Ovine Fetal Heart Research Articles

Capripoxviruses: Exploring the genetic relatedness between field and vaccine strains from Egypt.

Background and Aim:Lumpy skin disease (LSD) and sheep pox are economically important Capripoxvirus-induced diseases of cattle and sheep, respectively. Despite the extensive vaccination program adopted by Egyptian veterinary authorities, LSD and sheep pox are still prevalent and spread throughout the whole country. The current study was designed for molecular characterization and phylogenetic analysis of LSD virus (LSDV) and Sheep pox virus (SPPV) recovered from field cases in Egypt along with vaccinal strains to assess their genetic relatedness.Materials and Methods:Skin biopsies were collected from naturally infected cases of LSD in Ismailia (n=3 farms) and Beni-Suef (n=2 farms) Governorates and sheep pox in Beni-Suef (n=1 flock). Virus isolation was carried out on primary ovine fetal kidney and heart cell cultures. DNA was extracted from infected materials (skin lesions, infected cell cultures) as well as LSDV Neethling vaccine strain and Romanian SPPV vaccine strain. Polymerase chain reaction was performed using oligonucleotide primers targeting the entire open reading frame of G protein-coupled receptors (GPCR) gene and gene sequences were analyzed.Results:Virus isolation on primary ovine fetal kidney and heart cell culture revealed a cytopathic effect at the third passage characterized by rounding of infected cells and margination of nuclear chromatin. Comparative sequence analysis of GPCR gene revealed that Egyptian LSDV isolated from Ismailia and Beni-Suef shared 99:100% nucleotide and amino acid (AA) identities with each other. In comparison to the vaccinal strains, Egyptian LSDV isolates shared 98:99 nucleotide and AA identities with LSDV Neethling vaccine strain and 93:94% with SPPV Romanian vaccine strain. No differences at the nucleotide or AAs were observed between the SPPV vaccine and virulent strains (100% identity). Phylogenetic analyses revealed that LSDV Neethling vaccine strain is more related to field Egyptian LSDV and clustered within the LSDV group while Romanian SPPV vaccine strain clustered in a separate clade with SPPV field isolates.Conclusion:Comparative sequencing and phylogenetic analyses of the GPCR gene reveal a minimal genetic variation between LSDV field isolates from different locations and a close relationship between virulent field strains and homologous vaccines.

PW287 Identification of key microRNAs involved in regulation of ovine fetal heart development

PW287 Identification of key microRNAs involved in regulation of ovine fetal heart development

Thyroid hormone is required for growth adaptation to pressure load in the ovine fetal heart

Thyroid hormone exerts broad effects on the adult heart, but little is known regarding the role of thyroid hormone in the regulation of cardiac growth early in development and in response to pathophysiological conditions. To address this issue, we determined the effects of fetal thyroidectomy on cardiac growth and growth-related gene expression in control and pulmonary-artery-banded fetal sheep. Fetal thyroidectomy (THX) and/or placement of a restrictive pulmonary artery band (PAB) were performed at 126 ± 1 days of gestation (term, 145 days). Four groups of animals [n = 5-6 in each group; (i) control; (ii) fetal THX; (iii) fetal PAB; and (iv) fetal PAB + THX] were monitored for 1 week prior to being killed. Fetal heart rate was significantly lower in the two THX groups compared with the non-THX groups, while mean arterial blood pressure was similar among groups. Combined left and right ventricle free wall + septum weight, expressed per kilogram of fetal weight, was significantly increased in PAB (6.27 ± 0.85 g kg(-1)) compared with control animals (4.72 ± 0.12 g kg(-1)). Thyroidectomy significantly attenuated the increase in cardiac mass associated with PAB (4.94 ± 0.13 g kg(-1)), while THX alone had no detectable effect on heart mass (4.95 ± 0.27 g kg(-1)). The percentage of binucleated cardiomyocytes was significantly decreased in THX and PAB +THX groups (∼16%) compared with the non-THX groups (∼27%). No differences in levels of activated Akt, extracellular signal-regulated kinase or c-Jun N-terminal kinase were detected among the groups. Markers of cellular proliferation but not apoptosis or expression of growth-related genes were lower in the THX and THX+ PAB groups relative to thyroid-intact animals. These findings suggest that in the late-gestation fetal heart, thyroid hormone has important cellular growth functions in both physiological and pathophysiological states. Specifically, thyroid hormone is required for adaptive fetal cardiac growth in response to pressure overload.

