Autophagy is involved in ethanol-induced cardia bifida during chick cardiogenesis

Excess alcohol consumption during pregnancy has been acknowledged to increase the incidence of congenital disorders, especially the cardiovascular system. However, the mechanism involved in ethanol-induced cardiac malformation in prenatal fetus is still unknown. We demonstrated that ethanol exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Previously, we reported that autophagy was involved in heart tube formation. In this context, we demonstrated that ethanol exposure increased ATG7 and LC3 expression. mTOR was found to be inhibited by ethanol exposure. We activated autophagy using exogenous rapamycin (RAPA) and observed that it induced cardiac bifida and increased GATA5 expression. RAPA beads implantation experiments revealed that RAPA restricted ventricular myosin heavy chain (VMHC) expression. <i>In vitro</i> explant cultures of anterior primitive streak demonstrated that both ethanol and RAPA treatments could reduce cell differentiation and the spontaneous beating of cardiac precursor cells. In addition, the bead experiments showed that RAPA inhibited GATA5 expression during heart tube formation. Semiquantitative RT-PCR analysis indicated that BMP2 expression was increased while GATA4 expression was suppressed. In the embryos exposed to excess ethanol, BMP2, GATA4 and FGF8 expression was repressed. These genes are associated with cardiomyocyte differentiation, while heart tube fusion is associated with increased Wnt3a but reduced VEGF and Slit2 expression. Furthermore, the ethanol exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. In sum, our results revealed that disrupting autophagy and excess ROS generation are responsible for inducing abnormal cardiogenesis in ethanol-treated chick embryos.

妊娠期过量饮酒已被证实会提升先天性疾病的发病风险，尤以心血管系统受累最为突出。然而，乙醇诱发产前胎儿心脏畸形的具体分子机制仍未明确。本研究证实，在鸡胚原肠胚形成阶段暴露于乙醇，会增加双心畸形（cardia bifida）的发生率。此前本团队已报道自噬（autophagy）参与心管形成过程，在此研究背景下，我们发现乙醇暴露可上调ATG7与LC3的表达水平，且乙醇会抑制mTOR的活性。我们通过外源性雷帕霉素（rapamycin, RAPA）激活自噬，结果观察到其可诱发双心畸形，并上调GATA5的表达。雷帕霉素微球植入实验显示，雷帕霉素会抑制心室肌球蛋白重链（ventricular myosin heavy chain, VMHC）的表达。体外（in vitro）前原条外植体培养实验表明，乙醇与雷帕霉素处理均可降低心肌前体细胞的分化能力与自发搏动频率。此外，微球实验证实，在心脏管形成阶段，雷帕霉素会抑制GATA5的表达。半定量逆转录聚合酶链反应（semiquantitative RT-PCR）分析结果显示，BMP2的表达水平上调，而GATA4的表达被抑制。在过量乙醇暴露的鸡胚中，BMP2、GATA4与FGF8的表达均受到抑制。上述基因均与心肌细胞分化相关，而心管融合则与Wnt3a表达上调、VEGF及Slit2表达下调密切相关。进一步研究发现，乙醇暴露还会引发过量活性氧（reactive oxygen species, ROS）的产生，这可能会损伤发育中胚胎的心肌前体细胞。综上，本研究结果揭示，自噬功能紊乱与过量活性氧生成共同介导了乙醇处理鸡胚的心脏发生异常。