| Background and ObjectiveCongenital heart defects (CHDs) represent a common developmental anomaly and leading cause of mortality in newborns. The prevalence is9.3per1000live births in Asia, and8to10per1000live births in the United States. In spite of the advance together with the progress in early diagnosis and therapy made during the last decades, the etiology of CHD, caused by complicatied factors involved in cardiac malformation and dysfunction, remains largely obscure. However, there is increasing evidence that the occurrence of CHD may result from the interruption of early heart development triggered by an interplay among genetic, environmental and epigenetic risk factors.Recent cohort studies have reported that the environmental risk factors includes maternal cigarette smoke, maternal exposure to drugs and chemical reagent like formaldehyde, reproductive problems like abortion, viral infection as well as gestational hypertension. It has been reported in a cohort study that approximately28%of reproductive-aged women smoke cigarettes and20%continue to smoke during pregnancy in the United States.The maternal smoke during pregnancy is a known risk factor for the development of CHD, but the mechanism and regulation is still unclear. Moreover, there are still unknow whether the toxic components produced by cigarette smoke are engaged in this process. Nicotine, a primary additive and toxic component of cigarette smoke, has been widely regarded a key factor contributing to the smoke-associated injury of cardiovascular tissues. Nicotine induces angiogenesis and accelerates the growth of tumor and atheroma in association with increased neovascularization. However, whether nicotine affects cardiac differentiation and cardiogenesis during early development is remain unknown.Cardiomyogenesis is a complicated process with hierarchical interation including a great number of transcriptional factors, cardiac co-transfactors and the relevant signaling pathway. Genetic abnormalities of several promyogenic factors are associated with the development of CHD, such as the homedomain factor Nkx2.5, T-box factor Tbx5, zinc finger factor GATA4and myofilament myosin heavy chain (MHC). During the early development of embryo, those nuclear proteins involved in cardiac differentiation and maturation are expressed in mutant forms in early embryos leading to the defects of myogenesis in early phases of cardiovascular development, and play essential roles throught caridac differentiation derived from embryonic stem cells. The expression of promyogenic nuclear transcription factors is critical for differentiation of embryonic stem cells into myogenic cells. However, little is known about the impact of maternal smoke (one of the important environmental risk factors of CHD), as well as nicotine (toxic and additive component of cigarette smoke) on the expression of cardiac transcriptional factors during cardiogenesis.Epigenetic regulation is a heritable and invertilble modification capable of influencing gene expression without changing the primary DNA sequence. Epigenetic modification is an essential regualtion during the development, mainly including DNA methylation, Non-coding RNAs and histion modification. Several epigenetic modification has been reported to be involved in the cardiogenesis. Besides, the environmental risk factors of CHD have also been reported to affect the cardiogenesis through epigenetic modification. DNA methylation is one of the primary epigenetic modification, and it can regulate gene expression through affecting the DNA methylation level. Generally, low level of DNA methylation can increase gene expression while high level of DNA methylation inhibit relevant gene expression. Such modification takes place during genetic transcription process. When the enviroment affect maternity, the DNA methylation level may change with a following subsequent of genetic alteration in offspring. It has been reperted that the environmental risk factors affecting the maternity could alter the DNA methylation level of several cardiac transcritional factors during pregnancy. On the other hand, it is well-known that regulation of DNA methylation level could affect the genetic expression of cardiac transcritonal factors during cardiac differentiation leading the alteration of cardiac differentiation efficiency derived from stem cells. In this study, we