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Acta Biochimica et Biophysica Sinica 2009 41(1):86-96; doi:10.1093/abbs/gmn010
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

Effects of methotrexate on the developments of heart and vessel in zebrafish

Shuna Sun1, Yonghao Gui1,*, Yuexiang Wang2, Linxi Qian2, Xuefei Liu2, Qiu Jiang2 and Houyan Song2

1 Children's Hospital, Fudan University, Shanghai 201102, China
2 Department of Molecular Genetics, Shanghai Medical College and Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, 200032, China

* Corresponding author: Tel, 86-21-6403-7254; Fax, 86-21-6403-8992; E-mail, yhgui{at}shmu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
 References
 
Methotrexate (MTX), an antagonist of folic acid, can inhibit dihydrofolate reductase (DHFR) which is of great importance in the synthesis of tetrahydrofolic acid and embryonic development. In this study, we found that after being exposed to 1.5 mM MTX at 6–10 hours post-fertilization, zebrafish embryos fail to form normal cardiovascular system. In MTX-treated embryos, the morphological development of ventricle and atrium was disrupted, the cardiac twist was abnormal, the heart rate and ventricular shortening fraction were reduced, and the vascular development was disrupted. We also found that either microinjection with dhfr–gfp mRNA or treatment with folinic acid calcium salt pentahydrate (CF) could cause improved development in the heart and vessels in MTX-treated embryos, which proved that MTX induced the malformations by inhibiting DHFR. The transcript levels of genes such as hand2, mef2a, mef2c, and flk-1 were reduced in MTX-treated embryos. Compared with the MTX-treated group, the transcript levels of hand2, mef2a, mef2c, and flk-1 were increased in the MTX + dhfr–gfp mRNA-injected group and in the MTX + CF group. Our results indicated that the disrupted development of the heart and vessels in MTX-treated embryos is related to the reduced transcript levels of hand2, mef2a, mef2c, and flk-1.

Keywords    methotrexate; folic acid; heart; vessel; gene transcript level; zebrafish

Received: May 30, 2008; Accepted: September 16, 2008
   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
 References
 
The cardiovascular defects are the major cause of deaths, and the incidence of congenital heart defects (CHDs) is about 1% of live birth [1,2]. Recently, more and more studies have shown that folic acid dysfunction is related with CHD [3]. Methotrexate (MTX) is the antagonist of folic acid. MTX has multiple therapeutic uses including treatment for cancers, ectopic pregnancy, and autoimmune disorders. More frequent use of MTX may result in an increased number of exposures in pregnant women and their fetuses. Analysis of a large case–control study suggested that during early pregnancy, the use of MTX increased the risk of congenital heart disease in new-borne babies [4]. MTX can inhibit the function of dihydrofolate reductase (DHFR) that is critical for nucleotide synthesis and methylation, both of which play essential roles in embryonic development. DHFR structure is highly conserved in mammals. Zebrafish DHFR is about 60% identical to human DHFR. Similar structural and kinetic properties were revealed between zebrafish and human DHFR [5]. A better understanding of effects of MTX and DHFR on cardiovascular development may provide important clues to the study of the relationship between folic acid deficiency and CHD in human.

Folic acid dysfunction leading to malformations in cardiovascular system has been proved in different animal models, such as chick, mouse, and so on. These animal models showed delayed heart development [6], reduced thickness of cardiac ventricular compact walls, fewer embryonic myocardium [7], and conotruncal heart defects [8]. However, none of the fetuses in these studies can live to birth; few of these investigations referred to the transcript levels of genes and none was carried out with zebrafish. No investigation about the effects of MTX on zebrafish development was found in literatures. Zebrafish offers several distinct advantages for genetic and embryological studies including external fertilization, rapid development, and optical clarity of its embryos [9]. It is an attractive and widely used vertebrate model for studying cardiovascular development. Zebrafish is particularly suitable for our study because severe defects in the heart do not lead to immediate lethality as in many vertebrate models, and developing zebrafish can survive the first week of life without functional circulation [10].

