BMP4介导孕期糖皮质激素暴露对子代心脏发育和心功能印迹效应的机制研究

The interaction between intra- and external uterine environments with genesis will substantially influence the pregnancy outcome and the health of offspring. As the first-line treatment for the autoimmune diseases or preterm labor during pregnancy, pre-natal over-exposure to glucocorticoids (GC) programs some permanent pathological lesions on fetal organs and increase the morbidity of adult diseases. Our previous studies found that over exposure to GC during pregnancy compromised cardiac functions and increased the susceptibility of offspring’s myocardium to ischemia/reperfusion. Using MeDIP plus high-throughput second generation sequencing combined with bioinformatics analysis, bone morphogenetic protein-4 (BMP4) was suggested to contribute to this programming effect. In the present study, we will firstly determine the expression of BMP4 and its receptors and endogenous inhibitors in myocardium of fetal and adult offspring exposed to GC during pregnancy. Then we utilize several strains of Bmp4 conditional knock-out mice or transgenic mice to further study the mechanisms underlying the programming effects on cardiac development and functions induced by prenatal GC exposure. Finally, MeDIP-Seq, Bmp4 Bisulfite-Sequencing and ChIP-qPCR on Bmp4 promoter region using sperm or oocyte from F2 offspring, to dissect the mechanisms underlying transgenerational inheritance mediated by the epigenetic modification on BMP4 or other molecules. This study will provide new lines for the optimal usage of GC during pregnancy, and shed lights on the prevention and cure for congenital heart diseases caused by environmental risk factors.

妊娠期子宫内外环境与遗传的相互作用，是影响妊娠结局和子代健康的重要因素。糖皮质激素（GC）作为妊娠合并免疫性疾病和早产风险治疗的一线药物，会对子代多个器官系统产生印迹效应，危及胎儿健康。我们前期的研究发现，孕期GC暴露会导致子代大鼠心功能下降，心肌对缺血再灌注损伤的耐受性下降，并通过MeDIP-Seq发现骨形成蛋白BMP4可能在其中发挥重要作用。在此基础上，本课题首先在孕期GC暴露的小鼠模型上观察BMP4及其受体和内源性抑制物在不同发育阶段和成年心肌的表达变化，随后以BMP4心肌条件性敲除或过表达小鼠为模型，深入研究孕期GC暴露对子代心脏发育和心功能印迹效应的分子机制和信号通路，最后通过对子二代精、卵甲基化测序和染色质免疫共沉淀等方法阐明GC暴露对BMP4的表观遗传修饰承载印迹效应的跨代遗传规律，为指导妊娠期糖皮质激素合理用药，以及防治环境因素所致先天性心脏病提供理论依据和新的治疗靶点。

孕期宫内暴露于不良环境会导致子代心血管疾病的发生。糖皮质激素（GC）对于胎儿器官的正常发育和成熟至关重要，也是患有自身免疫性疾病孕妇的一线治疗药物。然而，孕期 GC的过度暴露可能会影响胎儿器官的发育，并导致出生后多种慢性疾病的易感性升高。我们之前的研究发现，在缺血再灌注后，孕期暴露于地塞米松 (DEX) 的大鼠子代的心功能明显受损。在本研究中，我们进一步揭示了孕期DEX暴露引起心肌细胞中骨形态发生蛋白 4 (Bmp4) 启动子发生DNA 高甲基化，显著抑制了心脏缺血诱导的转录因子 HIF-1alpha与Bmp4启动子的结合能力，阻碍缺血再灌注对BMP4的诱导，从而减弱BMP4对心肌细胞的保护作用，最终表现为心脏功能障碍。此外，我们采用了两种心肌特异性 Bmp4基因敲入小鼠模型，发现体内 BMP4的过表达可以挽救孕期GC暴露引起的心脏功能障碍。深入的机制研究表明，BMP4 通过p-Smad和Parkin依赖性方式减少线粒体 PGC-1alpha的表达，来保护心肌细胞免受线粒体自噬和凋亡引起的损伤。这些研究表明，由于孕期GC暴露导致心肌细胞中Bmp4 启动子高甲基化，使得缺氧诱导的 HIF-1alpha对Bmp4的反式激活受到抑制，增加了子代心脏对出生后“二次打击”的易感性。DNA甲基化抑制剂（例如5-AZA）预处理可能是孕期GC暴露对新生儿心脏功能编程效应的潜在治疗方法。