emphasise on the regulation of nicotine on DNA methylation level and genetic expression level of cardiac transcriptional factors during cardiac differentiaion and early cardiogenesis.DNA methylation is a type of chemical modifications of DNA that is stable over rounds of cell division but doesn’t cause changes in the underlying DNA sequence of the organism with the regulation of DNA methytransferase. During the methylation process,5-methylcytosine is a methylated form of the DNA base cytosine by the DNA methyltransferases that alter the morphology and structure of DNA and may be involved in the regulation of gene transcription. When cytosine is methylated, the DNA maintains the same sequence, but the expression of methylated genes can be altered. Generally, high level of DNA methylation repress the genetic expression while low level induce genetic expression. Therefore,5-methylcytosine and DNA methyltransferase, to some extent, could indicate the level of DNA methylation. Lots of cardiac specific transcriptional factors are involved in the process of cardiac differentiation and early cardiogenesis. Meanwhile, the environmental risk factors can regualte the relevant gene expression throught alteration of DNA methylation level.Mouse embryonic stem cells (mESCs) have self-renewal and pluripotency that can give rise to almost all cell types. mESCs can maintain self-renewal persistantly under certain culturing condition with stem cell medium (leukemia inhibitory factor, LIF), while can form embryoid bodies (EBs) with three germ layers including entoderm, mesoderm and ectoderm by hangingdrop cultruing systerm and differentiated medium without LIF. mESCs can develop into embryoid bodies by in vitro hanging drop culture. The differentiation of EBs is similar as early embryogenesis imitating the development of prophase and early gastrulation. Embryoid bodies derived from mESCs by hanging drop culture can differenate into spontaneous beating cardiomyocytes under two-dimension adherent culture with differentiation medium without LIF. Therefore, this in vitro cardiac differentiaiton model from embryoid bodies has been widely used in the study of cardiac differentiaion and cardiomyogenesis. So we use the in vitro cardiac differentiaiton model from embryoid bodies as cardiac differentiaiton model in the study to test the effect of nicotine on cardiac differentiation and relevant mechanism.This study used an in vivo embryogenesis model to investigate whether the maternal cigarette smoke and nicotine exposure during pregnancy affect the cardiac development in fetus; while an in vitro cardiac differentiaiton model derived from embryoid bodies to study whether nicotine exposure alters expression of promyogenic genes by regulating DNA methylation in developing embryoid bodies.Methods and ResultsPregnant Sprague-Dawley (SD) rats were randomly divided into control and experimental groups for exposure to cigarette smoke (15and20cigarettes/day) or nicotine (15and20mg/kg/day) during gestation. The impact of cigarette smoke and nicotine on the cardiac morphogenesis and function was tested by ultrasound examination in neonates delivered from rats with or without maternal cigarette smoke(15and20cigarettes/day) or nicotine exposure (15and20mg/kg/day) during the pregnancy. M-mode echocardiography revealed that both nicotine-exposed and smoking female rats could give birth to offspring whose hearts performed in a similar pattern as those from untreated control rats. Neonatal rats from the maternal nicotine exposure or smoke contracted regularly. Similar to the control, the majority of the newborn rats examined by echocardiography showed no major abnormality in M-mode echograms. No major change in birth rates and body weight were found between the control and smoke-or nicotine-exposed rats. However, Doppler assessment demonstrated that compared to the control, there were restricted blood flow signals in the neonatal hearts with the maternal nicotine exposure. Occasionally, cross-flow echo signals were found in the offspring with the maternal exposure to20cigarettes/day.Quantitative measurement of echo images showed that compared to that of the control rats, the left ventricular (LV) mass was significantly reduced in the newborn rats from the mothers exposed to20mg/kg/day nicotine. These offspring exhibited lower LV end-systolic volume (LVESV), LV end-diastolic