ENDOCRINE CONTROL OF FETAL CARDIOVASCULAR DYNAMICS

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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.