Kurt A. Bedell

Effect of Cortisol on Gene Expression of the Renin-Angiotensin System in Fetal Sheep

ABSTRACT: Components of the renin-angiotensin system have been found in a variety of tissues during fetal and postnatal life and appear to be developmentally regulated. We postulated that hormonal changes associated with parturition participate in the regulation of renin, angiotensinogen (Ao) and angiotensin type 1 receptor (AT1) gene expression. Cortisol, which increases rapidly in fetal blood before delivery, has been shown to influence the maturation of various systems in the developing fetus. To test the hypothesis that an increase in cortisol regulates fetal renin, Ao, and AT1 mRNA gene expression, we used Northern blot analysis to study the effects of an intraperitoneal infusion of cortisol (3 mg/h, 1 mL/h) for 48 h on the expression of these genes in twin ovine fetuses (n = 10 pairs) at 130-d gestation (term 145 d); one twin in each pair served as a saline-treated control (0.9% NaCl, 1 mL/h). Plasma cortisol levels were significantly higher in cortisol-treated fetuses (113 ± 23 nmol/dL) than in twin controls (4.6 ± 0.8 nmol/dL). Cortisol infusion significantly decreased AT1 receptor mRNA levels in kidney and liver by 24 ± 7% and 27 ± 8%, respectively, when compared with controls (p < 0.05), whereas in contrast, increased mRNA levels (p < 0.05) in heart right atrium (91 ± 23%) and ventricle (59 ± 20%). Renin mRNA levels decreased in renal cortex by 77 ± 13% (p < 0.05) in cortisol-treated animals compared with controls. Hepatic Ao mRNA levels decreased by 15 ± 5% in response to cortisol (p < 0.05), whereas no significant effect was seen on renal Ao gene expression. These findings demonstrate that cortisol exhibits tissue specific positive and negative regulation of renin, Ao and AT1 receptor gene expression during fetal life and may act as an important modulator of the renin-angiotensin system during parturition.

Development of baroreflex influences on heart rate variability in preterm infants

To investigate developmental changes in autonomic cardiovascular reflexes in preterm infants, we used autoregressive power spectral analysis to analyze the effect of upright tilting on heart rate variability in preterm infants. Twenty-eight infants were studied in a longitudinal fashion beginning at 28-32 weeks postconceptional age (postnatal age 1-5 weeks). Each week, heart rate variability in the supine position and after 45 degrees head-up tilt was analyzed by spectral analysis. With the initial study of each infant, there was no significant change in heart rate following head-up tilt compared with baseline (-0.5+/-0.9 bpm). However, linear regression analysis revealed that with increasing postnatal age, the change in heart rate in response to tilting became more positive (mean slope of regressions 0.45+/-0.12 bpm/week, P<0.005). The power spectral density of R-R interval variability in the low-(LF; 0.02-0.15 Hz) and high-(HF; 0.15-1.5 Hz) frequency ranges were obtained and the values normalized by dividing each component by the total power. For measurements obtained in the supine position, the LF/HF ratio progressively decreased with increasing postnatal age, indicating a maturational change in sympathovagal balance. We used the difference in the LF/HF ratio between tilt and the recumbent position as a measure of the change in autonomic input to the heart in response to unloading of the arterial baroreceptors. No significant change in these ratios were observed when infants were first studied between 28 and 32 weeks postconceptional age, suggesting that the cardiac baroreflex is poorly developed at this stage of development. However, with postnatal maturation, the LF component of the power spectrum became progressively larger with tilt relative to the basal state, such that the difference between LF/HF(tilt) and LF/HF(base) became progressively more positive (P <0.006). These findings suggest that in premature infants, cardiac baroreceptor reflexes become more functional with postnatal development.

