ENDOCRINE CONTROL OF FETAL CARDIOVASCULAR DYNAMICS

在胎儿中，心脏激素心钠素(ANF)是 对心血管功能和液体平衡的控制很重要 在正常和低氧条件下。胎儿心房和脑室 ANF在妊娠早期表达，且在心房的表达增加 而在脑室内则随着成熟而减少。这 应用提示阐明心脏的差异性调节 胎儿发育过程中特定的心钠素基因转录，以及 低氧诱导ANF的分子机制 在胎儿心脏中的表达。心房和心室的原代培养 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建议克隆绵羊并对其进行测序 ANF基因，并对绵羊ANF启动子的5‘- 侧翼序列。特定目标2将确定增强子/抑制子 介导ANF基因发育调控的元件 绵羊胎儿心房和心室肌细胞的转录。ANF 分泌物和mRNA将通过RIA和Northern印迹分析来确定， 响应。ANF启动子活性将在转染法中测定 使用包含全长或截短5‘-的DNA构建物的实验 羊心钠素启动子侧翼序列与荧光素酶的融合 记者基恩。特定目标3将研究低氧的影响 诱导胎儿缺氧诱导因子-1活性的研究 低氧条件下培养的房室肌细胞 条件。ANF多肽分泌、ANF和HIF-1的mRNA水平 量过了。缺氧诱导因子-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 用凝胶电泳法测定。具体目标4将 阐明HIF-1与ANF相互作用的发育性变化 低氧对胎儿心房室组织启动子活性的影响 肌细胞。总体而言，这些研究将检验这样的假设： 羊胎心房心钠素基因表达的发育规律 脑室是由于脑室的激活和发育阶段- 5‘-调控区中的特定增强子/抑制子元件 ANF基因。此外，缺氧诱导了胎儿心钠素基因的表达 心房和心室是通过诱导HIF-1激活而介导的 ANF启动子具有发育阶段特异性。建议数 研究将提供有关舱室控制的重要信息- 胎儿心脏心钠素的特异性转录及其相互作用 低氧与这种调节有关。此信息将提供 胎儿心血管疾病的诊断和治疗的科学依据 与压力相关的体液紊乱，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.