Uterine Lumen Space Restriction Decreases Nitric Oxide Synthase and Alters Its Relationship with Caveolins in the Ovine Fetal Heart.

Uterine Lumen Space Restriction Decreases Nitric Oxide Synthase and Alters Its Relationship with Caveolins in the Ovine Fetal Heart.

Cardiac corticosteroid receptors mediate the enlargement of the ovine fetal heart induced by chronic increases in maternal cortisol

Previous studies have demonstrated that modest, physiologically relevant increases in maternal cortisol in late gestation result in enlargement of the fetal heart. In this study, we investigated the role of mineralocorticoid receptor (MR) or glucocorticoid receptor (GR) in this enlargement. Ewes with single fetuses were randomly assigned at approximately 120 days of gestation to one of four groups: maternal cortisol infusion (1 mg/kg per day, cortisol); maternal cortisol infusion with fetal intrapericardial infusion of the MR antagonist (MRa) potassium canrenoate (600 microg/day; cortisol+MRa); maternal cortisol infusion with fetal intrapericardial infusion of the GR antagonist (GRa) mifepristone (50 microg/day, cortisol+GRa); and maternal saline infusion (control). At approximately 130 days of gestation, fetal heart to body weight ratio and right ventricular (RV) and left ventricular (LV) free wall thicknesses were increased in the cortisol group when compared with control group. Fetal hearts from the cortisol+MRa group weighed significantly less, with thinner LV, RV, and interventricular septum walls, when compared with the cortisol group. Fetal hearts from the cortisol+GRa group had significantly thinner RV walls than the cortisol group. Fetal arterial pressure and heart rate were not different among groups at 130 days. Picrosirius red staining of fetal hearts indicated that the increased size was not accompanied by cardiac fibrosis. These results suggest that physiologic increases in maternal cortisol in late gestation induce fetal cardiac enlargement via MR and, to a lesser extent, by GR, and indicate that the enlargement is not secondary to an increase in fetal blood pressure or an increase in fibrosis within the fetal heart.

Effects of gestational age and cortisol treatment on ovine fetal heart function in a novel biventricular Langendorff preparation