Metabolic Adaptation of the Fetal and Postnatal Ovine Heart: Regulatory Role of Hypoxia-Inducible Factors and Nuclear Respiratory Factor-1

Numerous metabolic adaptations occur in the heart after birth. Important transcription factors that regulate expression of the glycolytic and mitochondrial oxidative genes are hypoxia-inducible factors (HIF-1α and -2α) and nuclear respiratory factor-1 (NRF-1). The goal of this study was to examine expression of HIF-1α, HIF-2α, and NRF-1 and the genes they regulate in pre- and postnatal myocardium. Ovine right and left ventricular myocardium was obtained at four time points: 95 and 140 d gestation (term = 145 d) and 7 d and 8 wk postnatally. Steady-state mRNA and protein levels of HIF-1α and NRF-1 and protein levels of HIF-2α were measured along with mRNA of HIF-1α-regulated genes (aldolase A, α- and β-enolase, lactate dehydrogenase A, liver and muscle phosphofructokinase) and NRF-1-regulated genes (cytochrome c, Va subunit of cytochrome oxidase, and carnitine palmitoyltransferase I ). HIF-1α protein was present in fetal myocardium but dropped below detectable levels at 7 d postnatally. HIF-2α protein levels were similar at the four time points. Steady-state mRNA levels of α-enolase, lactate dehydrogenase A, and liver phosphofructokinase declined significantly postnatally. Aldolase A, β-enolase, and muscle phosphofructokinase mRNA levels increased postnatally. Steady-state mRNA and protein levels of NRF-1 decreased postnatally in contrast to the postnatal increases in cytochrome c, subunit Va of cytochrome oxidase, and carnitine palmitoyltransferase I mRNA levels. The in vivo postnatal regulation of enzymes encoding glycolytic and mitochondrial enzymes is complex. As transactivation response elements for the genes encoding metabolic enzymes continue to be characterized, studies using the fetal-to-postnatal metabolic transition of the heart will continue to help define the in vivo role of these transcription factors.

Angiotensin AT1 receptor blockade fails to attenuate pressure-overload cardiac hypertrophy in fetal sheep

We examined the hypothesis that endogenous angiotensin II and angiotensin type 1 (AT1) receptors participate in the development of fetal right ventricular hypertrophy by studying the effects of AT1 receptor blockade on cardiac growth in fetal sheep subjected to constrictive banding of the pulmonary artery (PA). Seven pairs of twin fetuses were studied beginning at 126 ± 1 days gestation (term = 145 days). One twin was given losartan (10 mg ⋅ kg-1 ⋅ day-1iv) for 7 consecutive days after PA banding, and the other twin served as a saline-treated, PA-banded control. Four additional pairs of twins served as sham-operated controls. Fetal heart rate (HR) and mean arterial blood pressure (MABP) were similar in the two groups of PA-banded animals before treatment and remained unchanged in the PA-banded control group. Losartan resulted in a significant decrease ( P < 0.05) in MABP between days 0 and 7, whereas HR was not affected. Total body weight of the losartan-treated animals was significantly less ( P < 0.05) than twin PA-banded controls and nonbanded fetuses. Right ventricle weight-to-body weight ratios were similar in saline (2.29 ± 0.34 g/kg) and losartan-treated (2.11 ± 0.15 g/kg) PA-banded animals and significantly greater than that in nonbanded fetuses (1.52 ± 0.07 g/kg). Similar differences were seen in the right ventricle weight-to-left ventricle weight ratios. Right and left ventricle AT1 receptor mRNA and protein expression were also similar among the three groups, as were AT2 receptor mRNA levels. These data suggest that endogenous angiotensin II does not contribute to the development of pressure overload-induced right ventricular hypertrophy during fetal life and that expression of angiotensin receptors is not altered by increased afterload in the ovine fetus.

Ontogeny and regulation of cardiac angiotensin types 1 and 2 receptors during fetal life in sheep.

