormal mRNA expression of these important cardiac markers in MiRaldh2 group,we believe that silencing ofRaldh2 could inhibit cardiomyocyte-like cells differentiation by affecting several cardiomyocyte-specific TFs.Indeed,several studies have revealed thatRaldh2 is co-regulated by cardiogenic TFsGata4,Tbx20,andRaldh2 activity of cardiac fibroblasts were reduced inTbx20 conditional knockout mice[34-35].Interestingly,networks comprised ofTbx20,Nkx2.5 andGata4 can regulate cardiac development,promote specification of chamber myocardium or affect septation[36-38].Our results showed that the expressions ofGata4 andNkx2.5 inRaldh2 silenced P19 cells were opposite during the differentiation,one reason of this result might be the interference of the regulation network comprised ofGata4 andNkx2.5.Moreover,α-mhcandCtntwere the direct transcriptional targets ofGata4[39].Gata4 can regulateHand2 expression during cardiovascular development,andGata4 conditional knockout mice showed a heart phenotype similar to that ofHand2 knockout mice including hypoplasia of the right ventricle[18].The results in our study showed that genesα-mhcandCtntwere downregulated whileHand2 were upregulated in MiRaldh2 group compared with P19 group.Taken together,the normal myocardium development depended on a stable gene regulatory network,andGata4 might be involved in the regulation of several cardiac markers above byRaldh2.

The limitation in this study is that there is a lack of cell functional and morphological data to reveal the effect ofRaldh2 silencing on the differentiation of P19 cells into cardiomyocytes.Therefore,our further study will focus on analyzing myocomma morphology,proliferation,apoptosis and spontaneous rhythmic contraction of P19 cells affected byRaldh2 silencing.

In conclusion,the current investigation demonstrated that knockdown ofRaldh2 in P19 cells could affect the expressions of several important cardiomyocyte-specific TFs such asGata4,Tef-1,N-myc,α-mhc,Ctnt,Nkx2.5 andHand2,which suggested that the silence ofRaldh2 may repress the differentiation of P19 cells into cardiomyocyte-like cells.Considering the essential role ofRaldh2 plays in RA metabolism,this study provides more evidence for the association between RA deficiency and cardiac malformations.However,as the specific mechanism of congenital heart abnormalities related to RA metabolism is unclear,further studies are needed to probe the role of RA underlying the regulation in the process of myocardium differentiation.