尼古丁抑制心肌分化和心脏早期发育及其相关机制研究
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摘要
研究背景与目的
先天性心脏病(Congenital heart defects, CHDs)是一种常见的发育缺陷疾病。流行病学的研究结果显示,该病在美国的新生儿中的发病率为0.8-1%,而在亚洲的发病率为0.93%,是导致新生儿死亡的主要疾病之一。随着医学研究的快速发展,先天性心脏病的早期诊断和治疗方法在过去的数十年间也得到不断的进步和改善,但该病是胚胎期心脏在母体内的发育停滞或缺陷所致,而引起胚胎期心脏发育畸形的原因非常复杂,所以该病的病因及发病机制仍旧不明确。目前的研究中,越来越多的证据表明先天性心脏病的发生是干扰和调节心脏早期发育过程的遗传、环境危险因子和表观遗传学修饰共同作用的结果。
来自流行病学的研究报道,诱发先天性心脏病的环境危险因子包括：妊娠早期,母亲有主动或被动吸烟史、有饮酒史、接触药物或甲醛等化学试剂、先兆流产、病毒感染及妊娠期高血压疾病等。一项回顾性队列研究报道显示,在美国处于生殖年龄的妇女中,吸烟者约占28%,其中有20%的吸烟妇女在怀孕后整个妊娠期仍旧继续吸烟。目前流行病学的研究中已有报道,妊娠期母亲吸烟是诱导先天性心脏病的危险因素,但这一结论仍然具有争议性而且对于其如何影响心脏发育和相关的调控机制也尚不清楚。吸烟过程中产生的毒性物质是否参与及如何影响该过程也仍未有报道。尼古丁作为香烟中的最主要的成瘾和毒性成分,在吸烟引起的心血管系统损伤和疾病中发挥着关键和主导作用。有研究报道尼古丁通过促血管生成的作用促进动脉粥样硬化并加速肿瘤的形成和进展,但尼古丁能否及如何影响心肌细胞分化和心脏早期发育的相关研究也尚未有报道。
心脏发育是一个复杂的级联调节过程,包括大量的转录因子,心肌共转录因子及其相关的信号通路共同参与和相互作用。抑制和干扰这些转录因子的表达能诱导心肌异常发育,造成心脏功能受损,这也是诱发先天性心脏病的遗传危险因素。大量研究报道,数个重要的心肌转录因子的基因突变或异常表达都与先天性心脏病相关,比如心脏特异性同源盒转录因子Nkx2.5,锌指转录因子结合蛋白GATA4、T盒基因Tbx5和肌球蛋白重链MHC,这些与心肌分化和发育相关的转录因子在早期胚胎中的基因突变和异常表达都能影响心肌分化并导致心脏发育的缺陷。在胚胎发育的早期,当胚胎干细胞向心肌细胞分化时,促心肌分化的核转录因子发挥关键作用。干扰这些重要的心肌转录因子的表达,能够抑制心肌细胞的生成,从而影响心脏的发育和心功能。妊娠期母体吸烟作为诱发先天性心脏病的重要环境危险因子及其主要毒性和成瘾性代谢产物,尼古丁,是否通过干预胚胎心脏发育过程中的心肌重要因子的表达从而诱发心脏发育异常这一发病机制也尚不清楚。
表观遗传学调控指在不改变细胞核DNA序列的情况下,通过对基因结构的修饰作用,基因功能发生可遗传性及可逆性的改变。表观遗传学修饰是生物发育过程中重要的调控方式,主要包括DNA甲基化、非编码RNA调控和组蛋白的修饰作用。研究显示,多种表观遗传学修饰作用参与了心脏发育的过程,而诱发先天性心脏病的环境危险因素可通过改变表观遗传学修饰作用从而影响心脏发育过程。DNA甲基化是主要的表观遗传学修饰途径之一,DNA甲基化水平的高低能调控基因的表达,通常低甲基化促进基因的表达,而高甲基化则抑制基因的表达。这种修饰途径发生在基因的转录过程,当亲代受到环境因素的影响下,DNA甲基化水平发生改变,从而影响子代基因的表达水平。已有研究报道,妊娠期影响亲代的环境危险因素能够改变子代心脏组织中某些心肌重要转录因子的甲基化水平。另外,大量的研究报道显示,在干细胞向心肌细胞分化过程中,通过影响分化过程中的某些心肌重要转录因子的甲基化水平,可调控其基因表达水平,从而影响干细胞向心肌分化的效率。在胚胎心脏发育的早期,尼古丁能否通过调控心肌分化和发育过程中重要转录因子的甲基化水平,进而影响其基因和蛋白的表达水平,并最终干扰心肌分化和发育,这正是本研究的所要探讨和研究的重点。
DNA表观遗传学修饰是指在DNA甲基化转移酶的作用下,胞嘧啶受甲基化后,在5号碳上附加一个甲基,形成5-甲基胞嘧啶,使DNA的型态、结构发生改变。当胞嘧啶被甲基化后,DNA的序列仍旧不变,但被甲基化的基因的表达则会改变,通常低甲基化增加基因的表达水平,而高甲基化则降低其表达水平。因此,5-甲基胞嘧啶和DNA甲基化转移酶的水平是检测DNA甲基化水平的重要指标。心肌分化和心脏发育过程中,需要大量的心肌特异转录因子的参与和调控,影响心脏发育的环境危险因素可通过表观遗传学修饰作用诱导DNA甲基化,从而改变心肌转录因子的表达。