Previous studies have shown that the expression of cardiac angiotensin II(ANG II) type 1 (AT1) and type 2 (AT2) receptors are developmentally regulated, although factors modulating these receptors have not been well investigated. The present study was designed 1) to characterize the ontogeny of cardiac AT1 and AT2 gene expression during the last third trimester of gestation in fetal sheep and newborn lambs, 2) to determine the influence of ANG II on modulating cardiac AT1 and AT2 gene expression during fetal life, and 3) to investigate the role of AT1 receptor activity on the regulation of AT1 and AT2 mRNA levels during fetal cardiac development. Using sheep AT1 and AT2 cDNA probes, we demonstrated that cardiac AT1 gene expression is relatively unchanged during fetal (90-135 d of gestation, term 145 d) and newborn life. In contrast, cardiac AT2 mRNA expression was high during fetal development and decreased rapidly after birth. Continuous i.v. infusion of ANG II (9.5 nM/h) for 24 h, which raised ANG II levels from 84± 9 to 210 ± 21 pg/mL had no effect on the expression of cardiac AT1 or AT2 mRNA, but increased adrenal and decreased liver AT1 mRNA levels. Administration of the AT1 receptor antagonist losartan (1.2 mg kg-1 h-1) significantly decreased arterial blood pressure in fetuses at 110- and 135-d, but not 95-d gestation. Except for increased AT1 receptor gene expression in the right atrium at 95-and 135-d gestation, and left ventricle at 110-d gestation, cardiac AT1 and AT2 mRNA levels were unaltered by AT1 receptor blockade. In summary, this study demonstrates that cardiac AT2 but not AT1 receptor gene expression is regulated by the transition from fetal to newborn life. Neither ANG II nor blockade of AT1 receptors significantly alter the expression of AT1 or AT2 mRNA in the fetal heart. Endogenous ANG II also appears to significantly contribute to the maintenance of blood pressure homeostasis during the final third of gestation in fetal lambs.

ONTOGENY AND REGULATION OF CARDIAC ANGIOTENSIN II RECEPTOR SUBTYPES (AT1 AND AT2) IN DEVELOPING SHEEP. † 205

ONTOGENY AND REGULATION OF CARDIAC ANGIOTENSIN II RECEPTOR SUBTYPES (AT1 AND AT2) IN DEVELOPING SHEEP. † 205

Efficacy of an Automated Multiple Emitter Whole-Room Ultraviolet-C Disinfection System Against Coronaviruses MHV and MERS-CoV.

Efficient and automated methods of disinfecting surfaces contaminated with the Middle Eastern respiratory syndrome coronavirus (MERS-CoV) may prevent the spread of the virus. Here we report the efficacy and use of an automated triple-emitter whole room UV-C disinfection system to inactivate mouse hepatitis virus, strain A59 (MHV-A59) and MERS-CoV viruses on surfaces with a >5 log10 reduction. Infect Control Hosp Epidemiol 2016;37:598–599

Thyroid Hormone Increases Eaat1 Expression in Rat Heart.

Background: Hyperthyroid induced cardiac hypertrophy is related to increased cardiac workload. These changes are associated with an upregulation of metabolic pathways associated with energy production. The malate/aspartate shuttle, necessary to transfer the reducing equivalents produced by glycolysis into the mitochondria, is increased 33% in hyperthyroid rats. Of the shuttles two inner membrane protein carriers, the aspartate-glutamate carrier is rate-limiting. The Excitatory Amino Acid Transporter, Type 1 (EAAT1) has recently been shown to function as a glutamate carrier in the malate/aspartate shuttle. We hypothesize that EAAT1 is upregulated by thyroid hormone.Methods: Adult Sprague-Dawley rats were infused with, tri-iodothyroxine (T3), propylthiouracil (PTU), or saline over a period of 8 days. Serum free T3 levels were measured. Rats were euthanized, hearts weighed, and tissue frozen. Northern Blot analysis was performed on total RNA using a unique 350 bp 32-P labeled EAAT1 ribonucleotide probe and normalized to 18S rRNA. A spectrophotometric assay of the malate/aspartate with glutamate and lactate as substrates was performed on isolated mitochondria. Results are displayed as oxidation rate/min/mg mitochondrial protein. Protein lysates from mitochondria were used for immunoblot analysis with human anti-EAAT1 IgG.Results: EAAT1 steady-state mRNA levels were increased in the T3-treated rats compared to controls (0.031+/−0.005 vs. 0.011+/−0.002; P<0.05), and decreased in PTU-treated rats vs. controls (0.0011+/−0.0002 vs. 0.0015+/−0.0001; P<0.05). EAAT1 mitochondrial protein levels were increased in T3-treated rats vs. controls (8.9+/−0.4 vs. 5.9+/−0.6; P<0.005). EAAT1 protein levels were below detection in PTU-treated rats. Malate/aspartate shuttle activity was unchanged by PTU infusion.Conclusions: Hyperthyroidism in rats is related to an increase in expression of both EAAT1 mRNA and protein in cardiomyocytes. This 49% increase in the rate-limiting aspartate-glutamate carrier of the malate/aspartate shuttle correlates with the observed increase in shuttle activity. The upregulation of EAAT1 by thyroid hormone may facilitate the cardiomyocyte response to hyperthyroidism and the associated increased metabolic demand placed upon the cell.