In this study, we treated zebrafish embryos with MTX and observed the malformations in the heart and vessels of MTX-treated embryos. The transcript levels of genes that are very important for cardiovascular development were detected by real-time polymerase chain reaction (PCR). dhfr-increased transcripting experiment and tetrahydrofolic-acid-rescuing experiment were also carried out to investigate how MTX induced the malformations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Statistical analysis
 Results
 Discussion
 Funding
 References
 
Materials
Folic acid antagonist, MTX of >99.0% purity (HPLC) was from Sigma (St. Louis, USA) (Product Number M8407). Folinic acid calcium salt pentahydrate (CF) of ≥99.0% purity (HPLC) was from Fluka (St. Louis, USA) (Product Number 47612). Wild-type AB line zebrafish was obtained from University of Oregon (Eugune, USA). The breeding facility was bought from Aquatic Habitats Corporation (Apopka, USA). Fish and embryos were maintained, collected, and staged as previously described [11,12].

Drug exposure
MTX was dissolved in 250 mM sodium bicarbonate to prepare the stock solution (200 mM, pH 7.8). To obtain a series of work solutions, stock solution was diluted with egg water (60 mg/l Instant Ocean salts, pH 7.2). Control group was treated with same concentration of sodium bicarbonate solution. CF was dissolved into egg water to a final concentration of 5 mM. Each group contained 50 embryos and after exposure, the embryos were transferred into the egg water.

Microinjection of dhfr–gfp mRNA
dhfr–gfp (green fluorescent protein) mRNA was synthesized using the mMESSAGE mMACHINE system (Ambion, Austin, USA) from the linearized pT7TS-dhfr–gfp plasmids (zebrafish dhfr, digested with EcoRI, transcribed with T7); dhfr–gfp mRNA was diluted to 40 µg/ml in solution (0.1% phenol red, 0.2 M KCl, pH 7.0). Embryos were divided into five groups. Embryos in each group were microinjected with 1, 3, 6, 9, or 12 nl of dhfr–gfp mRNA, respectively, into the blastomeres of one-to-two-cell-stage embryos. After the microinjection, the numbers of embryos with green fluorescence at 12 hours post-fertilization (hpf) were calculated under the fluorescent microscope and the numbers of embryos with malformation were calculated under the optical microscope at 48 hpf in each group.

Morphological observation and functional evaluation of the heart
The abnormal phenotypes of the heart were observed under microscope at 48 hpf and the numbers of abnormal cardiac embryos were calculated in each group. Embryos were removed and transferred to modified Tyrode's solution (136 mM NaCl, 5.4 mM KCl, 0.3 mM NaH2PO4, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose, pH 7.3) at 48, 54, 60, and 72 hpf to obtain the heart rate record and ventricular contractility analysis. Heart rates were measured under a dissecting microscope. The diastolic and systolic lengths of ventricle were measured with a TK-C1381 video camera (JVC, Tokyo, Japan). Ventricular shortening fraction (VSF) was calculated with the following formula according to the standard method previously described [13,14]: VSF = (ventricular length at diastole – ventricular length at systole)/ventricular length at diastole.

Microangiography
Fluorescein (2 – 5 nl) was micro-injected into the heart of living zebrafish embryos at 60 hpf. About 2 – 3 min later, vascular images were observed on a BX61 fluorescent microscope (Olympus, Tokyo, Japan) and collected with a DP70 digital camera (Olympus).

Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed with RNA anti-sense probes which were labeled with digoxigenin for the following genes: dihydrofolate reductase gene (dhfr; Accession No. NM 131775), ventricular myosin heavy chain (vmhc; Accession No. NM 001112733), atrial myosin heavy chain (amhc; Accession No. NM 198823), the heart and neural crest derivatives expressed transcript 2 (hand2; Accession No. NM 131626), myocyte-specific enhancer factor 2A (mef2a; Accession No. NM 131301), myocyte-specific enhancer factor 2C (mef2c; Accession No. NM 131312), and kinase insert domain receptor (flk-1; Accession No. NM 131472). Embryos older than 24 hpf were treated with 0.003% phenylthiourea (sigma) in egg water to inhibit pigment production. Whole-mount in situ hybridization (n = 20) was performed according to standard procedures [15]. Images were acquired using BX61 and SZX12 microscopes equipped with a DP70 digital camera.