小鼠胚胎干细胞(Mouse Embryonic Stem cells, mESCs),具有自我更新和多向分化潜能,在一定的培养条件下(干细胞培养基：含白血病抑制因子,Leukemia inhibitory factor, LIF)可以长期维持自我更新能力,而在分化培养基(不含LIF)和悬滴培养条件下可形成具有内、中、外3胚层结构的拟胚体(Embryoid Bodies,EBs)。这些拟胚体向心肌细胞的分化过程与早期胚胎发育过程相似,可以模拟胚胎发育早期原肠运动前期到早期的发育过程。小鼠胚胎干细胞通过悬滴法制备形成拟胚体后在分化培养基的作用下,能够贴壁自然分化,并形成具有自发性、节律性收缩的心肌细胞,拟胚体模拟心肌分化的模型被广泛的用于体外模型心肌分化的研究中。因此,本研究的体外实验部分选用小鼠胚胎干细胞制备成的拟胚体定向心肌细胞分化的模型,以检测尼古丁对心肌分化影响及其相关机制的研究。
本研究从体内早期胚胎发育的模型中,探讨妊娠期母亲被动吸烟和尼古丁干预这两种环境危险因子是否影响胎儿的心脏发育及其功能。此外,运用体外的拟胚体向心肌分化的细胞模型研究尼古丁在干细胞向心肌细胞分化过程中如何通过影响DNA甲基化水平和心肌转录因子的表达,并最终影响干细胞向心肌分化。
研究方法和结果
怀孕的Sprague-Dawley (SD)大鼠被随机分为对照组(相同体积的生理盐水)、吸烟处理组(15和20支烟/天)和尼古丁处理组(15和20mg/kg/天)。在整个妊娠期,怀孕的母鼠分别每天给予15或20支烟被动吸烟处理和15或20mg/kg尼古丁干预直至分娩(PND0),用超声心动图检测子代新生乳鼠的心脏形态结构及功能。超声心动图M-mode图像显示妊娠期吸烟和尼古丁处理组的子代新生乳鼠的心脏跳动的模式和频率与对照组相似,也没有出现异常的M-mode超声心动图。心脏重量(F=0.655,P=0.636)和心脏重量/体重(F=1.149,P=0.4)在各处理组间的差异没有统计学意义。但多普勒检测结果显示,与对照组相比,尼古丁处理组,新生乳鼠心脏的血流动力学图出现血流受限现象,而在20支烟/天处理组中则出现左右心室血流交错现象。
定量分析超声心动图检测所获得的心功能指标,结果显示：妊娠期母鼠给予20mg/kg/天尼古丁处理,子代新生乳鼠的左心室心肌质量指数(Left Ventricular Mass, LV Mass),左心室收缩末期容量(LV end-systolic volume, LVESV),左心室舒张末期容量(LV end-diastolic volume, LVEDV)和左心室舒张末期后壁厚度(Diastolic left ventricular posterior wall, LVPW;d),与对照组相比均显著降低,差异有统计学意义(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).20支烟/天尼古丁处理组,于对照组相比,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;s0.45±0.06vs0.53±0.07, P=0.026)也显著降低,差异均有统计学意义。
尼古丁和吸烟处理组的左心射血分数(Left ventricular ejection fraction, LVEF)和左心室短轴缩短率(Left ventricular fractional shortening, LVFS)与对照组相比,整体水平差异有统计学意义(LVEF,F=8.117,P=0.000；LVFS,F:8.917,P=0.000),但是这两个评价左心功能的重要指标在吸烟和尼古丁干预后,不但没有显著下降,反而在20支烟/天处理组中较对照组上升,差异有统计学意义(LVEF,88.32±4.59vs78.86±7.24,P=0.001；LVFS,56.99±7.54vs45.83±7.04,P=0.000).上述实验结果表明妊娠期孕鼠给予20支烟/天被动吸烟处理或20mg/kg/天的尼古丁处理致后代的左心功能发生异常,且尼古丁和被动吸烟干预产生一致的效应。
为了进一步研究尼古丁对心脏发育和心肌分化的影响,本研究用小鼠胚胎干细胞(mESCs)制备成拟胚体并向心肌细胞分化的过程中给予一系列的尼古丁处理,其中包括低浓度(0.01-1pM)和高浓度(100-1000μM),用MTT法检测其对干细胞增殖影响、PCR和Western blotting等方法检测在分化过程中心肌转录因子的表达水平。系列浓度梯度的尼古丁(0.01-1000μM),不影响悬滴法中拟胚体的形成,也不影响其形态和大小。MTT实验结果显示,mESC在分化培养基中,前三天处于指数生长期,培养第三天达到细胞增殖的高峰,第四天开始细胞增殖缓慢；说明运用悬滴法制备拟胚体的过程中,悬滴培养4天,细胞快速增殖形成拟胚体。有趣的是,在细胞增殖的高峰期(第3天),100μM尼古丁(P<0.01).100μM尼古丁+100gM六烃季胺(P<0.01)和100μM六烃季胺处理组(P<0.05)比对照组细胞数目显著增加,差异有统计学意义。此效应不能被六烃季胺阻断,说明该效应与尼古丁乙酰胆碱受体无关。
