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Fetal Physiological Programming
1Department of Physiology and Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, Australia; and 2Department of Pediatrics, University of Iowa, Iowa City, Iowa
Submitted 16 August 2004 ; accepted in final form 22 November 2004
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ABSTRACT |
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angiotensinogen; Glut-1 mRNA
By 4 days after birth, the right ventricular free wall (RVFW) mass of the lamb is reduced by nearly 50% (relative to the mass measured at 145–146 days gestation). At 4–6 wk of age, the numbers of myocytes in both left ventricular free walls (LVFWs) and RVFWs are less than the numbers found in the fetus at 145–146 days gestation (term 
150 days) (5).
Cortisol is a key fetal hormone that affects cellular and organ differentiation (8). High levels of cortisol occur as a result of labor, and synthetic steroids are used in the treatment of premature labor. Cortisol was hypertensinogenic in fetal sheep (33) and caused a reduction in fetal sheep cardiac DNA levels (25). Prenatal dexamethasone also increased the protein:DNA ratio in neonatal rat hearts (29). We postulated that high levels of cortisol would affect the rate of binucleation of fetal cardiac myocytes and/or their size and number.
Because there is extensive remodeling of the heart at birth, we also proposed that these effects of cortisol might be associated with altered expression of cardiac genes known to be associated in the adult with cardiac remodeling [i.e., genes of the cardiac renin-angiotensin system (RAS) and genes for the cardiac glucocorticoid and mineralocorticoid receptor]. Lindpaintner et al. (19) showed that pretreatment with dexamethasone stimulated angiotensinogen (Aogen) gene expression and efflux of Aogen from the adult rat heart. ANG II is known to cause cardiac hypertrophy and the signaling cascades known to be associated with myocyte growth and hypertrophy, i.e., the MAPK cascades and calcineurin may be involved. In isolated fetal sheep myocytes, insulin-like growth factor-I (IGF-I) and ANG II stimulate ERKs along with cellular proliferation, suggesting the ERK cascade is active in the fetal heart (30, 31). As we were examining the effects of cortisol on the cardiac RAS, we also looked at the effects of large-dose infusions of cortisol on levels of components of ERK 1/2, JNK, p38 MAPK cascades, and calcineurin.
Glut-1 is the major glucose transporter in the fetal rat heart. At birth, it is suppressed and in the adult Glut-4 becomes the major glucose transporter (27). Thyroid hormones control this switching in the perinatal period (7). Because cortisol, similar to the thyroid hormones, is involved in development, we thought it might also affect the level of expression of Glut-1.
Binucleated myocytes are terminally differentiated and cannot undergo cellular division (11, 18). Thus, at a given age of development, the proportion of cardiac myocytes that are uninucleated indicates the capacity of the developing heart to grow by hyperplasia. Up to 110 days gestation, the fetal sheep heart grows mainly by hyperplasia; after this time, ventricular free wall (VFW) mass increases by both hypertrophy and hyperplasia (5).
By 145–146 days gestation, most fetal sheep cardiac myocytes are binucleated (5). Therefore, the capacity of the sheep heart to grow by hyperplasia after birth is limited. In the pig, however, 90% of cardiac myocytes are mononuclear at birth (2) and after birth there is no loss of myocytes from the RVFW; LVFW myocyte numbers increase by 28% (2). However, in nearly all mammalian species, the developing heart ceases to grow by hyperplasia before or shortly after birth. Thus factors that alter the rate of terminal differentiation of cardiac myocytes could influence the number of myocytes present in the adult heart.
Studies in the neonatal pig showed that normal cardiac development depends on the integrity of the neonatal RAS (2, 3). From these experiments, it was not possible to determine whether blockade of the neonatal RAS simply reduced afterload and this led to underdevelopment of the heart or whether angiotensin played a role in cardiac myocyte development. However, blockade of the rat RAS for the first week of life decreased the number of proliferating myocytes by 23% (9).
