2019, Vol. 46
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, and in vivo treatment 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, and RALDH2, also known as aldehyde dehydrogenase 1 family, member A2 (ALDH1A2) gene plays an essential role in this metabolism.It was reported that Raldh2-/- 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 in Raldh2-/- mouse embryo, and the activity of RA was undetectable in epicardium as well as the adjacent myocardium[4, 6].However, the role of Raldh2 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 as Gata4, Tef-1, N-myc, α-mhc, Ctnt, Nkx2.5 and Hand2, 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 of Raldh2 on myocardial differentiation by using P19 cell model and testing the expressions of cardiac markers when Raldh2 was knocked down.
Materials and MethodsCell culture P19 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 experiments Protocol 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 1×106 cells/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 1×105 cells/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 of Raldh2 miRNA interference expression plasmids and determination of knockdown efficiency The sequences of four pairs of miRNA oligo (Tab 1) were designed and synthesized based on Raldh2 sequences.The oligos were annealed into double chains and inserted into miRNA expression vectors pcDNATM 6.2-GW/EmGFPsiR respectively using BLOCK-iTTM Pol 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 pcDNATM 6.2-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 four Raldh2 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.
| Oligo | Oligomeric single-stranded DNA sequence (5'to 3') |
| miRaldh2-1 | F:TGCTGTGGAAAGCCAGCCTCCTTGATGTTTTGGCCACTGACTGACATCAAGGACTGGCTTTCCA |
| R:CCTGTGGAAAGCCAGTCCTTGATGTCAGTCAGTGGCCAAAACATCAAGGAGGCTGGCTTTCCAC | |
| miRaldh2-2 | F:TGCTGTAACGTTGGAGAACACGGTAGGTTTTGGCCACTGACTGACCTACCGTGCTCCAACGTTA |
| R:CCTGTAACGTTGGAGCACGGTAGGTCAGTCAGTGGCCAAAACCTACCGTGTTCTCCAACGTTAC | |
| miRaldh2-3 | F:TGCTGAAAGCTTGCAGGAATGGCTTAGTTTTGGCCACTGACTGACTAAGCCATCTGCAAGCTTT |
| R:CCTGAAAGCTTGCAGATGGCTTAGTCAGTCAGTGGCCAAAACTAAGCCATTCCTGCAAGCTTTC | |
| miRaldh2-4 | F:TGCTGTACCCTGGCAGAATGTTGACGGTTTTGGCCACTGACTGACCGTCAACACTGCCAGGGTA |
| R:CCTGTACCCTGGCAGTGTTGACGGTCAGTCAGTGGCCAAAACCGTCAACATTCTGCCAGGGTAC | |
| Negative | F:TGCTGAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT |
| R:CCTGAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTC | |
| Four pairs of miRNA oligo were designed and synthesized based on Raldh2.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. | |
| Target gene | Sequence |
| Raldh2 | F:5'-CCAGCCACAGGAGAGCAAGT-3' |
| R:5'-GCTTGCAGGAATGGCTTACC-3' | |
| Nkx2.5 | F:5'-GACAAAGCCGAGACGGATGG-3 |
| R:5'-CTGTCGCTTGCACTTGTAGC-3' | |
| Hand2 | F:5'-CGCAGGACTCAGAGCATCAA-3' |
| R:5'-GCCAGCAGATCCATGAGGTA-3' | |
| Gata4 | F:5'- ACGGAAGCCCAAGAACCTG-3' |
| R:5'-GCTGCTGTGCCCATAGTGAG-3' | |
| Ctnt | F:5'-GAGCTACAGACTCTGATCGAGG-3' |
| R:5'-CCGCTCATTGCGAATACGC-3' | |
| α-mhc | F:5'-ACTCGCTGCGGTATTTCA-3' |
| R:5'-CCTCCTGCTCAATCCACC-3' | |
| Tef-1 | F:5'-AGAGCCCTGCCGAAAACATGG-3' |
| R:5'-TGGCTGTCCTGTCTGTATCAT-3' | |
| N-myc | F:5'-AGCAGCACAACTATGCTG-3' |
| R:5'-TTAGCAAGTCCGAGCGTGTTC-3' |
Construction of lentivirus expression vector with highest Raldh2 interference efficiency and multiplicity of infection (MOI) test The fragment of Raldh2 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 (2×104 cells 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 and Raldh2 miRNA P19 cells were plated in 6-well plates at a concentration of 5×105 cells per well.Lentivirus liquid of Raldh2 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.Stable Raldh2 silenced P19 cell lines were isolated and amplified after 14 days of selection in blasticidin-resistent condition.
Kill curve test P19 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)-A same value).
Statistical analysis Each 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's t test with Graphpad Prism 6.0 software.Differences were considered statistically significant at P < 0.05.
ResultsIdentification of the most suitable Raldh2 miRNA interference plasmids First, we detected the mRNA level of Raldh2 in P19 cells by qPCR.Then we constructed four interference plasmids specific to Raldh2.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 of Raldh2 by qPCR.Raldh2 mRNA expression levels were significantly reduced in 293T cells transfected with Raldh2 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 of Raldh2 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 the Raldh2 miRNA expression plasmid and the packaging plasmid suggested the successful packaging of lentivirus Lenti6.3-miRaldh2-2 (Fig 1B).
