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Endocrinology Vol. 147, No. 8 3643-3649
Copyright © 2006 by The Endocrine Society

Cortisol Stimulates Cell Cycle Activity in the Cardiomyocyte of the Sheep Fetus

G. D. Giraud, S. Louey, S. Jonker, J. Schultz and K. L. Thornburg

Heart Research Center (G.D.G., S.L., S.J., J.S., K.L.T.) and Departments of Medicine (Cardiovascular Medicine) (G.D.G., K.L.T.), Physiology and Pharmacology (G.D.G., S.J., K.L.T.), and Surgery (Cardiothoracic) (J.S.), Oregon Health & Science University, and Portland Veterans Affairs Medical Center (G.D.G.), Portland, Oregon 97239-3098

Address all correspondence and requests for reprints to: Kent L. Thornburg, Heart Research Center, L464, Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239-3098. E-mail: thornbur{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of cortisol in regulating cardiac myocyte growth in the near-term fetal sheep is unknown. We hypothesized that cortisol would suppress cardiomyocyte proliferation and stimulate cardiomyocyte binucleation and enlargement, signs of terminal differentiation. Cardiomyocyte dimensions and percent binucleation were determined in isolated cardiac myocytes from seven cortisol-treated and seven control fetuses; percentage of myocytes positive for Ki-67 was determined in an additional four cortisol-treated and four control hearts. Cortisol was infused into the circumflex coronary artery at subpressor rates (0.5 µg/kg·min, 7 d). Cortisol infusion had no hemodynamic effects, compared with controls or pretreatment conditions. Cortisol treatment increased heart weight (44.0 ± 8.7 g vs. control, 34.9 ± 9.1 g, P < 0.05). Heart to body weight ratio was greater in treated hearts, compared with controls (10.3 ± 1.9 vs. 7.7 ± 0.9 g/kg, P < 0.01). Ventricular myocyte length, width, and percent binucleation were not different between groups. The proportion of treated myocytes in the cell cycle staining for Ki-67 was higher in the left ventricle (5.5 ± 0.1 vs. 2.7 ± 0.4%, P < 0.005) and right ventricle (4.4 ± 0.4 vs. 3.7 ± 0.7%, P < 0.05), compared with controls. Wet weight to dry weight ratios from cortisol-treated and control hearts were not different. In conclusion, whereas cortisol infused into the fetal sheep heart has no effect on cardiomyocyte size or maturational state, it stimulates entry of cardiomyocytes in the cell cycle. Thus, increases in fetal heart mass associated with subpressor doses of cortisol are due to cardiomyocyte proliferation and not hypertrophic growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WITH MATURATION OF the ovine fetal hypothalamic pituitary adrenal axis over the last few weeks of gestation, plasma cortisol (hydrocortisone) levels rise continuously from approximately 10 to approximately 70 ng/ml until parturition (~145 d gestation) (1). It is well known that cortisol affects the differentiation and maturation of many tissues including lung, liver, skeletal muscle, and kidney (2, 3), but the role of corticosteroids in supporting cardiomyocyte maturation is not known. Understanding the mechanisms behind the maturation of the myocardium is important because hormonal action may regulate the set point for working myocyte numbers in the mature heart that has lifelong effects (4, 5). The myocardium of the fetal sheep expresses both the cytosolic mineralocorticoid (MR) and glucocorticoid receptors (GR) (6, 7). Glucocorticoids may also act through nonclassical receptor mechanisms as well (8). Glucocorticoids have been shown to underlie changes in the fetus that portend cardiovascular disease in adult life (9, 10). Thus, it would not be surprising if increasing plasma glucocorticoid concentrations affected the growth and maturation of the fetal myocardium not only during the last few weeks of prenatal life but in adult life as well.

Over the last two thirds of gestation (term, ~145 d), the fetal heart of the sheep grows in two primary phases as shown by Burrell et al. (11): 1) in early fetal life, all cardiomyocytes contain a single nucleus, replicate through cell division but increase little in size; and 2) beginning on about d 100, a fraction of the mononucleated cardiomyocytes go through so-called terminal differentiation, become binucleated through karyokinesis without cytokinesis, and cease to divide. However, binucleated cardiomyocytes are capable of hypertrophic growth and remain so throughout life. The rat heart goes through similar changes but the terminal differentiation process occurs about 8–14 d after birth. The role of corticosteroids in regulating these maturation stages has been little studied.