Structural and functional maturation of a number of fetal organs and physiological systems occurs in the immediate period prior to term, in association with the prepartum increase in fetal plasma cortisol concentration. At present, little is known about how myocardial sensitivity to adrenergic and muscarinic cholinergic stimulation changes as the fetus approaches term, nor the role of the prepartum increase in plasma cortisol concentration in mediating these changes. This study used a novel Langendorff, biventricular, ovine fetal heart preparation to investigate the effects of advancing gestation and cortisol treatment on myocardial sensitivity to adrenergic (isoprenaline) and muscarinic cholinergic (carbachol) stimulation. It was hypothesized that cortisol infusion would fully mimic developmental changes in myocardial responsiveness to adrenergic and cholinergic stimulation. Sixteen Welsh Mountain sheep fetuses were surgically prepared under general anaesthesia with vascular catheters. At 125 +/- 1 days gestational age (dGA; term, 145 dGA) fetuses were infused with saline vehicle (n= 7; Premature Control) or with cortisol (n= 4; 2-3 mg kg(-1) d(-1)i.v.; Premature Cortisol) for 5 days. The Term Control group (n= 5) comprised fetuses that were surgically prepared at 130 dGA and infused with vehicle for 5 days prior to delivery (n= 2), or that received no surgery (n= 3). Under terminal anaesthesia, Premature Control and Premature Cortisol fetuses were delivered at 130 dGA and Term Control fetuses between 135 and 143 dGA. Following exsanguination under anaesthesia, fetal hearts were mounted in the Langendorff preparation, allowing measurement of left ventricular (LV) developed pressure and right ventricular (RV) developed pressure, heart rate (HR), coronary perfusion pressure and perfusate distribution to the myocardium. Cortisol infusion elevated fetal plasma cortisol concentrations to values similar to those measured close to term (45.0 +/- 7.1 ng ml(-1)). Advancing gestational age, but not cortisol treatment, enhanced fetal LV developed pressure, RV developed pressure and HR responses to carbachol (P < 0.05). Advancing gestational age, but not cortisol treatment, suppressed fetal LV developed pressure, RV developed pressure and HR responses to isoprenaline (P < 0.05). Maximum doses of either carbachol or isoprenaline had no effect on coronary perfusate distribution. Changes in myocardial responsiveness to adrenergic and muscarinic cholinergic stimulation with advancing gestation provide mechanisms that contribute to the maturation of the cardiovascular system as the ovine fetus approaches term. These changes in myocardial responsiveness are not solely dependent on preparturient elevations in fetal plasma cortisol concentration.

Effect of cortisol on fetal ovine vascular angiotensin II receptors and contractility

The renin angiotensin system is important in the regulation of fetal blood pressure. This study investigated the expression of angiotensin AT 1 and AT 2 receptors in the ovine fetal heart, aorta and umbilical artery, and how these receptors are affected by cortisol. Cortisol infusion into the fetus has previously been shown to cause an increase in fetal blood pressure. We hypothesised that this effect of cortisol is mediated by upregulation of the angiotensin AT 1 receptor. Binding studies performed on tissues with intact endothelium demonstrated both receptor subtypes in the fetal aorta and right ventricle, although the latter contained mainly angiotensin AT 2 receptors. In contrast, only angiotensin AT 1 receptors were found in the umbilical artery. Cortisol infusion into fetuses (3 mg/day for 3–5 days) caused a physiological increase in plasma cortisol levels to 29±4 nM. This was associated with an increase in systolic pressure (57.8±1.7 vs. 52.2±1.5 mm Hg, P<0.05), but cortisol had no effect on the density or affinity of angiotensin receptors, nor on the in vitro contractile responses of carotid and umbilical arterial rings to 5-μM angiotensin II. In conclusion, this study has demonstrated differential expression of angiotensin AT 1 and AT 2 receptors in the different regions of the ovine fetal cardiovascular system and that the angiotensin AT 1 receptor is functional. The lack of any effect of low doses of cortisol on these receptors and on the contractility of isolated fetal vessels to angiotensin II suggests cortisol acts by other mechanisms to raise fetal arterial pressure.

Quantitative electron microscopic study of the hypoxic fetal sheep heart.

In order to determine the effects of chronic, high-altitude hypoxia on the ovine fetal heart, we exposed pregnant ewes to 3,820 m beginning at 30 days gestation. We previously showed that following approximately 110 days of hypoxia the fetal heart showed significant reduction in cardiac output (76% of control) and contractility, and elevated levels of citrate synthase and lactate dehydrogenase. To investigate ultrastructural influences on these observed physiologic changes at altitude, we hypothesized that the volume densities of myofibrils and mitochondria, and glycogen content would be reduced in the ovine fetal heart and that this may contribute to contraction and cardiac output deficits in hypoxia. Mitochondria and myofibril volume density were determined by standard point-counting techniques and glycogen content was determined by biochemical analysis. The glycogen content from the hypoxic right ventricle (4.8 +/- 0.3%) was significantly lower than in control right ventricle (6.8 +/- 0.5%) and both left ventricles (hypoxia, 7.2 +/- 0.5; control, 7.8 +/- 0. 4%). Total mitochondrial volume density was also significantly reduced following hypoxia (15.5 +/- 0.7%) compared to controls (16.9 +/- 0.4%). As is common in the ovine fetal heart, the myofibril volume density of the right ventricle from both groups was significantly higher than the left ventricle (RV, 58.6 +/- 1.6; LV 54.3 +/- 0.9%). However, it was not different between control and high altitude. In support of our hypothesis, we may speculate that deficits in the quantity of myocyte glycogen and mitochondria contribute to the observed reduction in cardiac output and contractility, despite the upregulation of citrate synthase and lactate dehydrogenase. In contrast, myofibril volume density was unchanged.