RNA extraction and semi-quantitative real-time PCR analysis
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNAs extracted from micro-dissected hearts of embryos of 48 hpf (n = 50) and 18 hpf (n = 50) were used to detect transcript levels of hand2, mef2a, and mef2c. RNAs extracted from embryos of 24 hpf (n = 50) and 36 hpf (n = 50) were used to detect the transcript level of flk-1. RNAs were reverse-transcribed using oligo-dT primer. SYBR green method was used to quantify cDNA. The sequence-specific primers were listed in Table 1. Real-time quantitative PCR was done using the ABI Prism 7000 Sequence Detection System (Perkin-Elmer Applied Biosystem, Inc.). Amplification conditions were identical for all reactions and consisted of: 95°C for 3 min, then 45 s at 95°C, annealing for 45 s at 56°C, and extension for 45 s at 60°C for 40 cycles. Relative expression levels of each gene were computed with respect to the amount of β-actin. Value of expression level of each gene was divided by β-actin in different individual cDNA samples. Results thus show each value as the mean ± SD of three separate semi-quantitative PCRs (n =3). Specificity of each reaction was controlled by melting curve analysis, which began at 50°C and increased to 95°C in 1°C increments.


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  		Table 1 Sequence-specific primers used in this study

 

   Statistical analysis
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
Results
 Discussion
 Funding
 References
 
All the data were presented as mean ± SD. Comparisons between groups were made with ANOVA (t-test with Bonferroni correction). A P-value <0.05 was considered statistically significant. All the data were analyzed with the SPSS 13.0 software (SPSS, USA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
Discussion
 Funding
 References
 
Expression of dhfr in zebrafish at 10 hpf
The result of in situ hybridization showed that at 10 hpf, dhfr expressed in whole organism that includes the head, the mesoderm, and the tail bud (Fig. 1).


Figure 1
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  		Fig. 1 Expression of dhfr in zebrafish at 10 hpf    Expression of dhfr was detected in whole organism as arrows indicated. (A) Left lateral view, head toward top. (B) Dorsal view, head toward top. a, head; b, mesoderm; c, the tail bud. Bar = 300 µm.

 
MTX-induced malformations of the heart and vessels in zebrafish
We found that exposure of embryos before 6 hpf to MTX cause embryo death or severe overall malformations (data not shown). When embryos after 10 hpf were exposed to MTX, less effects of MTX on embryonic development were observed (data not shown). The dose–response curve in 6–10 hpf exposing stage showed that treatment with 1.5 mM MTX induced evident cardiac malformations in about 78% embryos [Fig. 2(A)], but less injury in developments of the head, fins, and somites. Therefore, in following experiments, embryos exposed to 1.5 mM MTX at 6 – 10 hpf were defined as MTX-treated group. In the MTX-treated group, embryos developed abnormal cardiac morphology and pericardial edema at 48 hpf. Cardiac morphologic abnormalities included hypogenetic ventricle with dilating atrium, both ventricle and atrial dilating, or both ventricle and atrium hypogenetic as a linear tube [Fig. 3(D)–(F)]. In controls, the heart rate progressively increased with maturity, whereas in the MTX-treated group, the heart rate progressively decreased and was lower than in controls. Compared with the controls, the heart rate and VSF markedly decreased in every time point in the MTX-treated group [Fig. 2(B) and (C)]. Using microangiography, we detected vascular malformation in MTX-treated embryos. In controls, at 60 hpf, intersomitic vessels and cranial vessels can be clearly observed [Fig. 3(G) and (K)]. In MTX-treated embryos, vessels were unclear and even could not be observed [Fig. 3(H) and (L)].


Figure 2
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  		Fig. 2 Effects of MTX on the heart development of zebrafish    (A) Dose–response curve of cardiac malformation caused by MTX. (B, C) Effects of MTX on the heart rate and VSF of zebrafish embryos. (D, E, F) At 48 hpf, cardiac abnormal ratio, heart rate, and VSF in MTX-treated group, MTX + CF group, and MTX + dhfr–gfp mRNA group. Data are presented as mean ± SD (n = 50). *P < 0.05.