The administration of exogenous glucocorticoid to the sheep fetus leads to a general slowing of somatic growth (2, 12). However, the heart is affected by cortisol differently from most other fetal organs. It continues to grow in the presence of exogenous glucocorticoid. Therefore, the fetal heart weight to body weight ratio increases within a few days of exposure to increased plasma glucocorticoid levels. Corticosteroid infusion experiments in the fetus can elevate arterial blood pressure. In fact, the Nathanielsz group (13) has provided evidence that the normal slow elevation of arterial pressure requires a functional adrenal organ. Thus, most experiments that administer exogenous glucocorticoids are complicated by a dose-dependent hypertension (13, 14, 15).

Tangalakis et al. (16) found that a cortisol infusion (100 µg/h iv, 24 h) increased arterial blood pressure by 14–15% in the midterm fetal sheep. It is well known that an increased hemodynamic load will alter the maturational program of the myocardium through changes in the cardiomyocyte. Fetal hearts that have been subjected to increased systolic load have thicker ventricular chamber walls and more cardiomyocytes than unloaded age matched hearts (17); the right ventricle (RV) is more sensitive to afterload than the left ventricle (LV) because of right ventricular mechanical disadvantage owing to its larger meridional radius of curvature to wall thickness ratio (18, 19). Loading the fetal right ventricle leads to increased cardiomyocyte size, increased rate of binucleation, and cellular proliferation.

Lumbers et al. (6) found that a high-dose cortisol infusion (72.1 mg/d for ~60 h) into the near term fetus was associated with increased LV but not RV cardiomyocyte size and increased myocardial angiotensinogen mRNA levels. However, the infusion of cortisol was also accompanied by substantial increases in fetal blood pressure. Systolic, diastolic, and mean arterial pressures exceeded those of saline-infused control fetuses. Thus, cardiomyocytes were subjected to both increased cortisol levels and increased systolic load.

Slotkin et al. (20) found that administration of dexamethasone [0.2 or 0.8 mg/kg sc on embryonic d (E)17, E18, and E19] to pregnant rats decreased the total myocardial DNA content (microgram per organ) of offspring. They interpreted this finding as evidence for cardiomyocyte enlargement and decreased cardiomyocyte proliferation in the offspring. Contrary to that study, Torres et al. (21) found that prenatal exposure to maternally administered dexamethasone (48 µg/d from E17) increased cell proliferation in the late term and early neonatal rat heart. Rudolph et al. (22) infused cortisol directly into the left coronary artery of near-term fetal sheep (1.2 µg/min, 72–80 h). Blood pressures were not reported. Their experiments led to reduced LV DNA concentrations without affecting RV DNA concentrations. They concluded that cortisol inhibits myocyte replication, whereas stimulating growth dominated by hypertrophy.

Because of the varied effects of corticosteroid infusion experiments, the role of cortisol on cardiomyocyte growth remains controversial. The direct effects of cortisol on fetal cardiac myocyte growth when unencumbered by hypertension have not been studied. Based on the studies of others in sheep, we hypothesized that cortisol would increase cardiac mass in the sheep fetus by stimulating hypertrophy of cardiomyocytes, reduce hyperplastic growth, and stimulate accelerated terminal differentiation and binucleation of cardiomyocytes. To test this hypothesis, we infused cortisol at subpressor doses into the circulation of the near-term fetal sheep to differentiate the effects of cortisol from cardiac loading effects. We measured cardiac mass, cardiac myocyte dimensions, and the proportion of cardiac myocytes that were binucleated. We stained the cardiac myocytes for the Ki-67 nuclear antigen, which is present only in cells that are in the cell cycle (23). From these experiments, we were able to evaluate growth patterns of the fetal cardiac myocyte under conditions of increased levels of circulating cortisol well within the physiological range.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Surgery
Time-bred pregnant ewes (Ovis aries) of mixed Western breed were purchased from local farmers and brought to the laboratory pens several days before surgery to become accustomed to new surroundings. Guidelines established by the Department of Comparative Medicine, Oregon Health & Science University, for the care and use of sheep were followed according to the protocol as approved by the Institutional Animal Care and Use Committee. After a 24-h fasting period, sterile surgery was performed as described previously (24, 25, 26) on ewes of 122 ± 1 (mean ± SD) d gestation. Anesthesia was induced by administering an iv mixture of diazepam and ketamine; anesthesia was maintained using 1.0% halothane (Pittman-Moore, Washington Crossing, NJ) in a 70:30 mixture of oxygen and nitrous oxide.