Effects of long-term, high-altitude hypoxia on the capillarity of the ovine fetal heart.

To determine the effect of chronic hypoxia on myocardial capillarity, we exposed pregnant ewes to an altitude of 3,820 m from day 30 to day 139 of gestation and compared the fetus to low-altitude (approximately 300 m) controls. We hypothesized that capillarity would increase in the hypoxic myocardium to optimize oxygen and metabolite flux to hypoxic tissues. Fetal hearts were fixed by retrograde aortic perfusion and processed for microscopy and stereological evaluation. Fiber cross-sectional area and capillary density were measured and standardized to sarcomere length. Capillary volume density and capillary diameter were measured, capillary-to-fiber ratio and capillary length density were calculated, and the capillary anisotropy coefficient was obtained from a table of known values. Capillary-to-fiber ratio, capillary volume density, and the capillary anisotropy coefficient were not different between hypoxia and control groups. Capillary diameter was significantly larger in the right compared with the left ventricle of hypoxic but not control hearts; fiber cross-sectional area tended to be larger in the right ventricle of both groups, but this was not significant. As a result of larger fiber size, capillary density and capillary length density were significantly smaller in the right ventricle of hypoxic but not control fetal hearts. Contrary to our hypothesis, the ovine fetus does not show morphological adaptation in the myocardium after approximately 109 days of high-altitude hypoxic stress.

Effect of three hours of hypoxia on atrial natriuretic factor gene expression in the ovine fetal heart

OBJECTIVES: The current study investigated the effects of 3 hours of hypoxia on atrial natriuretic factor gene expression and peptide content in each of the four cardiac chambers of the near-term ovine fetus. STUDY DESIGN: Twenty-three chronically catheterized ovine fetuses at 125 to 129 days' gestation (term 145 days) were used for this study. Fetal hypoxia was induced for 3 hours in 12 fetuses by infusion of nitrogen into the maternal trachea. The remaining fetuses were used as controls. Fetal arterial Po2 and plasma atrial natriuretic factor concentrations were measured during hypoxia. At the end of the hypoxic period atrial natriuretic factor peptide contents and messenger ribonucleic acid levels in each cardiac chamber were determined by radioimmunoassay and Northern blot analysis, respectively. RESULTS: With infusion of nitrogen into the maternal trachea, fetal arterial Po2 was reduced within 30 minutes by an average of 8.0 ± 0.3 (SEM) mm Hg (p < 0.0001) and remained reduced at this level throughout the entire hypoxic period. Plasma atrial natriuretic factor concentrations increased by 1152 ± 212 pg/ml (p < 0.003) and the increase was sustained for the duration of hypoxia. Atrial natriuretic factor peptide and messenger ribonucleic acid levels were much higher in the atria than in the ventricles. Hypoxia did not result in alterations of atrial natriuretic factor peptide content or messenger ribonucleic acid abundance in each cardiac chamber. CONCLUSIONS: In the near-term ovine fetus, 3 hours of hypoxia resulted in greatly elevated plasma atrial natriuretic factor concentrations; this response was sustained for the duration of hypoxia. However, the increase was not associated with a detectable change in atrial natriuretic factor peptide content or an induction of atrial natriuretic factor gene expression in the atria and ventricles. (Am J Obstet Gynecol 1997;176:42-48.)