经3-D悬滴法培养后将拟胚体转移到细胞培养板中,在2-D的培养条件下进一步贴壁分化和生长。贴壁分化总共持续16天,期间给予系列浓度梯度的尼古丁处理。空白对照组的拟胚体在经1周贴壁分化后,可以在显微镜下观察到跳动的细胞,而在分化的第12天跳动的拟胚体的比例达到峰值。在尼古丁处理组(0.01-1000μM)跳动的拟胚体比例均是在第14天达到最高峰,而比对照组推迟两天。在分化的第12天,100μM尼古丁处理组,跳动的拟胚体的比例比对照组下降了61.59%(13.38±5.44vs.74.97-11.28,P=0.000),而1000μM尼古丁处理组比对照组下降了66.52%(8.445±1.42vs.74.97±11.28,P=0.000)。因此,高浓度(100-1000μM)尼古丁作用抑制并推迟拟胚体向心肌细胞分化的过程。
心脏发育需要能促进心肌细胞生成或抑制抗心肌细胞形成的核转录因子的作用。用实时定量PCR检测分化第12天的拟胚体中转录因子的信使RNA的表达水平,结果显示：小鼠胚胎干细胞干性维持基因Oct4和Nang,在尼古丁处理后,mRNA的相对表达量有增加的趋势,但是差异没有统计学意义(Oct4,F=0.313, P=0.863; Nanog, F=0.757, P=0.608)。相反,在100gM和1000μM尼古丁处理中,Tbx5和GATA4的mRNA表达水平比对照组均显著降低(Tbx5,F=3.209, P=0.043; GATA4, F=9.043, P=0.000),差异具有统计学意义。采用Western blot进一步检测Tbx5和GATA4在尼古丁处理后的拟胚体中的蛋白表达水平,结果显示：尼古丁剂量依赖性的下调Tbx5和GATA4的蛋白表达水平,差异有统计学意义(Tbx5,F=24.069,P=0.000; GATA4, F=9.303, P=0.001)。将获得的蛋白条带的灰密度值做相对定量分析,得到Tbx5和GATA4相对于看家基因GAPDH的相对表达量,结果再次显示：给予0.01-1000gM尼古丁剂量依赖性的下调Tbx5和GATA4的蛋白表达水平,高浓度组(100和1000μM)尼古丁处理对这两个转录因子的抑制作用强烈。
为了进一步阐明尼古丁对心肌分化的影响及相关的机制,实验中选用尼古丁乙酰胆碱受体拮抗剂,六烃季胺(Hexamethonium, Hexa),用于单独或者和尼古丁共同作用于拟胚体的分化过程。采用免疫荧光染色法特异检测经分化的拟胚体中GATA4P日性细胞数量。经过4天悬滴培养(Day4)的拟胚体,在显微镜下无法观察到跳动的心肌细胞,但是免疫荧光可检测到少量的GATA4阳性细胞的存在。在第四天分化早期的拟胚体中,每组GATA4阳性细胞的数量和大小相似,但是,经过8天的贴壁分化(Day12),各组GATA4阳性的细胞数量显著增加,并同时可观察拟胚体中出现节律性跳动的细胞。荧光显微镜镜检记录的图片显示,在第12天的拟胚体中,与对照组相比,尼古丁处理降低GATA4阳性细胞的数量,六烃季胺和尼古丁共同处理则轻微增加GATA4阳性细胞数量。
采用流式细胞仪定量分析GATA4阳性细胞数量,100μM尼古丁处理组GATA4阳性细胞数量比对照组显著降低8.80%,差异有统计学意义(8.27±2.26%vs.17.07±3.18%,P=0.005)。分化12天的拟胚体中,100gM尼古丁和100gM六烃季胺共同处理组与尼古丁单独处理组相比,GATA4细胞数量升高9.03%,差异有统计学意义(18.20±0.90%vs8.27±2.26%,P=0.003),说明尼古丁的抑制效应可被尼古丁乙酰胆碱受体拮抗剂部分阻断。
用实时定量PCR检测经分化12天的拟胚体中相关基因的mRNA表达水平,单独100μM尼古丁处理对Oct4和Tbx5mRNA的表达量的整体水平没有差异(Oct4, F=2.335,P=0.150; Tbx5, F=0.996, P=0.455);而Nanog和GATA4的表达量在各组中整体水平差异显著,均具有统计学意义(Nanog, F=5.273, P=0.027; GATA4, F=11.172, P=0.003)。Nanog的mRNA的表达量在尼古丁(100μM)和六烃季胺(1001μM)共同处理组比对照组显著升高(2.40±0.90vs.00±0.30,P=0.015),与尼古丁单独处理组相比也显著升高(2.40±0.90vs.0.7±0.58,P=0.001),差异均具有统计学意义。尼古丁乙酰胆碱受体拮抗剂同样也能阻断尼古丁抑制心肌转录因子GATA4表达的效应：GATA4的mRNA表达量经尼古丁处理后较对照组显著降低,差异有统计学意义(0.39±0.28vs.1.0±0.32,P=-0.035),然而尼古丁(100μM)和六烃季胺(100μM)共同处理组较对照组却显著升高(1.7±0.40vs.1.00±0.32,P=0.035),且与尼古丁单独处理组相比也显著升高,差异均具有统计学意义(1.70±0.40vs.0.3±0.28,P=0.001)。