volume dimension (LVEDV), and diastolic left ventricular posterior wall (LVPW;d);(LV Mass:14.85±2.26vs.29.91±2.52, P=0.000; LVESV:2.05±1.27vs.4.20±1.09,P=0.000; LVEDV:0.96±0.18vs.1.30±0.13, P=0.000; LVPW;d:0.44±0.06vs.0.53±0.07, P=0.01). Maternal cigarette smoke at20cigarettes/day also reduced LV mass, LVESV, LVEDV and LVPW;d in the newborn rats:LV Mass (21.28±2.49vs.29.91±2.52, P=0.000), LVESV (2.19±0.73vs.4.20±1.09, P=0.000), LVEDV (1.01±0.12vs.1.30±0.13P=0.001), LVPW;s,(0.45±0.06vs.0.53±0.07, P=0.026).However, the neonates from the nicotine-exposed or smoking female rats did not exhibit significant supressive effect on left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), on the contrary, both LVEF and LVFS sinificantly elevated in20cigarettes/day group comparing to control group (LVEF,88.32±4.59vs.78.86±7.24, P=0.001, LVFS,56.99±7.54vs.45.83±7.04, P=0.000). Taken together, the maternal exposure to nicotine (up to20mg/kg/day) or cigarette smoke (up to20cigarettes/day) induce the left ventriclar cardiac malformation and malfuction in the neonatal hearts.The high incidence of neonatal cardiac impairment following the maternal nicotine exposure and cigarette smoke points to the possibility that the nicotine exposure or smoke exerts an effect on embryonic stem cell growth and differentiation.To test this possibility, the stem cell survival and cardiac development were analyzed in EBs generated from murine ESCs treated with or without serial dosages including both clinically relevant (0.01-1μM) and pharmacological (100-1000μM) does of nicotine. MTT assay was conducted to test the proliferation and cytotoxicity, while PCR, Western bloting together with other assays were carried out to evaluate the expression level of certain cardiac transcriptional factors. Morphologically, treatment with nicotine at a concentration range up to1000μM did not cause significant changes in the sides and shapes of EBs developed in a "hanging-drop" ESC3-D culture system. The MTT assays demonstrated the MTT activity increased for the first3days of culture and then gradually declined or unchanged in the following days, indicating the presence of high levels of cell turnover during early EB formation. Interestingly, in EBs exposed to nicotine at100μM concentrations (P<0.01),100μM nicotine plus100μM Hexa (P<0.01),100μM Hexa (P<0.05), the MTT activity were significantly higher than that in untreated EBs. The nicotine-induced change in MTT activity could not blocked by Hexa, a specific inhibitor of nicotine receptors, suggesting that the nicotine-induced increase in MTT activity was independent of nicotine receptors.Following the3-D culture, EBs were transferred to petri dishes for further growth and differentiation adherently in2-D cultures. The effect of treatment on the cardiac differentiation of ESCs in developing EBs was detected for a total of16-day EB differentiation. Contractile cells spontaneously appeared in the cultures and became microscopically visible in the developing EBs after1weeks in control group. The percentage of beating EBs peaked at day12. In the nicotine-treated cultures, however, the contracting EBs emerged at day14, about2days later than the untreated EBs. The numbers of beating EBs decreased by61.59%(13.38±5.44vs.74.97±11.28, P=0.000) and66.52%(8.445±1.42vs.74.97±11.28, P=0.000), respectively when the EBs were treated with nicotine at the pharmacological concentrations (100and1000μM). Thus, persistent exposure to nicotine at a pharmacological concentration might retard the cardiomyogenic development of EBs.The initiation of cardiomyogenesis requires expression of nuclear factors that promote myogenic gene expression and/or suppression of other factors that prevent myogenesis. Further evaluation of nuclear factor mRNA in developing EBs by qRT-PCR showed that levels of Oct4and Nanog, two embryonic factors that de-differentiate stem cells, were not reduced by nicotine treatment, and instead to some degree, the nicotine exposure slightly increased the Oct4and Nanog mRNA contents in developing EBs. By contract, levels of Tbx5and GATA4mRNA were markedly reduced by nicotine treatment, in particular when the EBs were treated with nicotine at100μM or above(Tbx5, F=3.209, P=0.043; GATA4, F=9.043, P=0.000). Expression of the promyogenic factors Tbx5and GATA4in nicotine-treated EBs was further confirmed at protein levels by Western blot