Division of Pharmacology, Department of Basic Sciences, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, CA 92350, USA.
Cardiovasc Res. 2012 Feb 1;93(2):302-10. doi: 10.1093/cvr/cvr322. Epub 2011 Dec 2.
Hypoxia causes protein kinase C epsilon (PKCε) gene repression in foetal hearts, resulting in heightened cardiac susceptibility to ischaemic injury in offspring. We tested the hypothesis that hypoxia inducible factor 1 (HIF-1) and/or reactive oxygen species (ROS) mediate hypoxia-induced PKCε gene repression.
Hypoxia induced in vivo to pregnant rats, ex vivo to isolated foetal rat hearts, and in vitro in the rat embryonic ventricular myocyte cell line H9c2 resulted in a comparable decrease in PKCε protein and mRNA abundance in foetal hearts and H9c2 cells, which was associated with a significant increase in CpG methylation of the SP1-binding sites at the PKCε promoter. In H9c2 cells and foetal hearts, hypoxia caused nuclear accumulation of HIF-1α, which was inhibited by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole and 2-methoxy estradiol. The HIF-1α inhibitors had no significant effect on hypoxia-induced PKCε mRNA repression. Hypoxia produced a time-dependent increase in ROS production in H9c2 cells and foetal hearts that was blocked by ROS scavengers N-acetyl-cysteine or tempol. In accordance, N-acetyl-cysteine and tempol, but not apocynin, inhibited the hypoxic effect and restored PKCε protein and mRNA expression to the control values in foetal hearts and H9c2 cells. The ROS scavengers blocked hypoxia-induced CpG methylation of the SP1-binding sites, restored SP1 binding to the PKCε promoter, and abrogated the hypoxia-induced increase in the susceptibility of the heart to ischaemic injury in offspring.
The results demonstrate that hypoxia induces epigenetic repression of the PKCε gene through a NADPH oxidase-independent ROS-mediated pathway in the foetal heart, leading to heightened heart vulnerability to ischaemic injury in offspring.
缺氧导致胎儿心脏中蛋白激酶 C ɛ（PKCε）基因抑制，从而使后代心脏对缺血性损伤的敏感性增加。我们检验了这样一个假设，即缺氧诱导因子 1（HIF-1）和/或活性氧（ROS）介导缺氧诱导的 PKCε 基因抑制。
对怀孕大鼠进行体内、对分离的胎鼠心脏进行离体和对大鼠胚胎心室肌细胞系 H9c2 进行体外缺氧处理，导致胎鼠心脏和 H9c2 细胞中 PKCε 蛋白和 mRNA 丰度的相似下降，这与 PKCε 启动子 SP1 结合位点的 CpG 甲基化显著增加有关。在 H9c2 细胞和胎鼠心脏中，缺氧导致 HIF-1α 的核积累，这被 3-(5'-羟甲基-2'-呋喃基)-1-苯并吲哚和 2-甲氧基雌二醇抑制。HIF-1α 抑制剂对缺氧诱导的 PKCε mRNA 抑制没有显著影响。缺氧导致 H9c2 细胞和胎鼠心脏中 ROS 产生的时间依赖性增加，该增加被 ROS 清除剂 N-乙酰半胱氨酸或替莫泊芬阻断。相应地，N-乙酰半胱氨酸和替莫泊芬，而不是阿朴肉桂酸，抑制了缺氧的作用，并使胎鼠心脏和 H9c2 细胞中的 PKCε 蛋白和 mRNA 表达恢复到对照值。ROS 清除剂阻断了缺氧诱导的 SP1 结合位点的 CpG 甲基化，恢复了 SP1 与 PKCε 启动子的结合，并消除了缺氧诱导的后代心脏对缺血性损伤敏感性的增加。
研究结果表明，缺氧通过胎儿心脏中 NADPH 氧化酶独立的 ROS 介导途径诱导 PKCε 基因的表观遗传抑制，导致后代心脏对缺血性损伤的敏感性增加。