The abdomen was opened in the midline exposing the uterus. The uterus was incised and the superior portion of the fetus delivered through the uterine incision. The right jugular vein was cannulated with two 1.7-mm outer diameter polyvinyl catheters (V-8; Bolab, Lake Havasu City, AZ), and the catheters were advanced to the right atrium. One jugular vein catheter was used to measure right atrial pressure, whereas the second jugular vein catheter was used to withdraw blood samples. The aorta was cannulated via the right carotid artery using 1.3- and 1.7-mm outer diameter polyvinyl catheters (Bolab, V-5 and V-8), and the catheters were advanced to the junction of the brachiocephalic artery and aorta. One carotid artery catheter was used to measure aortic pressure, whereas the other was used to withdraw blood samples. The fetal heart was exposed through a left thoracotomy in the fifth intercostal space. A 1.3-mm outer diameter polyvinyl catheter with a 3.0-cm SILASTIC brand tip (outer diameter 1.7 mm; Dow Corning, Midland, MI) was placed in the coronary sinus via the left azygous vein. The pericardium was opened over the lower margin of the main pulmonary artery exposing the left atrial appendage, the proximal left anterior descending coronary artery and the proximal circumflex coronary artery. An ultrasonic flow probe (Crystal Biotech, Hopkinton, MA) was placed around the circumflex coronary artery near its origin. A 1.3-mm outer diameter polyvinyl catheter with a 1-cm SILASTIC brand tip (outer diameter 0.6 mm) was placed within the circumflex coronary artery near its origin. A 1.3-mm outer diameter polyvinyl catheter with a 1-cm SILASTIC brand tip with multiple side holes was placed into the anterior pericardial space via the pericardiotomy. The pericardium overlying the great vessels was left open, allowing passage of the catheters and flow probe connector cable. The fetal chest was closed in anatomic layers. A 1.7-mm outer diameter polyvinyl catheter was attached to the fetal skin and used to measure amniotic fluid pressure. All catheters were anchored to the fetal skin. The fetus was returned to the uterus and the uterus was closed. All catheters and the flow probe cable were passed through the ewe’s abdominal wall and tunneled to the ewe’s flank at which they were stored in a nylon pouch sutured to the skin. The abdomen was closed, and 1 million U penicillin G (Bristol-Myers Squibb, Princeton, NJ) was instilled into the amniotic space. Anesthesia was terminated and the ewe was allowed to recover. After surgery, ewes were placed in a clean pen for 6 ± 1 d before experiments were performed.

Experimental groups
The fetuses consisted of two groups, a cortisol-treated group (14 fetuses) and a control group (14 fetuses). The control group and the cortisol-treated fetuses were of the same gestational age. To perform the appropriate tests, the cortisol-treated group and the control group were subdivided.

From the cortisol group, 10 fetuses received a 0.5 µg/kg·min intracoronary cortisol infusion in which hemodynamic studies were performed before and daily for the 7 d of infusion. Hearts from seven of these fetuses were dissociated for myocyte measurements. Hearts from the remaining three fetuses were processed for wet weight to dry weight ratios. Four additional fetuses received 0.5 µg/kg·min iv cortisol infusion via the jugular vein for 2 d, and their hearts were dissociated for Ki-67 staining; the infusions for these fetuses commenced at the same age as the 7-d infusion fetuses. A 2-d infusion for cardiomyocyte proliferation studies was chosen to determine cell cycle responses early in the infusion period when perhaps the response might be greatest. We did not test the Ki-67 response at 7 d when the heart had already increased in mass. We reasoned that the absence of an increase in the percentage of cells staining positive for Ki-67 at 7 d of infusion would not rule out an earlier increase. Unlike angiotensin II, which is largely cleared by the coronary circulation (Thornburg, K.L., and G. D. Giraud, unpublished observations), cortisol uptake by the myocardium is low. Thus, we reasoned that venous infusion rates of cortisol that gave similar systemic concentrations for the 2-d Ki-67 experiments would have effects on the myocardium similar to those with direct circumflex infusion.

In the control group, seven of the 14 fetuses were matched for the coronary cortisol infusion animals. Of these, three received an intracoronary saline infusion as a sham infusion and four had occluded intracoronary infusion catheter that were not infused with saline. Because there were no differences between these two groups for any factor measured, the control data were pooled. Hemodynamic studies were performed on each of the 7 experimental days, and their hearts were dissociated for myocyte measurements; hearts from three uninstrumented fetuses were processed for wet weight to dry weight ratios. Four hearts from uninstrumented age-matched fetuses were prepared for Ki-67 staining.