蛋白免疫印迹法获得同上相似结果：尼古丁对GATA4的蛋白表达的抑制作用强烈而对Tbx5蛋白表达相对微弱。同样的,尼古丁对上述两个心肌转录因子蛋白水平的抑制作用可被尼古丁乙酰胆碱受体拮抗剂部分阻断。以上结果都说明尼古丁对心肌分化的抑制作用是通过抑制拟胚体分化过程中心肌转录因子的的mRNA和蛋白表达水平导致的,而且这种抑制效应可以被尼古丁乙酰胆碱受体拮抗剂阻断。
为了研究尼古丁在心肌分化早期的作用和机制,同时也进一步探讨其是否通过调控DNA甲基化的表观遗传学修饰作用来影响心肌重要转录因子的表达。本研究选用斑点杂交法(dot blot)和酶联免疫吸附测定法(ELISA)检测经12天分化的拟胚体中5-甲基胞嘧啶的表达水平并用western blot检测DNA甲基化转移酶(DNMT1和DNMT3B)的蛋白水平。
斑点杂交法结果显示,分化的第12天的拟胚体经尼古丁处理后,5-甲基胞嘧啶的表达显著升高,但尼古丁和六烃季胺共同处理时,其表达水平比尼古丁单独处理组降低。酶联免疫吸附测定法定量检测分化早期的拟胚体中5-甲基胞嘧啶的水平,该数据经统计分析,结果显示：5-甲基胞嘧啶的表达水平在各处理组中差异显著,具有统计学意义(F=31.170,P=0.000),经LSD方法多重比较结果显示：经尼古丁处理后,5-甲基胞嘧啶的水平比对照组显著升高,差异有统计学意义(26.37±1.56vs.18.48±1.51,P=0.000),然而当加入100μM六烃季胺和100μM尼古丁共同处理时,其表达量比尼古丁单独处理组却显著降低,差异具有统计学意义±(14.78±2.03vs.26.37±1.56, P=0.000)。酶联免疫吸附测定法所得的结果不仅再次验证尼古丁干预显著升高分化早期的拟胚体中5-甲基胞嘧啶的表达水平,也与前述dot blot的结果相一致,并进一步显示该抑制作用可以被尼古丁乙酰胆碱受体拮抗剂(六烃季胺)阻断,提示该受体参与了上述过程。
为了进一步研究尼古丁在拟胚体向心肌分化过程中,对DNA甲基化水平的影响,本实验采用western blot法检测两个重要的DNA甲基化转移酶(DNMT1和DNMT3B)的蛋白表达水平。蛋白条带灰密度分析结果显示：DNMT1和DNMT3B的蛋白表达水平,在各处理组间差异显著,具有统计学意义(DNMT1: F=29.881, P=0.000; DNMT3B:F=26.651, P=0.000)。DNMT1和DNMT3B的蛋白表达水平,在尼古丁单独处理组中比对照组显著升高,差异有统计学意义(DNMT1,1.13±0.08vs.0.52±0.11,P=0.000; DNMT3B,0.36±0.06vs.0.20±0.02,P=0.001)；但在100μM尼古丁和100μM六烃季胺共同处理组中与单独尼古丁组相比,两者的表达水平均没有差异(DNMT1,1.17±0.09vs.1.13±0.08,P=0.689; DNMT3,0.41±0.01vs.0.36±0.06,P=0.227)。
上述结果表明在拟胚体向心肌细胞分化的早期,尼古丁处理显著的升高DNMT1和DNMT3B的蛋白表达水平,但当同时加入尼古丁乙酰胆碱受体拮抗剂后,不能阻断该效用,尼古丁对DNMT1和DNMT3B的促进作用与对5-甲基胞嘧啶的作用是一致,提示在小鼠胚胎干细胞向心肌分化过程中,尼古丁干预能显著增加DNA甲基化水平。
结论
对体内胚胎心脏发育模型的研究结果显示,妊娠期持续给予吸烟和尼古丁处理诱发子代新生大鼠的心脏功能异常：孕期持续给予20支烟／天或者20mg/kg/天尼古丁干预显著降低左心室心肌质量指数(LV Mass),左心室收缩末期容量(LVESV),左心室舒张末期容量(LVEDV)和左心室舒张末期后壁厚度(LVPW;s)。
对体外拟胚体向心肌分化的细胞模型研究结果显示,持续给予尼古丁处理选择性抑制两个心肌转录因子(Tbx5和GATA4)的mRNA和蛋白水平并降低GATA4阳性的心肌祖细胞数量,从而抑制拟胚体向心肌细胞的分化。尼古丁对心肌分化的抑制作用可以被尼古丁乙酰胆碱受体抑制剂阻断,提示该受体参与了尼古丁抑制心肌分化的过程并起重要作用。但尼古丁处理后却能显著提高5-甲基胞嘧啶和DNA甲基化转移酶(DNMTl和DNMT3B)的表达水平,暗示在拟胚体向心肌分化的早期,尼古丁升高DNA甲基化的水平,使相关的心肌转录因子的启动子区域的甲基化水平升高,从而抑制该基因的表达。
本研究结果首次揭示长期、持续的吸烟和尼古丁干预抑制心脏的发育导致左心功能异常；在拟胚体向心肌分化过程中,尼古丁抑制心肌特异转录因子表达水平的同时上调DNA甲基化水平。本研究展示了先天性心脏病的环境危险因子是如何通过影响遗传因素和改变表观遗传学修饰作用,并最终诱导心脏发育异常；对CHD的病因和机制研究具有重要的意义。
Background and Objective
Congenital 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 Results
Pregnant 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.