analysis. The nicotine down-regulation of Tbx5and GATA4expression depended on the concentrations of nicotine (Tbx5, F=24.069, P=0.000; GATA4, F=9.303, P=0.001). Densitometry of the protein bands provided a semi-quantitative assessment of Tbx5and GATA4relative to the house-keeping protein GAPDH. EBs treated with nicotine at0.01-1000μM down regulate the expression of Tbx5and GATA4in dosage dependently manner,100μM or above showed the most prominent inhibition of the two promyogenic factor expression.In order to further clarify the impact of nicotine on cardiogenesis and relevant mechanism, Hexa, a general antagonist of nAChRs, was employed to treat the early EBs alone or combined with nicotine during cardiac differentiation. Immunofluorescence microscopy localized GATA4positive cells in EBs undergoing differentiation. Although no contractile cells were microscopically visible, a cluster of ESC-derived cells with positive immunofluorescence of GATA4was detected in early (day4) EBs. The sizes and numbers of GATA4-positive cell at day4EBs were similar among each group. However, when developing further into mature myocytes at day12, significantly increased numbers of GATA4-immunostained cells were found in the region with synchronized contraction. Comparing to the untreated group, nicotine treated EBs showed decreased numbers of GATA4-positive cell clusters at day12EBs. Addition of Hexa to nicotine exposure slightly increased numbers of GATA4-positive cells.Quantitative measurement by flow cytometry demonstrated that100μM nicotine treated EBs contained8.80%less GATA4-positive cells (8.27±2.26%vs.17.07±3.18%, P=0.005), while100μM nicotine co-treated with100μM Hexa contains9.03%more GATA4-positive cells than only nicotine-treated group (18.20±0.90%vs.8.27±2.26%, P=0.003).This nicotine inhibitory effect could be partially reversed by addition of the nicotine receptor inhibitor Hexa. In day12EBs, addition of Hexa (100μM) increased numbers of GATA4-positive cells in contractile EBs treated with nicotine(100μM). The Hexa treatment alone did not have the effect on the development of GATA4-positive cells in regular EBs without the nicotine exposure.Analysis of mRNA in the developing EBs by qPCR demonstrated that nicotine treatment did not inhibit, but to a certain extent increased, expression of the embryonic reprogramming factors Oct4and Nanog (Oct4, F=2.335, P=0.150; Nanog, F=5.273, P=0.027). Interestingly, addition of the general nAChRs antagonist Hexa in the EB cultures with or without nicotine markedly increased expression of Oct4and Nanog. The antagonist treatment also reversed the repressive effect of nicotine on GATA4mRNA but had a modest effect on Tbx5expression (GATA4, F=11.172, F=0.003; Tbx5, F=0.996, P=0.455).The mRNA expression level of Nanog in100μM nicotine and Hexa co-treated group is higher than that in control group (2.40±0.90vs.1.00±0.30, P=0.015), while significantly increased than only nicotine treated group (2.40±0.90vs.0.76±0.58, P=0.001). Than antagonist of nAchRs can also block the inhibitive effect of nicotine on GATA4expression:100μM Nicotine treatment significantly down-regulate the GATA4mRNA level (0.39±0.28vs.1.00±0.32, P=0.035); while100μM nicotine and Hexa co-treated group up-regulate the GATA4level compare to control group(2.40±0.90vs.1.00±0.30, P=0.015) or100μM nicotine-treated group(2.40±0.90vs.0.76±0.58,P=0.001).Western blot analysis further confirmed that nicotine treatment inhibited mainly expression of GATA4protein and to a much less extent Tbx5protein. Again, the nicotine inhibitory effect on protein expression of the two transcription factor was blocked partially by addition of Hexa. Taken together, the nicotine effect occurred on the transcriptional levels of cardiomyogenic gene expression in the early EBs, and it could be reversed by inhibition of nAChRs.To further study the effect and relevant mechenism of nicotine on early cardiac differentiation, as well as to investigate whether nicotine regulate the expression of cardiac transcriptional factors through the epigenetic modification of DNA methylation, in the study, we use dot blot and Enzyme-linked immuno sorbent assay analysis (ELISA) to test5-methylcytosine while use western blot to test DNA methyltransferase (DNMTs, DNMT1and DNMT3B) in day12-differentiated EBs treated with or without nicotine in the presence or absence of Hexa.The dot blot analysis