 


Figure 3
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  		Fig. 3 Morphological observation of heart and vessels    (A–F) Morphological observation of hearts. (G–J) Development of somatic vessels which was observed under fluorescent microscope. (K–N) Development of cranial vessels which was detected by fluorescein microangiography. (A, G, and K) Control group, (B, I, and M) MTX–dhfr–gfp mRNA-injected group, (C, J and N) MTX + CF-treated group, (D, E, F, H, and L) MTX-treated group. (A'–F') Conceptual diagram of A–F. In K, L, M, and N, arrows indicate cranial vessel. V, ventricle; A, atrium; Se, somatic vessels. Bars = 150 µm. A–N: left lateral views, heads toward left.

 
Microinjecting dhfr–gfp mRNA induced increase in transcript level of dhfr
Curves in Fig. 4(A) and (B) indicated that when microinjection volume was 6 nl, GFPs were observed in about 94% of embryos and number of malformation was only about 10%. When the microinjection volume was <6 nl, the number of embryos with GFPs was obviously reduced. When the microinjection volume was >6 nl, the number of embryos with malformation was markedly increased. So, we select 6 nl as the microinjecting volume in following experiments. In controls, no green fluorescence was observed. However, green fluorescence was detected in dhfr–gfp mRNA-injected groups, which indicated that microinjecting dhfr–gfp mRNA results in increased transcript of dhfr and GFP and induces increase in transcript level of dhfr.


Figure 4
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  		Fig. 4 Microinjecting dhfr–gfp mRNA induced an increase in transcript level of dhfr    (A) Number of embryos with green fluorescence with different dhfr–gfp mRNA-injected volumes. (B) Number of abnormal embryos after being injected with different quantities of dhfr–gfp mRNA. (C, D) Control group at 18 hpf, observed under microscope. (E, F) MTX + dhfr–gfp mRNA-injected group at 18 hpf (volume of microinjection was 6 nl) under fluorescent microscope. Bar = 600 µm.

 
Increasing transcript level of dhfr and CF treatment can rescue MTX-induced malformations of the heart and vessels
Compared with group which was only exposed to MTX, the MTX + CF group (treated with 1.5 mM MTX + 5 mM CF at 6–10 hpf) and the MTX + dhfr–gfp mRNA group have improved development in the heart. In the MTX + CF group and MTX + dhfr–gfp mRNA group, ventricle and atrium appeared morphologically normal [Fig. 3(B) and (C)], cardiac abnormal percentage was lower, heart rate was higher, and VSF was increased [Fig. 2(D)–(F)]. Microangiographically, we also observed clear images of intersomitic and cranial vessels in both the MTX + CF and MTX + dhfr–gfp mRNA groups [Fig. 3(I), (J), (M), and (N)].

Effects of MTX on transcript levels of vmhc, amhc, hand2, mef2a, mef2c, flk-1
Results of in situ hybridization with vmhc and amhc at 48 hpf showed that the transcript levels of vmhc and amhc in the MTX-treated group appeared normal [Fig. 5(E) and (F)]. The results of in situ hybridization with vmhc and amhc at 20 hpf showed that in the control group, primitive heart tubes on each side of midline fused to form circular heart tube [Fig. 5(C)]. But in MTX-treated embryos, primitive heart tubes remained on both sides of midline without fusing, which demonstrated delayed development in the heart [Fig. 5(G)]. Results of in situ hybridization with vmhc and amhc at 48 hpf showed that compared with controls, cardiac twist was disrupted in MTX-treated embryos [Fig. 5(D) and (H)].


Figure 5
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  		Fig. 5 Expression pattern of vmhc and amhc detected by in situ hybridization    (A–D) Control group and (E–H) MTX-treated group. (A, E) Expression of vmhc at 48 hpf, (B, F) expression of amhc at 48 hpf, (C, G) expression of amhc + vmhc at 20 hpf, (D, H) expression of amhc + vmhc at 48 hpf. A, B, D, E, F, and H: ventral views, heads to the top. C and G: dorsal views, tails to the top. Arrows in C and G indicate primitive heart tubes. V, ventrical; A, atrium. Bar in A, B, C, E, F, G: 300 µm. Bar in D and H: 150 µm.