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METHODS |
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With the use of methods already described (21), fetal sheep underwent surgery at 121 days gestation (term = 150 days) to implant vascular catheters. General anesthesia was induced with maternal intravenous sodium thiopentone (1 g, Abbott Australasia, Kurnell, NSW, Australia) and maintained using 1–2% halothane (Fluothane, Zeneca Pharmaceuticals, Melbourne, Victoria, Australia) in oxygen. Polyvinyl catheters (1.0-mm ID, 1.5-mm OD) were inserted into a fetal femoral artery and both tarsal veins and a 1.5-mm ID, 2.7-mm OD catheter was placed in the amniotic cavity. After surgery and for the next 2 days, 600 mg of procaine penicillin (Ilium Propen, Troy Laboratories, Smithfield, NSW, Australia) and 80 mg of gentamycin sulphate (Gentam, Troy Laboratories) were given intramuscularly to the ewe and to the fetus via the amniotic catheter. On day 3 following surgery, all ewes were given 150 mg of medroxyprogesterone acetate itramuscularly (Depo-Provera, Upjohn, Rydalmere, NSW, Australia) to prolong the time before ewes went into labor.
Experimental protocol. Experiments began 5 or more days after surgery when maternal food and fluid intake had returned to preoperative levels. Fetuses were 128–130 days old when the experiments finished. Fetuses were given either hydrocortisone sodium succinate (72.1 mg/day, n = 9; Solu-Cortef, Pharmacia & Upjohn) in 0.15 M saline or 0.15 M saline alone (n = 8) at an intravenous infusion rate of 0.66 ml/h for 2–3 days. Infusions were stopped at 72 h or earlier (mean time 61 ± 3.5 h, SE) if ewes showed behavioral or physical signs of going into labor.
Fetal arterial blood samples (5 ml) were collected into heparinized syringes 15 min before the infusions began, at 1 h, 48 h, and immediately before death (56–72 h). Arterial PO2 and pH corrected to 39.5°C were measured immediately using a blood gas analyzer (ABL-715, Radiometer, Copenhagen, Denmark). Hematocrit was measured using a Hettich centrifuge. The remaining blood was centrifuged at 1,100 g for 10 min at 4°C, and the plasma was stored at –20°C. Plasma cortisol was measured using a coated tube radioimmunoassay (Spectria, Orion Diagnostica, Espoo, Finland) after separation from cortisol-binding proteins using a dichloromethane extraction process (4).
Fetal arterial and amniotic pressures and heart rate were measured in five fetuses (3 cortisol infused and 2 saline infused) using disposable transducers (Biotrans 11 dome, Critical Assist Group, Sydney, Australia) connected to a Grass Polygraph recorder (model 79E). Measurements were made for 1 h on the final day of the study, 2–3 h before the animals were killed. Heart rate was determined from the beat-to-beat interval, and blood pressure was corrected for amniotic pressure.
Ewes and their fetuses were killed at 128–130 days gestation by an intravenous injection of pentobarbitone sodium (100 mg/kg body wt; Lethabarb, Virbac Australia, Peakhurst, Australia) to the ewe. A postmortem was performed immediately. Fetuses were removed quickly and weighed. Hearts, liver, lungs, and kidneys were weighed, and the free walls of the right and left ventricles (RVFW and LVFW), the atria, and the interventricular septum were dissected out and weighed. Small blocks of RVFW and LVFW were dissected from midway between the atrioventricular groove and the apex. The remaining tissue was frozen in liquid nitrogen and stored at –80°C for later analysis.
Preparation of cardiac myocytes.
The small blocks of myocardium (1 cm3, extending from the epicardium to the endocardium) were sliced (Vibroslicer 752, Campden Instruments, speed 9) into 400-µm-thick sections and myocytes were isolated using a method already described (5). Slices of RVFW and LVFWs were immersed in cold Ca-Krebs (118 mM NaCl, 4.75 mM KCl, 1.18 mM KH2PO4, 246 mM MgSO4·7H2O, 84 mM NaHCO3, 2.5 mM CaCl2, and 100 mM glucose; pH 7.0) that had been aerated with carbogen (5% CO2 in oxygen, BOC gases, Lidcombe, NSW, Australia) for at least 20 min previously. One to two tissue slices from each ventricle were digested for 14 min at 37°C with collagenase (305 U/ml, Type 2, Worthington Biochemical) in Krebs solution (3 mg/ml), bubbled with carbogen. The digestion was stopped and cells were treated with relaxing solution and ethidium bromide (5).