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| A:Raldh2 mRNA levels in 293T cells transfected with Raldh2 miRNAs.Expression of Raldh2 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 with Raldh2 miRNA (5×). Fig 1 The evaluation of Raldh2 expression knocked down by the interference vectors |
Establishment of MiRaldh2 group, the P19 stable cell line harboring Raldh2 miRNA In pLenti6.3-miRaldh2-2 infected 293T cells, the number of expressing GFP cells were 16 and 14 in 10-6 dilution 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 1.5×108 TU/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 and Raldh2 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 of Raldh2 in MiRaldh2 group comparing with P19 group (Fig 3B).Our qPCR results showed that the silencing efficiency of Raldh2 miRNA was approximately 91% (t=25.52, P < 0.0001, 95% CI:0.81-1.01).
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| Fig 2 P19 cells were infected with Lenti6.3-miRaldh2-2 at different MOIs for 96 (×5) |
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| 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 of Raldh2 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 of Raldh2 miRNA stable P19 cell line (MiRaldh2 group) |
Relative expression of cardiac markers in Raldh2 silenced P19 cells The qPCR analysis was carried out to evaluate whether or not repression of Raldh2 could be correlated with different levels of expression of cardiac specific TFs (Fig 4).Current in vitro differentiation 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 like Gata4 and Nkx2.5 can regulate cardiac chamber morphogenesis and might be involved in the formation of cardiomyocytes [13-14].Tef-1, N-myc, α-mhc, Ctnt and Hand2 are also known as regulators of heart-specific gene expression as their essential roles in cardiomyocyte proliferation [14-16].
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| qPCR analysis of mRNA levels of Gata4, Tef-1, N-myc, α-mhc, Ctnt, Nkx2.5 and Hand2.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 of Raldh2 restrains cardiomyocyte differentiation of P19 cells |
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 4th to the 7th day 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 of Gata4, Tef-1, N-myc, α-mhc and Ctnt in siRaldh2 group were remarkably decreased while Nkx2.5 and Hand2 were significantly increased during the differentiation.
The mRNA levels of Gata4 were expressed in both MiRaldh2 group and P19 group, and the expression of Gata4 from 0 to the 14th day was consistently lower in MiRaldh2 group than P19 group (Fig 4).In detail, relatively low ratio of Gata4 mRNA expression were detected in MiRaldh2 group (5%) in contrast with P19 group on day 0.Moreover, in the early stage of cell differentiation, the Gata4 mRNA level was lower in MiRaldh2 group, and it was remarkably decreased from the 2nd to 14th day 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, 7th and 14th day respectively when compared with that of P19 group at the same time points.Notably, the Gata4 mRNA level in MiRaldh2 group on the 7th day was the lowest during the progress of cell differentiation.
In contrast with P19 group, the expression of Tef-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 7th day 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, the N-myc mRNA 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 of N-myc was 50% of that on day 4 in MiRaldh2 group (t=5.161, P=0.0141).As for α-mhc, the mRNA levels were also reduced in MiRaldh2 group.Indeed, compared with P19 group, α-mhc was remarkably down-regulated in MiRaldh2 group on day 4, 7 and 14, and the minimal level was only 0.20±0.02 on the 7th day (t=17.76, P < 0.000 1) compared with P19 group.And the expression of Ctnt in MiRaldh2 group was also lower than that of P19 group from the 4th to the 7th day (Fig 4).
Unlike Gata4, Tef-1, N-myc, α-mhc and Ctnt, the results showed that the expressions of Nkx2.5 and Hand2 in MiRaldh2 group were dramatically higher than those of P19 group.Nkx2.5 is known as one of the earliest cardiac progenitor cell markers.With Nkx2.5 haploinsufficiency, mice would exhibit atrial septal defects accompanied with decreased cardiomyocyte proliferation[17].In our study, on day 0, the expression of Nkx2.5 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 of Nkx2.5 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 of Hand2 might be related to CHDs [18].Our study showed that during the whole 14 days, the relative mRNA levels of Hand2 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 4th to the 7th is 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.
DiscussionCHD 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 development in vitro, 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 by Raldh2 is a critical step in retinoic acid metabolism, we investigated the expression changes of cardiac markers in myocardial differentiation when silencing Raldh2 on P19 cell model.
We successfully constructed a miRNA interference vector to suppress endogenous Raldh2 expression in P19 cells in this study.To explore the effect of Raldh2 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 of Raldh2 would generally downregulate the expression of Gata4, Tef-1, N-myc, α-mhc and Ctnt, but upregulate the expression of Nkx2.5 and Hand2, suggesting that repression of Raldh2 might bring negative effects on the cardiomyocyte-like cells differentiation of P19 cells by interfering cardiac TFs.