Conclusions
The 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 wall
Besieds, 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.
先天性心脏病(Congenital heart defects, CHDs)是一种常见的发育缺陷疾病。流行病学的研究结果显示,该病在美国的新生儿中的发病率为0.8-1%,而在亚洲的发病率为0.93%,是导致新生儿死亡的主要疾病之一。随着医学研究的快速发展,先天性心脏病的早期诊断和治疗方法在过去的数十年间也得到不断的进步和改善,但该病是胚胎期心脏在母体内的发育停滞或缺陷所致,而引起胚胎期心脏发育畸形的原因非常复杂,所以该病的病因及发病机制仍旧不明确。目前的研究中,越来越多的证据表明先天性心脏病的发生是干扰和调节心脏早期发育过程的遗传、环境危险因子和表观遗传学修饰共同作用的结果。
来自流行病学的研究报道,诱发先天性心脏病的环境危险因子包括：妊娠早期,母亲有主动或被动吸烟史、有饮酒史、接触药物或甲醛等化学试剂、先兆流产、病毒感染及妊娠期高血压疾病等。一项回顾性队列研究报道显示,在美国处于生殖年龄的妇女中,吸烟者约占28%,其中有20%的吸烟妇女在怀孕后整个妊娠期仍旧继续吸烟。目前流行病学的研究中已有报道,妊娠期母亲吸烟是诱导先天性心脏病的危险因素,但这一结论仍然具有争议性而且对于其如何影响心脏发育和相关的调控机制也尚不清楚。吸烟过程中产生的毒性物质是否参与及如何影响该过程也仍未有报道。尼古丁作为香烟中的最主要的成瘾和毒性成分,在吸烟引起的心血管系统损伤和疾病中发挥着关键和主导作用。有研究报道尼古丁通过促血管生成的作用促进动脉粥样硬化并加速肿瘤的形成和进展,但尼古丁能否及如何影响心肌细胞分化和心脏早期发育的相关研究也尚未有报道。
心脏发育是一个复杂的级联调节过程,包括大量的转录因子,心肌共转录因子及其相关的信号通路共同参与和相互作用。抑制和干扰这些转录因子的表达能诱导心肌异常发育,造成心脏功能受损,这也是诱发先天性心脏病的遗传危险因素。大量研究报道,数个重要的心肌转录因子的基因突变或异常表达都与先天性心脏病相关,比如心脏特异性同源盒转录因子Nkx2.5,锌指转录因子结合蛋白GATA4、T盒基因Tbx5和肌球蛋白重链MHC,这些与心肌分化和发育相关的转录因子在早期胚胎中的基因突变和异常表达都能影响心肌分化并导致心脏发育的缺陷。在胚胎发育的早期,当胚胎干细胞向心肌细胞分化时,促心肌分化的核转录因子发挥关键作用。干扰这些重要的心肌转录因子的表达,能够抑制心肌细胞的生成,从而影响心脏的发育和心功能。妊娠期母体吸烟作为诱发先天性心脏病的重要环境危险因子及其主要毒性和成瘾性代谢产物,尼古丁,是否通过干预胚胎心脏发育过程中的心肌重要因子的表达从而诱发心脏发育异常这一发病机制也尚不清楚。
表观遗传学调控指在不改变细胞核DNA序列的情况下,通过对基因结构的修饰作用,基因功能发生可遗传性及可逆性的改变。表观遗传学修饰是生物发育过程中重要的调控方式,主要包括DNA甲基化、非编码RNA调控和组蛋白的修饰作用。研究显示,多种表观遗传学修饰作用参与了心脏发育的过程,而诱发先天性心脏病的环境危险因素可通过改变表观遗传学修饰作用从而影响心脏发育过程。DNA甲基化是主要的表观遗传学修饰途径之一,DNA甲基化水平的高低能调控基因的表达,通常低甲基化促进基因的表达,而高甲基化则抑制基因的表达。这种修饰途径发生在基因的转录过程,当亲代受到环境因素的影响下,DNA甲基化水平发生改变,从而影响子代基因的表达水平。已有研究报道,妊娠期影响亲代的环境危险因素能够改变子代心脏组织中某些心肌重要转录因子的甲基化水平。另外,大量的研究报道显示,在干细胞向心肌细胞分化过程中,通过影响分化过程中的某些心肌重要转录因子的甲基化水平,可调控其基因表达水平,从而影响干细胞向心肌分化的效率。在胚胎心脏发育的早期,尼古丁能否通过调控心肌分化和发育过程中重要转录因子的甲基化水平,进而影响其基因和蛋白的表达水平,并最终干扰心肌分化和发育,这正是本研究的所要探讨和研究的重点。
DNA表观遗传学修饰是指在DNA甲基化转移酶的作用下,胞嘧啶受甲基化后,在5号碳上附加一个甲基,形成5-甲基胞嘧啶,使DNA的型态、结构发生改变。当胞嘧啶被甲基化后,DNA的序列仍旧不变,但被甲基化的基因的表达则会改变,通常低甲基化增加基因的表达水平,而高甲基化则降低其表达水平。因此,5-甲基胞嘧啶和DNA甲基化转移酶的水平是检测DNA甲基化水平的重要指标。心肌分化和心脏发育过程中,需要大量的心肌特异转录因子的参与和调控,影响心脏发育的环境危险因素可通过表观遗传学修饰作用诱导DNA甲基化,从而改变心肌转录因子的表达。