Cardiac Hypertrophy Is Not Attenuated by Angiotensin Type 2 (AT2) Receptor Blockade in Fetal Sheep |[bull]| 139

We have previously shown in pulmonary artery (PA) banded fetal sheep that angiotensin II type 1 (AT1) receptor blockade fails to attenuate the development of right ventricular hypertrophy (RVH) and does not alter expression of cardiac AT1 or AT2 mRNA (Am J Physiol 42:R1501-8, 1997). Because AT2 mRNA is expressed in higher abundance in fetal compared to neonatal heart, and because there is evidence suggesting these receptors are involved in cellular growth and differentiation, we tested the hypothesis that endogenous angiotensin II and AT2 receptors participate in the development of fetal RVH. Three groups of fetuses were studied beginning at 126 d gestation(term 145 d) and were given either the AT2 receptor antagonist PD 123319 (5 mg/kg/d iv) for 7 consecutive days (n=5), PA banding plus PD 123319 (n=5), or sham surgery alone (n=4). Fetal heart rate and arterial pressure were similar among groups before and after the treatment period. Body weight and left ventricle weight/body weight at the end of study were also similar among groups. However, RV weight/body weight was significantly greater (p<0.05) in PA banded animals (2.38±.07 g/kg) than in PD alone (1.83±.14 g/kg) or sham fetuses (1.72±.07). Northern blot analysis revealed that PD alone decreased cardiac AT1 mRNA levels (55±7% and 73±13% of control (100%) in RV and LV, respectively). Similar decreases were seen in the RV (76±16%) and LV (71±9%) of PA banded animals. No changes in cardiac AT2 mRNA levels were detected in any group. These data suggest that in the ovine fetus, AT2 receptors do not contribute to the maintenance of blood pressure or the development of pressure-overload RV hypertrophy. In addition, expression of cardiac AT1 but not AT2 mRNA is altered by AT2 receptor blockade.

Angiotensin II Type 1 (AT1) Receptor Expression and Regulation in Human Fetal Lung In Vitro 305

Specific angiotensin II (ANG II) receptors are present early in gestation and may play a role in organ growth and development. To investigate regulation of the AT1 receptor in human lung, we first characterized changes in AT1 mRNA levels in midtrimester human fetal lung explants, which spontaneously differentiate in culture. Northern blot analysis revealed that during four days in explant culture in serum-free medium, AT1 mRNA levels decreased to 19±3, 10±3, 8±2 and 5±2% (days 1, 2, 3, 4, respectively, all p<0.05) of control levels (day 0). The decrease in AT1 mRNA after 48 h in culture also occurred in the presence of 5% fetal calf serum. Immunostaining demonstrated a mesenchymal distribution of the AT1 receptor with limited expression in vascular smooth muscle. AT1 mRNA levels were unaltered after 48 h in culture with ANG II (10-5 to 10-9 M), the AT1 receptor antagonist losartan (10-5 to 10-9 M), or cortisol (10-6 and 10-9 M). Finally, abundance of AT1 mRNA was similarly decreased by incubation in 2% or 20% oxygen. These findings indicate that AT1 mRNA and protein are present in midtrimester fetal lung and that AT1 mRNA levels decrease in association with spontaneous differentiation of human fetal lung in vitro. AT1 mRNA in human fetal lung explants is not regulated by ligand, receptor antagonist, cortisol, or oxygen.