Cells were examined with a Leica TCS NT laser-scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) equipped with an argon ion laser and coupled to an inverted microscope with a C-Apochromat (x63) water-immersion objective lens. Fluorescence was excited at 488 nm and the FITC absorbance setting was selected to detect fluorescence over the range 520–675 nm.
The proportions of uninucleated and binucleated cardiac myocytes in the RVFW and LVFW from each fetus were determined by counting 200 cells from each ventricle.
To measure the volumes of individual myocytes, undamaged uninucleated or binucleated myocytes were selected. Only myocytes with visible striations in the cytoplasm, a clearly defined cell membrane, and no evidence of cytoplasmic vacuoles were measured (Fig. 1) (5).
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20 times during optical scanning. The cross-sectional areas were measured using the computer program ImageJ (http://rsb.info.nih.gov). To measure the area of each myocyte accurately, the cell had to be distinguished from background. Image threshold was calculated using methods already described (5). For each individual myocyte, the cell volume was calculated from these areas using the following formula (5):
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where MV = volume of the cell (µm3), A = cross-sectional area of cell (µm2), n = number of sections, Zt = depth of cell (µm), and Zt/(n – 1) = distance between each section.
The approximate number of myocytes per gram VFW was calculated using the following formula:
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where AvMV = average volume of myocytes (µm3), specific gravity of ventricular myocardium = 1.06 and 1 µm3 = 10–12 ml.
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Overall, 18.5 ± 11.4 (SD) cells were counted from each heart (n = 14). The reproducibility of this method of measuring cell volume has been tested by three investigators who measured the volumes of five individual myocytes independently, with a coefficient of variation of 2.5% (5).
Real-time PCR. Real-time PCR was used to measure expression of Aogen, AT1 and AT2 receptors, Glut-1, glucocorticoid receptor (GR), mineralocorticoid receptor (MR) mRNAs, and the endogenous reference 18S rRNA in both VFWs. Total RNA was extracted from 0.4–0.6 g of VFW using a modified acid-guanidinium phenol chloroform method (10). The extracted total RNA was treated with DNase and then reverse transcribed using a TaqMan Reverse Transcription Reagents kit (Applied Biosystems) and methods described previously (12). To assess genomic contamination of cDNA, control reactions with no reverse transcriptase were included in a separate reverse-transcription reaction for all samples.
Real-time PCR reactions were carried out using an ABI PRISM 7700 Sequence Detector (Applied Biosystems) in 25 µl containing either 5 or 50 ng of cDNA. Primers and probes were designed using Primer Express Version 1.0 (Applied Biosystems; Table 1), and their sequences and preliminary experiments to determine appropriate concentrations and optimal conditions for use are described elsewhere (12). Reactions were multiplexed with the target gene and 18S gene expression detected in each well and primers limited for 18S.
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CT value for each unknown and calibrator sample. The average calibrator CT was subtracted from each unknown CT (CT), and finally the cardiac expression of the genes studied, relative to 18S, and relative to the calibrator sample, was evaluated using the expression 2–CT. The intra-assay coefficients of variation for the mRNAs studied were: Aogen 5.3% (n = 5), AT1 receptor 4.1% (n = 5), AT2 receptor 11.6% (n = 4), Glut-1 6.5% (n = 5), GR 12.5% (n = 4), and MR 19.2% (n = 5). Measurement of MAPK signaling proteins and calcineurin. Primary antibodies to ERK1/2, pERK 1/2, JNK, pJNK (Santa Cruz Biotechnology, Santa Cruz, CA), P38, pP38 (Cell Signaling Technology, Beverly, MA), and calcineurin (catalytic A subunit; BD Pharmigen, San Diego, CA) were used. Immunoblots were prepared as described previously (23). Briefly, left and right ventricular myocardium was homogenized in the presence of protease inhibitors and then sonicated for 20 s. After centrifugation, 20 µg of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat milk protein for 1 h and then incubated in primary antibody overnight at 5°C. After being washed, incubation of secondary antibody conjugated with horseradish peroxidase at room temperature was performed for 1 h. The secondary antibody was detected using the Pierce SuperSignal Kit, films were digitized, and signals were quantitated using National Institutes of Health (NIH) Image (Wayne Rasband, NIH). Serial protein dilutions were tested with each antibody to make sure that signals were in the linear range for the added protein.