 
Results of in situ hybridization showed that in controls at 18 hpf, hand2 expressed in mesoderm [Fig. 6(A)] and mef2a and mef2c highly expressed in somites [Fig. 6(E) and (I)]. At 48 hpf, hand2 expression in the heart was visible and more intensive in atrioventrical canal [Fig. 7(A)]. The expression of mef2a and mef2c in the heart of controls were also observed at 48 hpf [Fig. 7(E) and (I)]. Compared with controls, the transcript level of hand2 in MTX-treated embryos were reduced in mesoderm or in the heart [Figs. 6(B) and 7(B)], and the transcript levels of mef2a and mef2c were decreased in somites or in the heart [see (F) and (J) in Figs. 6 and 7]. In controls, at 24 hpf, flk-1 expressed at forming vasculature in trunk axial and crania [Fig. 8(A) and (E)], and at 36 hpf, flk-1 expression in trunk axial extended to intersomitic region [Fig. 8(I)]. In MTX-treated embryos, the transcript level of flk-1 at 24 hpf was reduced [Fig. 8(B) and (F)] and at 36 hpf, flk-1 expression was absent in intersomitic region [Fig. 8(J)].


Figure 6
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  		Fig. 6 Effects of MTX on expressions of hand2, mef2a, and mef2c detected by in situ hybridization at 18 hpf    (A–D) Expressions of hand2, (E–H) expressions of mef2a, (I–L) expression of mef2c. (A, E, and I) Control group, (B, F, and J) MTX-treated group, (C, G, and K) MTX + dhfr–gfp mRNA-injected group. (D, H, and L) MTX + CF-treated group. A–D: left lateral views, heads toward top. E–L: dorsal views, heads toward top. A–D: arrows indicate the mesoderm. E–L: arrows indicate the somites. Scale bars: 300 µm.

 


Figure 7
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  		Fig. 7 Effects of MTX on expressions of hand2, mef2a, and mef2c of zebrafish heart detected by in situ hybridization at 48 hpf    (A–D) Expressions of hand2, (E–H) expressions of mef2a, (I–L) expression of mef2c. (A, E, and I) Control group, (B, F, and J) MTX-treated group, (C, G, and K) MTX + dhfr–gfp mRNA-injected group. (D, H, and L) MTX + CF-treated group. A–L: ventral views, heads toward top. Arrows indicate the hearts. Scale bars: 150 µm.

 


Figure 8
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  		Fig. 8 Effects of MTX on expression of flk-1 detected by in situ hybridization    (A–D) Expression of flk-1 in somatic vessels at 24 hpf, (E–H) expression of flk-1 in cranial vessels at 24 hpf, (I–L) expression of flk-1 in somatic vessels at 36 hpf. (A, E, and I) control group, (B, F, and J) MTX treated group, (C, G, and K) MTX + dhfr–gfp mRNA injected group. (D, H and L) MTX + CF-treated group. (A–D, I–L) Left lateral view, head toward left. (E–H) Views from the top. Arrows in (A–D) indicated the vessels in trunk axes. Arrows in (E–H) indicated the vessels in crania. Arrows in (I–L) indicated the vessels in intersomites. Bars = 150 µm.

 
Real-time PCR analysis also showed that in the MTX-treated group, the transcript levels of hand2, mef2a, and mef2c in whole embryos at 18 hpf and in the heart at 48 hpf were reduced [Fig. 9(A) and (B)]. The transcript levels of flk-1 were decreased in MTX-treated embryos at both 24 and 36 hpf [Fig. 9(C)].


Figure 9
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  		Fig. 9 Expressions of genes detected by real-time PCR    (A) Expressions of hand2, mef2a, and mef2c at 18 hpf. (B) Expressions of hand2, mef2a, and mef2c in zebrafish heart at 48 hpf. (C) Expressions of flk-1 at 24 and 36 hpf. Data are expressed as the mean ± SD of three separate quantitative PCR (n =3). {bigtriangleup}P  < 0.05 compared with control group, {star}P < 0.05 compared with MTX-treated group, *P < 0.05 compared with MTX-treated group.