Ontogeny of insulin-like growth factor-I and -II gene expression in ovine fetal heart

Insulin-like growth factors (IGF)-I and -II have been implicated in growth and differentiation during embryonic and fetal development. To examine the role of the IGFs in growth of the fetal heart, we determined the gene expression of IGF-I and IGF-II in the four cardiac chambers of the ovine fetus from 58 to 146 days' gestation (term = 147 days). Total RNA was obtained from the cardiac chambers, analyzed by Northern blot, and hybridized to ovine specific cDNA probes for IGF-I and IGF-II. The resulting autoradiograms were subjected to light densitometry, and the intensity of the IGF signals was normalized to the respective 28S ribosomal RNA signals. In the atria and ventricles, IGF-I mRNA abundance was very low throughout the gestational period studied, whereas IGF-II mRNA levels were higher and readily detectable. In the atria, IGF-I mRNA was very low at 60 days' gestation and appeared to increase gradually toward term. Abundance of IGF-II mRNA was high at 60 days, increased further until 120 days, and decreased slightly toward term. In contrast, in the ventricles, IGF-I mRNA increased from 60 to 100 days and then declined moderately at term. Levels of IGF-II mRNA in the ventricles were high at 60 days and decreased progressively to low levels at term. No difference in IGF-I or IGF-II mRNA levels was noted between the right and left atria or right and left ventricles. These results suggest that developmental patterns for IGF-I and IGF-II gene expression exist in the ovine fetal heart, and the patterns differ between the atria and ventricles. Further, these gestational trends differ from those for atrial natriuretic factor (ANF) found in our previous studies, indicating that expression of the ANF gene in the fetal heart may not be associated with cardiac growth and differentiation.

Induction of vascular endothelial growth factor gene expression by hypoxia in the ovine fetal heart

Induction of vascular endothelial growth factor gene expression by hypoxia in the ovine fetal heart

Gene expression of insulin-like growth factor-I and -II in ovine fetal heart during development

Gene expression of insulin-like growth factor-I and -II in ovine fetal heart during development

Gene expression of atrial natriuretic factor in ovine fetal heart during development.

The present study quantified the abundance of atrial natriuretic factor (ANF) messenger RNA (mRNA) and determined the developmental pattern of ANF gene expression in the four cardiac chambers of the ovine fetus during the last two-thirds of gestation. Twenty-one fetuses from 13 time-dated pregnant ewes at gestational ages of 60-145 days were used for this study. Total RNA from fetal atria and ventricles was extracted and ANF mRNA was analyzed by Northern blotting. The ANF mRNA signal was quantified by light densitometry. The abundance of ANF mRNA in the cardiac chambers across gestational ages was analyzed by linear regression analysis and one-way analysis of variance. Atrial natriuretic factor mRNA was much more abundant in the atria than in the ventricles of all fetuses at each gestational age studied. Atrial ANF mRNA levels were lowest in the younger fetuses at 60 days and increased with advancing gestation. Ventricular ANF mRNA levels were highest in fetuses at 60 days and decreased to almost nondetectable levels near term. No difference in ANF mRNA abundance was noted between the right and left atria or the right and left ventricles at each gestational age. A developmental pattern of ANF gene expression is demonstrated in the ovine fetal heart during the last two-thirds of gestation. This pattern shows that atrial ANF mRNA abundance increases while ventricular abundance decreases as the fetus matures. Expression of the ANF gene in the fetal period may be regulated developmentally or induced by cardiovascular changes in utero.