Statistical analysis. Statistical Package for the Social Sciences (SPSS, Chicago, IL) was used to determine means and standard errors of means. Data were compared using either Student's t-tests or Wilcoxon and Mann-Whitney nonparametric tests. Linear regression analysis was performed using Graph Pad Prism version 1.03 (GraphPad Software).
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RESULTS |
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Fetal blood pressure, heart rate, and blood gases. In five fetuses (3 cortisol and 2 saline infused), arterial pressure was measured to confirm our previous findings (15, 33) that cortisol causes a rise in fetal arterial pressure (Table 4). Blood pressure was increased in cortisol-infused fetuses compared with saline-infused fetuses (P < 0.05; Table 4). At the end of the infusion, systolic blood pressure was positively related to plasma cortisol concentration (r = 0.9, n = 5, P < 0.04). There was no difference in fetal heart rate between the two groups.
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Cardiac myocyte volume and number of nuclei. As expected at this stage of gestation in the fetal sheep, approximately half of the cardiac myocytes examined were uninucleated (57 ± 4% in RVFW and 54 ± 3% in the LVFW of saline-infused fetuses). The proportions of RVFW or LVFW myocytes that were uninucleated (53 ± 2% in RVFW and 54 ± 4% in LVFW) were no different in cortisol-treated fetuses from the proportions measured in untreated fetuses.
In saline-infused fetal sheep, multinucleated cardiac myocytes were larger than uninucleated myocytes (P < 0.005, RVFW and P < 0.001, LVFW) and RVFW uninucleated cells were larger than LVFW uninucleated myocytes (P < 0.01). The increase in the cell volumes of RVFW uninucleated myocytes of cortisol-treated fetuses (4,375 ± 80 µm3, n = 49) was not significant (P < 0.1) when compared with 3,929 ± 193 µm3 (n = 44) from saline-infused fetuses. The increase in LVFW uninucleated myocyte volumes associated with cortisol treatment was, however, significant (P < 0.001; Fig. 2).
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As expected, it was estimated that there were more cardiac myocytes per gram LVFW than per gram RVFW (P < 0.001), so that overall there were more cardiac myocytes in the LVFW than in the RVFW (P < 0.001). Cortisol had no effect on myocyte numbers; the estimated number of myocytes x 109 was 0.18 ± 0.006/g RVFW, n = 8; the estimated total number was 0.92 ± 0.07 x 109. In cortisol-treated fetuses, the number of myocytes x 109/g LVFW was 0.24 ± 0.028, n = 6; the estimated total number was 1.48 ± 0.21 x 109. In saline-treated fetuses, the estimated number of myocytes x 109/g RVFW was 0.2 ± 0.029, n = 5 and the estimated total number was 1.03 ± 0.11 x 109. In this group, the number of myocytes x 109/g LVFW was 0.29 ± 0.038, n = 4 and the estimated total number was 1.9 ± 0.13 x 109.
Effects of cortisol on the expression of components of fetal cardiac RAS and other cardiac genes. In both groups, levels of Aogen, AT1, AT2, Glut-1, GR, and MR mRNA were similar in the RVFW and LVFW (Table 5). RVFW and LVFW Aogen mRNA levels were both increased in cortisol-infused fetal sheep (P < 0.02; Table 5). There were positive correlations between LVFW and RVFW Aogen mRNA levels and the last measured plasma cortisol level (r = 0.83, n = 14, P = 0.0003 and r = 0.61, n = 16, P < 0.02, respectively; Fig. 3). Neither cardiac AT1 nor AT2 receptor mRNA levels were changed in cortisol-treated fetuses.
