Effect of EglN1 knockdown on smooth muscle expression

Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects other organs at a distance. We developed mouse models to ask if inhibition of EglN1, which senses oxygen and regulates the HIF transcription factor, could suffice to mediate local and remote ischemic preconditioning. We used microarrays to detail the global expression changes induced when the oxygen sensor EglN1 is genetically deleted from skeletal muscle cells. We also used microarrays to assess the transcriptome alterations that occur in mouse hearts with pharmacologic inhibition of EglN1, using the EglN inhibitor FG-4497. We generated mice with a tamoxifen-inducible model of EglN1 loss using a floxxed EglN1 locus with skeletal muscle-specific CRE recombinase. WT mice and mice with the floxxed EglN1 locus were exposed to tamoxifen. Mouse skeletal muscle was isolated for RNA extraction and hybridization on Affymetrix microarrays. In a related experiment, mice were treated with the EglN inhibitor FG-4497, and RNA from their heart tissue was analyzed by microarray for transcriptome alterations compared to control hearts.

缺血预处理（ischemic preconditioning）是指短暂的亚致死性缺血可使机体对后续更长时间的缺血性损伤产生保护作用的现象。远程缺血预处理（remote ischemic preconditioning, RIPC）则是指某一器官的缺血可对远隔部位的其他器官产生保护效应。本研究构建了小鼠模型，旨在探究感知氧分压并调控缺氧诱导因子（hypoxia-inducible factor, HIF）转录因子的EglN1的抑制作用，是否足以介导局部及远程缺血预处理效应。我们通过基因芯片技术详细分析了从骨骼肌细胞中遗传敲除氧感受器EglN1后诱导的全基因组表达变化。此外，我们使用EglN抑制剂FG-4497对小鼠进行药物处理，通过基因芯片检测小鼠心脏的转录组改变情况。我们利用携带有骨骼肌特异性CRE重组酶的floxed EglN1位点，构建了他莫昔芬诱导型EglN1敲除小鼠模型。将野生型（wild type, WT）小鼠及携带floxed EglN1位点的小鼠予以他莫昔芬处理，分离小鼠骨骼肌组织以提取RNA，并在Affymetrix基因芯片上进行杂交检测。在另一相关实验中，我们对小鼠给予EglN抑制剂FG-4497处理，提取其心脏组织RNA，通过基因芯片分析转录组变化，并与对照组心脏组织进行比对。