小鼠胚胎干细胞(Mouse Embryonic Stem cells, mESCs),具有自我更新和多向分化潜能,在一定的培养条件下(干细胞培养基：含白血病抑制因子,Leukemia inhibitory factor, LIF)可以长期维持自我更新能力,而在分化培养基(不含LIF)和悬滴培养条件下可形成具有内、中、外3胚层结构的拟胚体(Embryoid Bodies,EBs)。这些拟胚体向心肌细胞的分化过程与早期胚胎发育过程相似,可以模拟胚胎发育早期原肠运动前期到早期的发育过程。小鼠胚胎干细胞通过悬滴法制备形成拟胚体后在分化培养基的作用下,能够贴壁自然分化,并形成具有自发性、节律性收缩的心肌细胞,拟胚体模拟心肌分化的模型被广泛的用于体外模型心肌分化的研究中。因此,本研究的体外实验部分选用小鼠胚胎干细胞制备成的拟胚体定向心肌细胞分化的模型,以检测尼古丁对心肌分化影响及其相关机制的研究。
本研究从体内早期胚胎发育的模型中,探讨妊娠期母亲被动吸烟和尼古丁干预这两种环境危险因子是否影响胎儿的心脏发育及其功能。此外,运用体外的拟胚体向心肌分化的细胞模型研究尼古丁在干细胞向心肌细胞分化过程中如何通过影响DNA甲基化水平和心肌转录因子的表达,并最终影响干细胞向心肌分化。
研究方法和结果
怀孕的Sprague-Dawley (SD)大鼠被随机分为对照组(相同体积的生理盐水)、吸烟处理组(15和20支烟/天)和尼古丁处理组(15和20mg/kg/天)。在整个妊娠期,怀孕的母鼠分别每天给予15或20支烟被动吸烟处理和15或20mg/kg尼古丁干预直至分娩(PND0),用超声心动图检测子代新生乳鼠的心脏形态结构及功能。超声心动图M-mode图像显示妊娠期吸烟和尼古丁处理组的子代新生乳鼠的心脏跳动的模式和频率与对照组相似,也没有出现异常的M-mode超声心动图。心脏重量(F=0.655,P=0.636)和心脏重量/体重(F=1.149,P=0.4)在各处理组间的差异没有统计学意义。但多普勒检测结果显示,与对照组相比,尼古丁处理组,新生乳鼠心脏的血流动力学图出现血流受限现象,而在20支烟/天处理组中则出现左右心室血流交错现象。
定量分析超声心动图检测所获得的心功能指标,结果显示：妊娠期母鼠给予20mg/kg/天尼古丁处理,子代新生乳鼠的左心室心肌质量指数(Left Ventricular Mass, LV Mass),左心室收缩末期容量(LV end-systolic volume, LVESV),左心室舒张末期容量(LV end-diastolic volume, LVEDV)和左心室舒张末期后壁厚度(Diastolic left ventricular posterior wall, LVPW;d),与对照组相比均显著降低,差异有统计学意义(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).20支烟/天尼古丁处理组,于对照组相比,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;s0.45±0.06vs0.53±0.07, P=0.026)也显著降低,差异均有统计学意义。
尼古丁和吸烟处理组的左心射血分数(Left ventricular ejection fraction, LVEF)和左心室短轴缩短率(Left ventricular fractional shortening, LVFS)与对照组相比,整体水平差异有统计学意义(LVEF,F=8.117,P=0.000；LVFS,F:8.917,P=0.000),但是这两个评价左心功能的重要指标在吸烟和尼古丁干预后,不但没有显著下降,反而在20支烟/天处理组中较对照组上升,差异有统计学意义(LVEF,88.32±4.59vs78.86±7.24,P=0.001；LVFS,56.99±7.54vs45.83±7.04,P=0.000).上述实验结果表明妊娠期孕鼠给予20支烟/天被动吸烟处理或20mg/kg/天的尼古丁处理致后代的左心功能发生异常,且尼古丁和被动吸烟干预产生一致的效应。
为了进一步研究尼古丁对心脏发育和心肌分化的影响,本研究用小鼠胚胎干细胞(mESCs)制备成拟胚体并向心肌细胞分化的过程中给予一系列的尼古丁处理,其中包括低浓度(0.01-1pM)和高浓度(100-1000μM),用MTT法检测其对干细胞增殖影响、PCR和Western blotting等方法检测在分化过程中心肌转录因子的表达水平。系列浓度梯度的尼古丁(0.01-1000μM),不影响悬滴法中拟胚体的形成,也不影响其形态和大小。MTT实验结果显示,mESC在分化培养基中,前三天处于指数生长期,培养第三天达到细胞增殖的高峰,第四天开始细胞增殖缓慢；说明运用悬滴法制备拟胚体的过程中,悬滴培养4天,细胞快速增殖形成拟胚体。有趣的是,在细胞增殖的高峰期(第3天),100μM尼古丁(P<0.01).100μM尼古丁+100gM六烃季胺(P<0.01)和100μM六烃季胺处理组(P<0.05)比对照组细胞数目显著增加,差异有统计学意义。此效应不能被六烃季胺阻断,说明该效应与尼古丁乙酰胆碱受体无关。