 
Increased transcript level of dhfr and CF treatment results in increase in transcript levels of hand2, mef2a, mef2c, and flk-1 in MTX-treated embryos
Compared with the MTX-treated group, in the MTX + CF and MTX + dhfr–gfp mRNA groups increased transcript levels of hand2 in mesoderm at 18 hpf and in the heart at 48 hpf [see (C) and (D) in Figs. 6 and 7] were found. Transcript levels of mef2a and mef2c in somites at 18 hpf and in the heart at 48 hpf were higher in both MTX + CF and MTX + dhfr–gfp mRNA groups than in the MTX-treated group [see (G), (H), (K), and (L) in Figs. 6 and 7]. Transcript levels of flk-1 in MTX + CF and MTX + dhfr–gfp mRNA groups were found to be increased in trunk axial vessels and cranial vessels at 24 hpf and the transcripts were visible in intersomitic vessels at 36 hpf [Fig. 8(C), (D), (G), (H), (K), and (L)]. Real-time PCR analysis also showed that transcript levels of hand2, mef2a, mef2c, and flk-1 in both MTX + CF embryos and MTX + dhfr–gfp mRNA embryos were increased compared with those in the MTX-treated group [Fig. 9(C)].


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
Funding
 References
 
MTX can tightly bind to DHFR and powerfully inhibit this enzyme, leading to a marked reduction in the synthesis of tetrahydrofolic acid and decreased production of N5,N10-methyl-tetrahydrofolic acid. As a result, important biological functions such as nucleotide synthesis and methylation reactions are compromised. By this way, biological functions of folic acid are blocked. In zebrafish, from 1–4 somites to long-pec stage, dhfr is expressed in whole organism (neural plate, tail bud, central nervous system, intermediate cell mass of mesoderm, optic tectum, retina, pectoral fin musculature, the heart, muscle, liver, spleen, spinal cord, and pharyngeal arch) (http://zfin.org, ZFIN ID: ZDB-FIG-050630-6067, 128, 6110, 2433, 7081) [5]. In this paper, we also observed that at 6–10 hpf, dhfr is expressed in whole organism including the head, mesoderm, and tail bud.

In zebrafish, 6 – 10 hpf is in gastrula period (5.25–10 hpf), which is a crucial stage for cardiac development. Therefore, extrinsic factors can strongly affect the cardiac development in this stage. In our experiments, MTX affected both the morphological and functional developments of the heart in zebrafish. We found that exposing zebrafish embryos to MTX at 6 – 10 hpf can induce obvious cardiac malformations. We also found that in the MTX-treated group, the heart rate and VSF decreased, indicating cardiac ventricular contractility was impaired. Using microangiography, we detected that MTX disrupted the development of vessels. We further detected the transcript levels of some genes which plays important roles in cardiovascular development.

Zebrafish vmhc labels ventricular myocardium and amhc labels myocardium of atrium [15]. vmhc and amhc are important for cardiac morphogenesis [16]. hand2 is a basic helix–loop–helix transcription factor. Functional analysis has shown that hand2 is involved in the development of the heart and vasculature [17]. hand2 can promote ventricular cardiomyocyte expansion [18]. Dysfunction of hand2 was associated with ventricular hypoplasia and cardiac growth delay [19,20]. In zebrafish, hand2 plays pivotal roles in cardiac morphogenesis and cardiac-specific transcription [21]. mef2a and mef2c belong to MEF2 family. Dysfunction of MEF2 causes chamber dilation and mechanical dysfunction during the course of cardiac development [22]. mef2c have fundamental role in ventricular myocyte development [23]. Inactivation of mef2c causes cardiac developmental arrest [24]. In zebrafish, mef2a controls cardiac ventricular contractility [13]. flk-1, the receptor of VEGF, is one of the earliest markers for angioblasts and indispensable for migration of angioblasts from ventral mesoderm to midline [25]. flk-1 is crucial for further formation of vascular system, including dorsal artery, axial vein, cranial vessels, and intersomitic vessels [26].

