High oxygen prevents fetal lethality due to lack of catecholamines.

The catecholamine norepinephrine is required for fetal survival, but its essential function is unknown. When catecholamine-deficient [tyrosine hydroxylase (Th) null] mouse fetuses die at embryonic day (E)13.5-14.5, they resemble wild-type (wt) fetuses exposed to hypoxia. They exhibit bradycardia (28% reduction in heart rate), thin ventricular myocardium (20% reduction in tissue), epicardial detachment, and death with vascular congestion, hemorrhage, and edema. At E12.5, before the appearance of morphological deficits, catecholamine-deficient fetuses are preferentially killed by experimentally induced hypoxia and have lower tissue Po(2) levels than wt siblings. By microarray analysis (http://www.ncbi.nlm.nih.gov/geo; accession no. GSE10341), hypoxia-inducible factor-1 target genes are induced to a greater extent in null fetuses than in wt siblings, supporting the notion that mutants experience lower oxygen tension or have an enhanced response to hypoxia. Hypoxia induces a 13-fold increase in plasma norepinephrine levels, which would be expected to increase heart rate, thereby improving oxygen delivery in wt mice. Surprisingly, increasing maternal oxygen (inspired O(2) 33 or 63%) prevents the effects of catecholamine deficiency, restoring heart rate, myocardial tissue, and survival of Th null fetuses to wt levels. We suggest that norepinephrine mediates fetal survival by maintaining oxygen homeostasis.

Early fetal hypoxia leads to growth restriction and myocardial thinning.

Hypoxia is necessary for fetal development; however, excess hypoxia is detrimental. Hypoxia has been extensively studied in the near-term fetus, but less is known about earlier fetal effects. The purpose of this study was to determine the window of vulnerability to severe hypoxia, what organ system(s) is most sensitive, and why hypoxic fetuses die. We induced hypoxia by reducing maternal-inspired O2 from 21% to 8%, which decreased fetal tissue oxygenation assessed by pimonidazole binding. The mouse fetus was most vulnerable in midgestation: 24 h of hypoxia killed 89% of embryonic day 13.5 (E13.5) fetuses, but only 5% of E11.5 and 51% of E17.5 fetuses. Sublethal hypoxia at E12.5 caused growth restriction, reducing fetal weight by 26% and protein by 45%. Hypoxia induced HIF-1 target genes, including vascular endothelial growth factor (Vegf), erythropoietin, glucose transporter-1 and insulin-like growth factor binding protein-1 (Igfbp-1), which has been implicated in human intrauterine growth restriction (IUGR). Hypoxia severely compromised the cardiovascular system. Signs of heart failure, including loss of yolk sac circulation, hemorrhage, and edema, were caused by 18-24 h of hypoxia. Hypoxia induced ventricular dilation and myocardial hypoplasia, decreasing ventricular tissue by 50% and proliferation by 21% in vivo and by 40% in isolated cultured hearts. Epicardial detachment was the first sign of hypoxic damage in the heart, although expression of epicardially derived mitogens, such as FGF2, FGF9, and Wnt9b was not reduced. We propose that hypoxia compromises the fetus through myocardial hypoplasia and reduced heart rate.

Beta1-adrenergic receptors maintain fetal heart rate and survival.

Beta-adrenergic receptor (betaAR) activation has been shown to maintain heart rate during hypoxia and to rescue the fetus from the fetal lethality that occurs in the absence of norepinephrine. This study examines whether the same subtype of betaAR is responsible for survival and heart rate regulation. It also investigates which betaARs are located on the early fetal heart and whether they can be directly activated during hypoxia. Cultured E12.5 mouse fetuses were treated with subtype-specific betaAR antagonists to pharmacologically block betaARs during a hypoxic insult. Hypoxia alone reduced heart rate by 35-40% compared to prehypoxic levels. During hypoxia, heart rate was further reduced by 31% in the presence of a beta(1)AR antagonist, CGP20712A, at 100 nM, but not with a beta2 (ICI118551)- or a beta3 (SR59230A)-specific antagonist at 100 nM. Survival in utero was also mediated by beta1ARs. A beta1 partial agonist, xamoterol, rescued 74% of catecholamine-deficient (tyrosine-hydroxylase-null) pups to birth, a survival rate equivalent to that with a nonspecific betaAR agonist, isoproterenol (87%). Receptor autoradiography showed that beta1ARs were only found on the mouse heart at E12.5, while beta2ARs were localized to the liver and vasculature. To determine if the response to hypoxia was intrinsic to the heart, isolated fetal hearts were incubated under hypoxic conditions in the presence of a betaAR agonist. Heart rate was reduced to 25-30% by hypoxia alone, but was restored to 63% of prehypoxic levels with 100 nM isoproterenol. Restoration was completely prevented if beta1ARs were blocked with CGP20712A at 300 nM, a concentration that blocks beta1ARs, but not beta2- or beta3ARs. Our results demonstrate that beta1ARs are located on the heart of early fetal mice and that beta1AR stimulation maintains fetal heart rate during hypoxia and mediates survival in vivo.

