ENDOCRINE CONTROL OF FETAL CARDIOVASCULAR DYNAMICS

在胎儿中，心脏激素心钠素（ANF）是 在控制心血管功能和体液平衡方面很重要 在正常和缺氧条件下。 胎儿的心房和心室 在妊娠早期表达ANF，并且在心房表达增加 而脑室中的则随成熟而降低。 这 本申请提出阐明心脏的差异调节， 胎儿发育期间的室特异性ANF基因转录，以及 缺氧诱导心钠素产生的分子机制 在胎儿心脏中的表达。 心房和心室的原代培养 来自妊娠60至145天的绵羊胎儿的肌细胞将用于 这些研究。 具体目标1建议克隆和测序绵羊 ANF基因，并表征绵羊ANF启动子，包括5 '- 侧翼序列 特异性目标2将识别增强子/阻遏子 ANF基因发育调控元件 在羊胎儿心房和心室肌细胞中的转录。 ANF 通过RIA和北方印迹分析测定分泌和mRNA， resp. ANF启动子活性将在转染中测定 使用含有全长或截短的5 '- 与荧光素酶融合的绵羊ANF启动子的侧翼序列 报告基因 具体目标3将研究缺氧的影响 对胎儿缺氧诱导因子1（HIF-1）活性的诱导作用 心房和心室肌细胞通过在低氧环境中孵育细胞 条件 ANF肽分泌、ANF和HIF-1 mRNA水平将被检测到。 测定了 HIF-1的丰度及其与其结合的特异性 绵羊ANF 5 '-侧翼区上的序列特异性位点将是 通过电泳迁移率变动分析测定。 具体目标4将 阐明HIF-1和ANF相互作用的发育变化 缺氧诱导胎儿心房和心室肌细胞启动子活性 肌细胞 总的来说，这些研究将测试假设， 心钠素基因在绵羊胎儿心房发育过程中的表达规律 心室是由于激活室-和发育阶段- 特异性增强子/阻遏子元件在5 '-调控区的 ANF基因。 缺氧诱导胎儿心钠素基因表达， 心房和心室通过诱导HIF-1介导，HIF-1激活 以发育阶段特异性方式表达ANF启动子。 拟议 研究将提供有关控制腔室的重要资料， 胎儿心脏特异性心钠素转录及其相互作用 缺氧与此调节。 这些信息将提供 为胎儿心血管疾病的诊断和治疗提供科学依据， 与压力有关的体液紊乱，如缺氧。

In the fetus the cardiac hormone atrial natriuretic factor (ANF) is important in the control of cardiovascular function and fluid balance under normal and hypoxic conditions. The fetal atria and ventricles express ANF in early gestation, and expression in the atria increases while that in the ventricles decreases with maturation. This application proposes to elucidate the differential regulation of cardiac chamber-specific ANF gene transcription during fetal development, and the molecular mechanisms underlying the hypoxic induction of ANF expression in fetal heart. Primary cultures of atrial and ventricular myocytes from ovine fetuses at 60 to 145 days gestation will be used for these studies. Specific Aim 1 proposes to clone and sequence the ovine ANF gene, and to characterize the ovine ANF promoter including the 5'- flanking sequences. Specific Aim 2 will identify the enhancer/repressor elements which mediate the developmental regulation of ANF gene transcription in ovine fetal atrial and ventricular myocytes. ANF secretion and mRNA will be determined by RIA and Northern blot analysis, resp. ANF promoter activity will be determined in transfection experiments using DNA constructs containing full length or truncated 5'- flanking sequences of the ovine ANF promoter fused to the luciferase reporter gene. Specific Aim 3 will investigate the effects of hypoxia on induction of hypoxia-inducible factor 1 (HIF-1) activity in fetal atrial and ventricular myocytes by incubating the cells in hypoxic conditions. ANF peptide secretion, ANF and HIF-1 mRNA levels will be measured. The abundance and binding specificity of HIF-1 to its sequence-specific site on the ovine ANF 5'-flanking region will be determined by electrophoretic mobility shift assay. Specific Aim 4 will elucidate the developmental change in interaction between HIF-1 and ANF promoter activity induced by hypoxia in fetal atrial and ventricular myocytes. Overall these studies will test the hypothesis that the developmental pattern of ANF gene expression in ovine fetal atria and ventricles is due to activation of chamber- and developmental stage- specific enhancer/repressor elements in the 5'- regulatory region of the ANF gene. Further, the hypoxia induced ANF gene expression in fetal atria and ventricles is mediated by induction of HIF-1 which activates the ANF promoter in a developmental stage-specific manner. The proposed studies will provide important information on the control of chamber- specific ANF transcription in the fetal heart and the interaction of hypoxia with this regulation. This information will provide the scientific basis for diagnosis and treatment of fetal cardiovascular and fluid disorders associated with stress such as hypoxia.