Developmental changes in atrial natriuretic factor content and localization of its messenger ribonucleic acid in ovine fetal heart

The current study investigated the developmental changes in atrial natriuretic factor peptide content and messenger ribonucleic acid localization in the atria and ventricles of the ovine fetus throughout the second half of gestation. Ovine fetuses from 67 to 146 days' gestation (term 147 days) and newborn lambs were used for the study. Tissue atrial natriuretic factor contents were determined by radioimmunoassay, and atrial natriuretic factor messenger ribonucleic acid distribution was determined by in situ hybridization. In fetal atria, atrial natriuretic factor peptide levels were much greater than those in the ventricles. The levels in the atria increased with advancing gestation from 70 to 140 days, reflecting an increase in weight of the atrial chambers. A similar trend was not observed in the ventricles. In the atria, atrial natriuretic factor peptide content (per unit protein) reached high levels at 100 to 110 days' gestation; this was associated with an increase in level of atrial natriuretic factor gene expression. In the ventricles, atrial natriuretic factor peptide content and gene expression were very low throughout the second half of gestation, except for a peak in content that occurred at 100 days. Atrial natriuretic factor messenger ribonucleic acid abundance was much greater in the atria than in the ventricles in fetuses from 90 to 130 days' gestation. The distribution of atrial natriuretic factor messenger ribonucleic acid was homogeneous throughout the thickness of the atria and ventricles of the fetal heart. During the second half of gestation in the ovine fetus, the expression of atrial natriuretic factor messenger ribonucleic acid in the atria and ventricles paralleled the appearance of the peptide.

Multiple receptor contributions to ovine fetal cardiovascular responses to vasopressin.

Selective AVP V1 receptor antagonist and V2 receptor agonist treatments were used to study V1 and V2 receptor contributions to AVP-induced effects on ovine fetal blood pressure, heart rate, and renal function. The results indicate that V1 receptors do not contribute to late gestation fetal renal responses to AVP. The pressor response to AVP was abolished by V1 receptor blockade while the heart rate response was not affected (Fig. 1). This separation of the blood pressure and heart rate responses to AVP has been noted previously, and indicates that AVP-induced fetal bradycardia is not a reflex response to increased blood pressure. A direct V1 receptor-mediated effect on the heart also appears unlikely. The absence of a dDAVP effect on fetal heart rate indicates that AVP-induced bradycardia is not a V2 receptor-mediated event. An AVP effect on oxytocin receptors appears unlikely due to the antioxytocic action of the Manning Compound. Demonstration of AVP V1a receptors in and around the brainstem cardiovascular control centers supports the view that circulating AVP can modulate vagal outflow via effects in the area postrema. However, if Manning Compound infusion blocks fetal V1a receptor-mediated vascular responses and V1b receptor-mediated ACTH release, then blockade of the heart rate response to AVP also would have been expected. In summary, the fetal heart rate response to AVP is not dependent on AVP-induced increases in blood pressure, V1 receptors blocked by the Manning Compound, or V2 receptors stimulated by dDAVP. We conclude that AVP contributes to fetal heart rate regulation, and the effect is not mediated by any known population of AVP receptors.

Effect of thyroid status on lung and heart beta-adrenergic receptors in fetal and newborn sheep.

The effect of altered thyroid status on the development of beta-adrenergic receptor (BAR) density and affinity was investigated in ovine fetal and newborn heart and lung. Fetal (119-121 days gestation) and newborn (2-3 days of age) sheep underwent either thyroidectomy alone, thyroidectomy plus infusion of a large dose of T3 or sham operation. Eight days later BAR were measured in heart and lung using the tritiated radioligand dihydroalprenolol. There was no apparent effect of altered thyroid status on fetal heart or lung BAR density or affinity. In contrast, the newborns thyroidectomized and infused with T3 had a 96% increase in heart BAR density and an 83% increase in lung BAR compared to the thyroidectomized only or sham-operated newborns which were similar. These results suggest that in the near term ovine fetus heart and lung tissue BAR are neither dependent on nor responsive to thyroid hormones, whereas in the newborn period heart and lung BAR are highly responsive to thyroid hormones.