In our study, abnormal cardiac developments in MTX-treated embryos were observed, including dilation or dysplasia of ventricle and atrium, delayed development of the heart, abnormal cardiac twist, and impaired contractility of ventricle. Abnormal formations of cranial vessels and intersomitic vessels in MTX-treated embryos were also found. Due to the central roles of vmhc, amhc, hand2, mef2a, mef2c, and flk-1 in cardiac morphogenesis, cardiac developmental process, ventricular contractility, and formation of vessels, the transcript levels of these genes that play vital roles in cardiovascular formation were detected.

In situ hybridization analysis showed that transcript levels of vmhc or amhc appeared normal in MTX-treated embryos at 48 hpf, suggesting that myocyte differentiations of ventricle and atrium were not affected by MTX. But in MTX-treated embryos, expressions of vmhc + amhc at 20 hpf demonstrated developmental retardation in the heart and expressions of vmhc + amhc at 48 hpf showed abnormal cardiac twist. In MTX-treated embryos, reduced transcript levels of hand2, mef2c, and mef2a were detected at 18 hpf, which indicated MTX affected the transcript levels of these genes in the early developmental stage. At 48 hpf, development of the heart in zebrafish is nearly completed. Cardiac looping and differentiation of ventricle and atrium are finished. Decreased transcript levels of hand2, mef2c, and mef2a in the heart of MTX-treated embryos were detected. Decreased transcript levels of hand2, mef2c, and mef2a may contribute to cardiac abnormalities. At 24 hpf, expressions of flk-1 were observed at midline in both the control and MTX-treated groups, which demonstrated that MTX did not disrupt the migration of angioblasts to midline. At 36 hpf, flk-1 expression was absent in intersomitic vessels, which suggested that angioblasts could not constitute the intersomitic vessels in trunk. Malformation of vessels induced by MTX may be related to the reduced transcript level of flk-1.

To prove that MTX-induced malformations of the heart and vessels have relationship with decreased transcript levels of hand2, mef2c, mef2a, and flk-1, the dhfr-increased transcripting experiment and tetrahydrofolic-acid-rescuing experiment were designed. As well known, MTX can inhibit functions of DHFR and results in down-production of tetrahydrofolic acid. If increased transcript levels of dhfr and giving tetrahydrofolic acid can rescue cardiovascular malformations and increase transcript levels of hand2, mef2c, mef2a, and flk-1 in MTX embryos, the conclusion that reduced transcript levels of hand2, mef2c, mef2a, and flk-1 were related with MTX-induced malformations in the heart and vessels was confirmed. In the dhfr-increased transcripting experiment, the dhfr–gfp mRNA was synthesized and microinjected into fertilized egg. dhfr–gfp mRNA can express both dhfr and gfp in vivo. So if the expression of gfp was detected, the expression of dhfr is proved. After being microinjected with dhfr–gfp mRNA, gfp expression was observed in embryos, which demonstrated microinjecting dhfr–gfp mRNA resulted in increased transcript level of dhfr. In tetrahydrofolic-acid-rescuing experiment, exogenous tetrahydrofolic acid was given by exposing zebrafish embryos to CF solution. CF can be converted to tetrahydrofolic acid in vivo and it is more stable than tetrahydrofolic acid when dissolved in water. We found that in the MTX + dhfr mRNA and MTX + CF-treated groups, cardiac abnormal ratio was decreased, malformations in the heart were rescued, and the heart rate and VSF were increased. The transcript levels of hand2, mef2a, mef2c, and flk-1 were increased both in the MTX + dhfr mRNA and MTX + CF-treated groups. Then we confirmed that malformations of the heart and vessels caused by MTX were associated with reduced transcript levels of hand2, mef2c, mef2a, and flk-1.

Our present findings were expected to provide important clues to investigations about adverse effects of folic acid dysfunction on cardiovascular development in human fetus. However, it is well known that many genes that are very important for cardiovasular development have combinatorial interactions, and the expressions of genes can be regulated by several factors. So, the mechanisms by which folic acid dysfunction induces reduced expression levels of these genes that were detected in our study need further investigation.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
References
 
This work was supported by grant from the National Natural Science Foundation of China (No. 39730470).


   Acknowledgements
 
We thank Dong Yongxin and Zhang Lifeng for their assistance. We are also grateful to the members of the Song Houyan's lab (Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University) for their advice and support.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
 References
 

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