Induction of tyrosine hydroxylase in the locus coeruleus of transgenic mice in response to stress or nicotine treatment: lack of activation of tyrosine hydroxylase promoter activity.

Prolonged stress or chronic nicotine administration leads to induction of tyrosine hydroxylase (TH) in adrenal medulla and locus coeruleus (LC) of the rat. In this study we use mice that express a transgene encoding 4.5 kb of TH gene 5'-flanking region fused upstream of the reporter gene, human alkaline phosphatase (hAP) to test whether TH gene promoter activity is stimulated by immobilization stress, cold exposure or nicotine administration in adrenal medulla and LC. TH-hAP transgene expression is increased in response to all three stimuli in the adrenal medulla. In contrast, TH-hAP expression does not increase in response to either immobilization stress or nicotine administration in the LC and only a small induction of LC TH-hAP mRNA is observed in response to cold exposure. TH mRNA is induced 2-3 fold and TH activity is increased significantly by all three stimuli in both the adrenal and LC. These results support the hypothesis that TH expression is induced by stress or nicotine treatment in both the adrenal medulla and LC of the mouse. The induction in the adrenal is dependent primarily on transcriptional mechanisms, whereas that in the LC is apparently dependent primarily on post-transcriptional mechanisms.

Tyrosine hydroxylase transcription depends primarily on cAMP response element activity, regardless of the type of inducing stimulus.

In neurons and neuroendocrine cells, tyrosine hydroxylase (TH) gene expression is induced by stimuli that elevate cAMP, by depolarization, and by hypoxia. Using these stimuli, we examined TH promoter mutants, cAMP response element binding protein (CREB) phosphorylation site mutants, and transcriptional interference with dominant negative transcription factors to assess the relative contributions of CREB/AP-1 family members to the regulation of basal and inducible TH transcription in PC12 cells. We found that basal transcription depends on transcription factor activity at the partial dyad (-17 bp), CRE (-45 bp), and AP1 (-205 bp) elements. Induced transcription is regulated primarily by activity at the CRE, with only small contributions from the AP1 or hypoxia response element 1 (HRE1; -225 bp) elements, regardless of inducing stimulus. CREB, ATF-1, and CREMtau all mediate CRE-dependent transcription, with CREB and CREMtau being more effective than ATF-1. Phosphorylation of CREB on Ser133, but not on Ser142 or Ser143, is required for induced transcription, regardless of inducing stimulus.

Catecholamines act via a beta-adrenergic receptor to maintain fetal heart rate and survival.

Mice lacking catecholamines die before birth, some with cardiovascular abnormalities. To investigate the role of catecholamines in development, embryonic day 12.5 (E12.5) fetuses were cultured and heart rate monitored. Under optimal oxygenation, wild-type and catecholamine-deficient fetuses had the same initial heart rate (200-220 beats/min), which decreased by 15% in wild-type fetuses during 50 min of culture. During the same culture period, catecholamine-deficient fetuses dropped their heart rate by 35%. Hypoxia reduced heart rate of wild-type fetuses by 35-40% in culture and by 20% in utero, assessed by echocardiography. However, catecholamine-deficient fetuses exhibited greater hypoxia-induced bradycardia, reducing their heart rate by 70-75% in culture. Isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, reversed this extreme bradycardia, restoring the rate of catecholamine-deficient fetuses to that of nonmutant siblings. Moreover, isoproterenol rescued 100% of catecholamine-deficient pups to birth in a dose-dependent, stereo-specific manner when administered in the dam's drinking water. An alpha-AR agonist was without effect. When wild-type fetuses were cultured with adrenoreceptor antagonists to create pharmacological nulls, blockade of alpha-ARs with 10 microM phentolamine or beta-ARs with 10 microM bupranolol alone or in combination did not reduce heart rate under optimal oxygenation. However, when combined with hypoxia, beta-AR blockade reduced heart rate by 35%. In contrast, the muscarinic blocker atropine and the alpha-AR antagonist phentolamine had no effect. These data suggest that beta-ARs mediate survival in vivo and regulate heart rate in culture. We hypothesize that norepinephrine, acting through beta-ARs, maintains fetal heart rate during periods of transient hypoxia that occur throughout gestation, and that catecholamine-deficient fetuses die because they cannot withstand hypoxia-induced bradycardia.