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Immunoblotting of hypertrophic signaling proteins. Expressions of myocardial total and phosphorylated (activated) ERK1/2, JNK, P38, as well as calcineurin were similar in cortisol-treated and saline-infused fetal sheep (Fig. 4).
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DISCUSSION |
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Cortisol did not affect the rate at which myocytes became terminally differentiated as proportion of RVFW and LVFW cardiac myocytes that were binucleated was similar in the two groups of fetuses and similar to others of the same gestational age reported in our previous study (5).
As expected, both RVFW uni- and binucleated cardiac myocytes were larger than LVFW myocytes and binucleated myocytes were 40–50% larger than uninucleated myocytes (Fig. 2) (5). Cortisol treatment was not associated with a marked increase in the size of RVFW cardiac myocytes, although there was 30% increase in the volumes of both uni- and binucleated LVFW cardiac myocytes (Fig. 2). This increase in LVFW myocyte volume may have been a direct effect of cortisol but it could also have resulted from its blood pressure raising effects, or the fact that the cortisol-treated group had lower arterial oxygen tensions at the beginning of the experiment and higher hematocrits by the end. If these factors were responsible for hypertrophy of LVFW myocytes, then there should have been a similar hypertrophic effect on RVFW myocytes (1). Cortisol does not appear, therefore, to be responsible for the marked increase in RVFW uninucleated cardiac myocyte size from 3,634 ± 789 µm3 at 145–146 days to 6,836 ± 737 µm3 at 4 days after birth (5).
Cortisol-treated fetuses had bigger heart weights relative to body weights and the ratios of the masses of RVFW and LVFWs to body weight were greater (Table 3). The increase in these cardiac to body weight ratios appeared to be related to plasma cortisol levels. Also, there were no changes in the estimated number of myocytes in the VFWs in cortisol-treated fetuses. It is unlikely, therefore, that the high levels of cortisol associated with parturition play a role in the massive reduction in RVFW myocyte number that occurs shortly after birth (from 1.26 ± 0.1 x 109 at 145–146 days to 0.6 ± 0.03 x 109 at 4 days) (5).
Two other major findings were that cortisol treatment was associated with a dose-dependent upregulation of cardiac Aogen gene expression and downregulation of Glut-1 gene expression. Because the 5'-flanking region of the Aogen gene has several glucocorticoid response elements (GREs) (22), cortisol probably directly affected transcription. This would explain the dose-dependent nature of the increase in Aogen mRNA (Fig. 2). In another study on the effects of a lower dose of cortisol (145 ± 45 nM) on tissue RASs, we found that there was a direct relationship between plasma cortisol levels and left ventricular Aogen mRNA (r = 0.71, P = 0.02, n = 10). In that study as there were only a small number of samples from the RV, this relationship could not be determined.
Glucocorticoids and other steroids induce hepatic Aogen production (22). There is strong evidence that the RAS is important for normal cardiac development. In the rat, in which the heart grows after birth by both hyperplasia in the first week of life and by hypertrophy, inhibition of angiotensin-converting enzyme inhibited cardiac myocyte hyperplasia and apoptosis. Because the proportion of cells undergoing apoptosis was 10-fold less than that of proliferating cells, the net effect was a reduction in heart and body weight and in survival rate (9). Beinlich et al. (3) also reported that treatment of neonatal pigs with enalapril reduced cardiac growth, in particular that of the LVFW, but suggested that the lower load on the LVFW may have affected its growth.
It is not known if the beneficial effects on cardiac growth and development depend on the integrity of the renal/circulating RAS or components of a cardiac RAS. At birth, plasma renin levels rise dramatically, regardless of the mechanism of delivery of the newborn (20). Because renin is not normally expressed in the fetal sheep heart (Lumbers ER, unpublished observations) but small amounts of active renin can be detected (Lumbers ER, unpublished observations), it would seem that if the cardiac RAS plays a role in neonatal cardiac growth, renal renin is required. In the rat, the glycoforms of renin that are taken up by the heart are those that have a relatively short half-life in the circulation (17). Lindpaintner et al. (19) found that dexamethasone stimulated cardiac Aogen gene expression and release of Aogen from the isolated, perfused adult heart but we are the first that we know of to have shown that high levels of cortisol stimulate expression of cardiac Aogen in the fetal heart.