经3-D悬滴法培养后将拟胚体转移到细胞培养板中,在2-D的培养条件下进一步贴壁分化和生长。贴壁分化总共持续16天,期间给予系列浓度梯度的尼古丁处理。空白对照组的拟胚体在经1周贴壁分化后,可以在显微镜下观察到跳动的细胞,而在分化的第12天跳动的拟胚体的比例达到峰值。在尼古丁处理组(0.01-1000μM)跳动的拟胚体比例均是在第14天达到最高峰,而比对照组推迟两天。在分化的第12天,100μM尼古丁处理组,跳动的拟胚体的比例比对照组下降了61.59%(13.38±5.44vs.74.97-11.28,P=0.000),而1000μM尼古丁处理组比对照组下降了66.52%(8.445±1.42vs.74.97±11.28,P=0.000)。因此,高浓度(100-1000μM)尼古丁作用抑制并推迟拟胚体向心肌细胞分化的过程。
心脏发育需要能促进心肌细胞生成或抑制抗心肌细胞形成的核转录因子的作用。用实时定量PCR检测分化第12天的拟胚体中转录因子的信使RNA的表达水平,结果显示：小鼠胚胎干细胞干性维持基因Oct4和Nang,在尼古丁处理后,mRNA的相对表达量有增加的趋势,但是差异没有统计学意义(Oct4,F=0.313, P=0.863; Nanog, F=0.757, P=0.608)。相反,在100gM和1000μM尼古丁处理中,Tbx5和GATA4的mRNA表达水平比对照组均显著降低(Tbx5,F=3.209, P=0.043; GATA4, F=9.043, P=0.000),差异具有统计学意义。采用Western blot进一步检测Tbx5和GATA4在尼古丁处理后的拟胚体中的蛋白表达水平,结果显示：尼古丁剂量依赖性的下调Tbx5和GATA4的蛋白表达水平,差异有统计学意义(Tbx5,F=24.069,P=0.000; GATA4, F=9.303, P=0.001)。将获得的蛋白条带的灰密度值做相对定量分析,得到Tbx5和GATA4相对于看家基因GAPDH的相对表达量,结果再次显示：给予0.01-1000gM尼古丁剂量依赖性的下调Tbx5和GATA4的蛋白表达水平,高浓度组(100和1000μM)尼古丁处理对这两个转录因子的抑制作用强烈。
为了进一步阐明尼古丁对心肌分化的影响及相关的机制,实验中选用尼古丁乙酰胆碱受体拮抗剂,六烃季胺(Hexamethonium, Hexa),用于单独或者和尼古丁共同作用于拟胚体的分化过程。采用免疫荧光染色法特异检测经分化的拟胚体中GATA4P日性细胞数量。经过4天悬滴培养(Day4)的拟胚体,在显微镜下无法观察到跳动的心肌细胞,但是免疫荧光可检测到少量的GATA4阳性细胞的存在。在第四天分化早期的拟胚体中,每组GATA4阳性细胞的数量和大小相似,但是,经过8天的贴壁分化(Day12),各组GATA4阳性的细胞数量显著增加,并同时可观察拟胚体中出现节律性跳动的细胞。荧光显微镜镜检记录的图片显示,在第12天的拟胚体中,与对照组相比,尼古丁处理降低GATA4阳性细胞的数量,六烃季胺和尼古丁共同处理则轻微增加GATA4阳性细胞数量。
采用流式细胞仪定量分析GATA4阳性细胞数量,100μM尼古丁处理组GATA4阳性细胞数量比对照组显著降低8.80%,差异有统计学意义(8.27±2.26%vs.17.07±3.18%,P=0.005)。分化12天的拟胚体中,100gM尼古丁和100gM六烃季胺共同处理组与尼古丁单独处理组相比,GATA4细胞数量升高9.03%,差异有统计学意义(18.20±0.90%vs8.27±2.26%,P=0.003),说明尼古丁的抑制效应可被尼古丁乙酰胆碱受体拮抗剂部分阻断。
用实时定量PCR检测经分化12天的拟胚体中相关基因的mRNA表达水平,单独100μM尼古丁处理对Oct4和Tbx5mRNA的表达量的整体水平没有差异(Oct4, F=2.335,P=0.150; Tbx5, F=0.996, P=0.455);而Nanog和GATA4的表达量在各组中整体水平差异显著,均具有统计学意义(Nanog, F=5.273, P=0.027; GATA4, F=11.172, P=0.003)。Nanog的mRNA的表达量在尼古丁(100μM)和六烃季胺(1001μM)共同处理组比对照组显著升高(2.40±0.90vs.00±0.30,P=0.015),与尼古丁单独处理组相比也显著升高(2.40±0.90vs.0.7±0.58,P=0.001),差异均具有统计学意义。尼古丁乙酰胆碱受体拮抗剂同样也能阻断尼古丁抑制心肌转录因子GATA4表达的效应：GATA4的mRNA表达量经尼古丁处理后较对照组显著降低,差异有统计学意义(0.39±0.28vs.1.0±0.32,P=-0.035),然而尼古丁(100μM)和六烃季胺(100μM)共同处理组较对照组却显著升高(1.7±0.40vs.1.00±0.32,P=0.035),且与尼古丁单独处理组相比也显著升高,差异均具有统计学意义(1.70±0.40vs.0.3±0.28,P=0.001)。
蛋白免疫印迹法获得同上相似结果：尼古丁对GATA4的蛋白表达的抑制作用强烈而对Tbx5蛋白表达相对微弱。同样的,尼古丁对上述两个心肌转录因子蛋白水平的抑制作用可被尼古丁乙酰胆碱受体拮抗剂部分阻断。以上结果都说明尼古丁对心肌分化的抑制作用是通过抑制拟胚体分化过程中心肌转录因子的的mRNA和蛋白表达水平导致的,而且这种抑制效应可以被尼古丁乙酰胆碱受体拮抗剂阻断。