showed that increased expression level of5-methylcytosine in nicotine-exposed group comparing to control group, but in nicointe-and Hexa-exposed group, the expression level decreased, suggest the reversion of nicotine effect by it’s nAchRs. The data from ELISA assay was statistically analysed by ONE-WAY ANOVA, and LSD was used in th multiple comparion. The expression level of5-methylcytosine had significantly change beteween groups(F=31.170, P=0.000). Resluts from LSD statisticlly analysis showed that simimlar results as the dot blot assay:There was an increased expression level of5-methylcytosine in nicotine-exposed group comparing to control group (26.37±1.56vs.18.48±1.51, P=0.000) but decreased while treated with both nicotine and Hexa (14.78±2.03vs.26.37±1.56, P=0.000). Both dot blot and ELISA assay showed nicotine exposure sinificantly increased the expression of5-methylcytosine in early cardiac differentiation from mouse embryoic bodies. The effect could also be block by nAchRs antagonist, suggesting the involvment of nAchRs.In order to study the effect of nicotine on DNA methylation during cardiac differentiation of EBs, western blotting was conducted to test the protein expression level of two primary DNA methyltransferases (DNMT1and DNMT3B). The quantifitive data from western blot bands was statistically analysed by ONE-WAY ANOVA, and LSD was used in th multiple comparion. The expression level of DNMT1and DNMT3B had significantly change beteween groups (DNMT1: F=29.881, P=0.000; DNMT3B:F=26.651, P=0.000). Resluts from LSD statisticlly analysis showed that there was an increased expression level of DNMT1and DNMT3B (DNMT1,1.13±0.08vs.0.52±0.11, P=0.000; DNMT3B,0.36±0.06vs.0.20±0.02, P<0.001); but no change in100μM nicoitne and100μM Hex exposed group when compare to nicotine-exposed group (DNMT1,1.17±0.09vs.1.13±0.08, P=0.689; DNMT3B,0.41±0.01vs.0.36±0.06, P=0.227).Taken together, nicotine exposure significantly increased the expression of DNMT1and DNMT3B in early cardiac differentiation from mouse embryoic bodies. The accelerating effect of nicotine on DNMT methytransferases (DNMT1and DNMT3B) as well as5-methylcytosine are consistent. The increased expression of5-methylcytosine and DNMT methytransferases (DNMT1and DNMT3B) indicated the higher DNA methylation level after nicoitne treatment during early cardiac differentiaon.ConclusionsThe study on early cardiogenesis on animal model shows that sustained cigarette smoke and nicotine exposure during pregnancy induce cardiac malfunction in rat offspring. The whole maternal exposure to smoke from20cigarettes daily, or20mg/kg nicotine per day, significantly reduced left ventricular mass, LV end-systolic volume, LV end-diastolic volume and diastolic left ventricular posterior wallBesieds, the study on in vitro cardiac differentiaiton model derived from embryoid bodies display that persistent exposure to nicotine selectively inhibits expression of two cardiomyogenic genes, GATA4and Tbx5at both mRNA and protein levels and reduces the number of GATA4-positive cardiac progenitor cells, resulting in attenuation of cardiac differentiation in early embryoid bodies. The repressive effects of nicotine on cardiac differentiation were attenuated by nicotinic acetylcholine receptors (nAChRs) inhibitor, suggesting the involvement and regulation of nAChRs in the adverse impact of nicotine on cardiac differentiation. However, nicotine exposure result in up-regulation of5-methylcytosine and DNA methytransferases (DNMT1and DNMT3B) indicating the activation or increased level of DNA methylation in the CpG insland of certain cardiac transcriptional facors’ protomer area during the early cardiac differentiaon.The data resulting from the current experiments show the first evidence that long-term cigarettet smoke or nicotine exposure exerts an inhibitory effect on cardiac development and nicotine inhibit expression of promyogenic transcriptional factors during the cardiac differentiation derived from mouse embryoid bodies. The nicotine-mediated down-regulation of promyogenic factors but up-regulation of DNA methylaion show how the environmental risk factor of CHD interact with genetic risk factor and the epigenetic regulation resulting in the occurrence of cardiac malformation. Those findings of this study may contribute to the pathogenesis of CHDs. |
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