These cortisol-induced changes in cardiac Aogen gene expression were not accompanied by any changes in levels of ANG II receptor subtype mRNA. The genes for both angiotensin receptor subtypes have GREs (16). We previously found that the dominant angiotensin receptor subtype in fetal ventricular myocardium is the AT2 receptor subtype, the function of which is largely unknown (6). AT1 receptors are present in the ventricular myocardium but their density is much lower. The density of cardiac AT2 receptors declines with increasing gestation (6). In younger fetal sheep (118 days), low doses of cortisol (29 ± 4 nM) had no effect on the density of aortic, carotid, and RVFW AT1 and AT2 receptors (15). RVFW and LVFW receptor densities were similar (6) but the response of fetal LVFW AT1 and AT2 receptors to low-dose cortisol was not studied. Because high doses of cortisol had no effect on the mRNA levels of either RVFW or LVFW AT1 and AT2 receptors, the perinatal fall in cardiac ANG II receptors (28) and in AT2 receptor mRNA levels (26) does not depend on the high levels of cortisol occurring as a result of parturition.
Glucose is a major source of energy for cardiac muscle and Glut-1 is the major glucose transporter in the cardiac myocytes of the fetal rat. In the adult rat, Glut-4 becomes the major transporter (27). Santalucia et al. (27) suggest that Sp1 is the major transcriptional factor promoting Glut-1 expression during fetal life and that it is downregulated during neonatal life. In myoblasts, products of Sp3 transcription inhibit Glut-1 expression (14). There is no evidence linking regulation of Sp1, Sp3, or Glut-1 expression to cortisol, although it has been shown that thyroidectomy prevents the neonatal repression of Glut-1 expression (7), suggesting that these transcription factors controlling Glut-1 expression may be regulated by hormones. Our finding that Glut-1 mRNA levels were significantly reduced in the LVFW and lower in the RVFW of cortisol-treated fetal sheep may indicate that high levels of cortisol in the perinatal period also modulate Glut-1 expression in the heart at birth.
We also examined the expression of GR and MR. There is strong evidence that mineralocorticoids influence cardiac remodelling and fibrosis of the adult heart, which has led to a revival of the use of aldosterone antagonists and development of new antagonists for the treatment of congestive cardiac failure (35). However, we found that high doses of cortisol did not affect mRNA levels of either GR or MR.
The three MAPK cascades were studied (Fig. 4). The lack of myocardial ERK, JNK, and P38 activation in the present study is perhaps not surprising as we found no evidence for cortisol-induced cardiomyocyte proliferation. In addition, glucocorticoids decrease MAPK activity possibly by inhibiting phosphorylation or by upregulating the MAPK phosphatases (which in turn dephosphorylates ERK, JNK, and P38) (13, 24).
Calcineurin-mediated translocation of NFAT transcription factors is a central regulator of cardiac hypertrophic growth. In cell lines, dexamethasone rapidly increases calcineurin activity through activation of IP3-dependent calcium stores (34). In rats, mineralocorticoid-induced cardiac hypertrophy is associated with increased myocardial calcineurin activity (32). We initially postulated that cortisol, which exerts both mineralocorticoid and glucocorticoid activities, would be associated with increased calcineurin levels. However, our data fail to support this. Interestingly, myocardial calcineurin levels were not altered in pressure and volume overload models of fetal cardiac hypertrophy (Seger JL, unpublished observations), suggesting calcineurin may have little role in the heart until postnatal life.
In summary, high doses of cortisol such as might occur in early postnatal life do not alter the rate of terminal differentiation of cardiac myocytes nor affect their number. There is evidence of a hypertrophic effect of cortisol on LVFW myocytes. Cardiac Aogen mRNA levels were increased in cortisol-treated animals and Glut-1 levels were depressed. The potential effects of increased levels of Aogen in the fetal sheep heart are unknown. Our findings also suggest that cortisol may play a role in neonatal suppression of Glut-1 gene expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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