为了研究尼古丁在心肌分化早期的作用和机制,同时也进一步探讨其是否通过调控DNA甲基化的表观遗传学修饰作用来影响心肌重要转录因子的表达。本研究选用斑点杂交法(dot blot)和酶联免疫吸附测定法(ELISA)检测经12天分化的拟胚体中5-甲基胞嘧啶的表达水平并用western blot检测DNA甲基化转移酶(DNMT1和DNMT3B)的蛋白水平。
斑点杂交法结果显示,分化的第12天的拟胚体经尼古丁处理后,5-甲基胞嘧啶的表达显著升高,但尼古丁和六烃季胺共同处理时,其表达水平比尼古丁单独处理组降低。酶联免疫吸附测定法定量检测分化早期的拟胚体中5-甲基胞嘧啶的水平,该数据经统计分析,结果显示：5-甲基胞嘧啶的表达水平在各处理组中差异显著,具有统计学意义(F=31.170,P=0.000),经LSD方法多重比较结果显示：经尼古丁处理后,5-甲基胞嘧啶的水平比对照组显著升高,差异有统计学意义(26.37±1.56vs.18.48±1.51,P=0.000),然而当加入100μM六烃季胺和100μM尼古丁共同处理时,其表达量比尼古丁单独处理组却显著降低,差异具有统计学意义±(14.78±2.03vs.26.37±1.56, P=0.000)。酶联免疫吸附测定法所得的结果不仅再次验证尼古丁干预显著升高分化早期的拟胚体中5-甲基胞嘧啶的表达水平,也与前述dot blot的结果相一致,并进一步显示该抑制作用可以被尼古丁乙酰胆碱受体拮抗剂(六烃季胺)阻断,提示该受体参与了上述过程。
为了进一步研究尼古丁在拟胚体向心肌分化过程中,对DNA甲基化水平的影响,本实验采用western blot法检测两个重要的DNA甲基化转移酶(DNMT1和DNMT3B)的蛋白表达水平。蛋白条带灰密度分析结果显示：DNMT1和DNMT3B的蛋白表达水平,在各处理组间差异显著,具有统计学意义(DNMT1: F=29.881, P=0.000; DNMT3B:F=26.651, P=0.000)。DNMT1和DNMT3B的蛋白表达水平,在尼古丁单独处理组中比对照组显著升高,差异有统计学意义(DNMT1,1.13±0.08vs.0.52±0.11,P=0.000; DNMT3B,0.36±0.06vs.0.20±0.02,P=0.001)；但在100μM尼古丁和100μM六烃季胺共同处理组中与单独尼古丁组相比,两者的表达水平均没有差异(DNMT1,1.17±0.09vs.1.13±0.08,P=0.689; DNMT3,0.41±0.01vs.0.36±0.06,P=0.227)。
上述结果表明在拟胚体向心肌细胞分化的早期,尼古丁处理显著的升高DNMT1和DNMT3B的蛋白表达水平,但当同时加入尼古丁乙酰胆碱受体拮抗剂后,不能阻断该效用,尼古丁对DNMT1和DNMT3B的促进作用与对5-甲基胞嘧啶的作用是一致,提示在小鼠胚胎干细胞向心肌分化过程中,尼古丁干预能显著增加DNA甲基化水平。
结论
对体内胚胎心脏发育模型的研究结果显示,妊娠期持续给予吸烟和尼古丁处理诱发子代新生大鼠的心脏功能异常：孕期持续给予20支烟／天或者20mg/kg/天尼古丁干预显著降低左心室心肌质量指数(LV Mass),左心室收缩末期容量(LVESV),左心室舒张末期容量(LVEDV)和左心室舒张末期后壁厚度(LVPW;s)。
对体外拟胚体向心肌分化的细胞模型研究结果显示,持续给予尼古丁处理选择性抑制两个心肌转录因子(Tbx5和GATA4)的mRNA和蛋白水平并降低GATA4阳性的心肌祖细胞数量,从而抑制拟胚体向心肌细胞的分化。尼古丁对心肌分化的抑制作用可以被尼古丁乙酰胆碱受体抑制剂阻断,提示该受体参与了尼古丁抑制心肌分化的过程并起重要作用。但尼古丁处理后却能显著提高5-甲基胞嘧啶和DNA甲基化转移酶(DNMTl和DNMT3B)的表达水平,暗示在拟胚体向心肌分化的早期,尼古丁升高DNA甲基化的水平,使相关的心肌转录因子的启动子区域的甲基化水平升高,从而抑制该基因的表达。
本研究结果首次揭示长期、持续的吸烟和尼古丁干预抑制心脏的发育导致左心功能异常；在拟胚体向心肌分化过程中,尼古丁抑制心肌特异转录因子表达水平的同时上调DNA甲基化水平。本研究展示了先天性心脏病的环境危险因子是如何通过影响遗传因素和改变表观遗传学修饰作用,并最终诱导心脏发育异常；对CHD的病因和机制研究具有重要的意义。
Background and Objective
Congenital 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 Results
Pregnant 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.
Conclusions
The 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 wall
Besieds, 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.
引文
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