Knockdown of Raldh2 may inhibit cardiomyocyte-like cells development Cardiomyocyte-specific TFs including Gata4, Tef-1, N-myc, α-mhc, Ctnt, Nkx2.5 and Hand2 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 many Gata4 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 overexpressing Gata4 might inhibit Raldh2 expression whereas depleting Gata4 would increases Raldh2 expression[26].Moreover, it was reported that knockdown of Raldh2 was sufficient to inhibit myocardial proliferation and decrease the numbers of Gata4-positive cardiomyocytes after ventricular resection in zebrafish, which suggested the potential interactions between Raldh2 and Gata4 [27].In this study, after repressing the expression of Raldh2 in P19 cells, the Gata4 mRNA levels were dramatically decreased and lower than control group (P < 0.001), suggesting that the downregulation of Raldh2 might break the balance between Raldh2 and Gata4 and consequently inhibit the differentiation of P19 cells into cardiomyocyte-like cells.
We also found that cardiac markers including Tef-1, N-myc, α-mhc and Ctnt were downregulated by means of knockdown of Raldh2.It was reported that Tef-1 knockout could result in defective heart development and embryonic lethal phenotypes in mice, histological examination revealed that the ventricular wall in the heart of Tef-1 knockout mouse embryos was abnormally thin with a reduced number of trabeculae[28].Our study showed that Tef-1 and α-mhc mRNA were downregulated in Raldh2 silenced P19 cells.Interestingly, N-myc is considered to be a marker of compact myocardium and participates in convergent obligate pathways of cardiomyocyte maturation.Moreover, cell culture studies have demonstrated that N-myc promotes cell cycle progression at the G1/S-phase, and N-myc mutant 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 of α-mhc was related with atrial septal defect, hypertrophic cardiomyopathy and dilated cardiomyopathy[30-31].Consequently, the significantly low expression of N-myc in our study demonstrated that cardiomyocyte-like cells proliferation was repressed in Raldh2 silenced P19 cells.Indeed, as an important indicator of myocardial function, Ctnt was downregulated in Raldh2 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 that Nkx2.5 plays a central role in progenitor proliferation and outflow tract morphogenesis interaction with Smad1, which suggested that abnormal expression of Nkx2.5 may cause several cardiac malformations[32].Our data showed that silencing Raldh2 could upregulate Nkx2.5 expression, which demonstrated that silencing Raldh2 could inhibit the cardiomyocyte-like cells differentiation at an early stage.In addition, genetic studies have revealed that precise dosage of Hand2 is essential for normal cardiomyocyte development and morphogenesis.Moreover, over dosage of Hand2 could cause hypertrophy of the myocardium in the adult mouse, and trisomy of the Hand2 gene directly causes congenital heart disease[18, 33].These studies show that overexpression of Hand2 may cause heart defects.Different from the general downregulations of Gata4, Tef-1, N-myc, α-mhc and Ctnt, the expressions of Hand2 and Nkx2.5 mRNA in MiRaldh2 group were significantly upregulated, demonstrating that silencing of Raldh2 might have a potential negative effect on cardiomyocyte-like cells development.
Gata4 might play an essential role in the regulation of other cardiac markers by Raldh2 Based on the evidence that abnormal mRNA expression of these important cardiac markers in MiRaldh2 group, we believe that silencing of Raldh2 could inhibit cardiomyocyte-like cells differentiation by affecting several cardiomyocyte-specific TFs.Indeed, several studies have revealed that Raldh2 is co-regulated by cardiogenic TFs Gata4, Tbx20, and Raldh2 activity of cardiac fibroblasts were reduced in Tbx20 conditional knockout mice[34-35].Interestingly, networks comprised of Tbx20, Nkx2.5 and Gata4 can regulate cardiac development, promote specification of chamber myocardium or affect septation[36-38].Our results showed that the expressions of Gata4 and Nkx2.5 in Raldh2 silenced P19 cells were opposite during the differentiation, one reason of this result might be the interference of the regulation network comprised of Gata4 and Nkx2.5.Moreover, α-mhc and Ctnt were the direct transcriptional targets of Gata4 [39].Gata4 can regulate Hand2 expression during cardiovascular development, and Gata4 conditional knockout mice showed a heart phenotype similar to that of Hand2 knockout mice including hypoplasia of the right ventricle[18].The results in our study showed that genes α-mhc and Ctnt were downregulated while Hand2 were upregulated in MiRaldh2 group compared with P19 group.Taken together, the normal myocardium development depended on a stable gene regulatory network, and Gata4 might be involved in the regulation of several cardiac markers above by Raldh2.
The limitation in this study is that there is a lack of cell functional and morphological data to reveal the effect of Raldh2 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 by Raldh2 silencing.
In conclusion, the current investigation demonstrated that knockdown of Raldh2 in P19 cells could affect the expressions of several important cardiomyocyte-specific TFs such as Gata4, Tef-1, N-myc, α-mhc, Ctnt, Nkx2.5 and Hand2, which suggested that the silence of Raldh2 may repress the differentiation of P19 cells into cardiomyocyte-like cells.Considering the essential role of Raldh2 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.
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