Experimental protocol
Laboratory procedure.
On the day of the experiment, ewes were placed in a stanchion cart and allowed free access to water and food. Hydrostatic pressures from the amniotic fluid space, pericardial space, right atrium, and aorta were measured using Transpac pressure transducers (Abbott Critical Care Systems, Chicago, IL) calibrated daily using a mercury manometer. All vascular pressures were referenced to pericardial pressure. The ultrasonic flow probes were connected to an ultrasonic flow meter (Triton Technology, San Diego, CA). The outputs from the flow meter and pressure transducers were connected to a Gould RS2000 eight-channel polygraph (Cleveland, OH). Arterial pH, PCO2 (partial pressure of carbon dioxide), and PO2 (partial pressure of oxygen) values were determined using a blood gas analyzer (Model 1306; Instrumentation Laboratories, Lexington, MA) corrected to 39 C. Arterial oxygen content was determined using an Instrumentation Laboratories cooximeter (Model 382).

Experimental protocol.
Baseline measurements of hydrostatic pressures, heart rate, and aortic blood gases were made. A 3-ml arterial blood sample was collected for measuring plasma cortisol concentrations by RIA. Cortisol solution or normal saline was infused using a portable continuous infusion pump (Dak Med, Buffalo, NY). Cortisol was infused into the circumflex coronary artery infusion at a rate of 0.5 µg/kg·min, estimated on the basis of a 3-kg fetus, the approximate average weight at this gestational age. This dose and infusion rate had no effect on right atrial or aortic pressure in pilot experiments. After the daily experiment, the ewe was returned to its pen. Each ewe was taken to the study room daily and measurements of fetal arterial blood gases and hemodynamic parameters made. The pump was checked daily for appropriate flow rate of cortisol solution or normal saline.

Heart collection
After the final study, the ewe and fetus were anesthetized with iv pentobarbital. The ewe’s abdomen and uterus were opened exposing the fetus. Three thousand units of heparin were injected into the umbilical vein followed by 3 ml of saturated KCl solution to stop the fetal heart in diastole. The fetus was removed from the uterus and weighed. The fetal heart was removed and trimmed of excess tissue in a standardized fashion, blotted dry, and weighed. In addition, the fetuses were dissected to confirm catheter position. The ewe was then killed with sodium pentobarbital.

Cardiac myocyte isolation procedure
Myocytes from hearts were dissociated using collagenase and protease as described previously (27, 28). The hearts were hung from a perfusion apparatus via the aorta and perfused by a series of solutions (39 C, oxygenated) retrogradely through the coronary arteries. Hearts were perfused for 5 min with oxygenated low calcium buffer [no calcium added; 140 mM NaCl, 5 mm KCl, 1 mM MgCl2.6H2O, 10 mM glucose, 10 mM HEPES (pH adjusted to 7.35 with NaOH)]; 10 min with 50 µM Ca-Tyrodes solution containing collagenase (type II, 300 U/ml, Worthington, Lakewood, NJ), protease (type XIV, 10 mg in 60 ml; Sigma, St. Louis, MO), and albumin (0.1%); 5 min with a high potassium (KB) solution [74 mM glutamic acid, 30 mM KCl, 30 mM KH2PO4, 20 mM taurine, 1.5 mM MgSO4, 0.5 mM EGTA, 10 mM HEPES, 10 mM glucose (pH adjusted to 7.37 using KOH)]. Portions of the RV free wall the LV free wall area receiving local cortisol infusion were separately removed and the chunks gently agitated in KB solution to release the cells. Isolated cells were filtered through a nylon mesh, which removed tissue chunks resulting in a cell slurry containing greater than 95% isolated individual cells (29). In addition to cardiac myocytes, endothelial cells, fibroblasts, and red cells were also present, but these cells do not interfere with measurements in this study. Isolated cells were divided into aliquots. One aliquot tube was fixed with 1.5% glutaraldehyde in monophosphate buffer (pH 7.4) for staining for Ki-67 and one was refrigerated for determination of myocyte dimensions. Freshly isolated cardiac myocytes (<2 h old) were measured for length and width using calibrated image analysis software (Optimas, Seattle, WA) at x400 phase microscopy (Zeiss Axiophot; Bartels and Stout, Bellevue, WA); 100 myocytes were measured without regard for nucleation. In addition, nucleation was determined in 300 myocytes to calculate a percent binucleation.

Ki-67 staining
Fixed myocytes were dried onto slides at a density of approximately 600 cells/cm2. Cells were fixed onto the slides by immersion in acetone for 30 min at 4 C. The cells were then permeablized in boiling sodium citrate [0.01 M (pH 6.0)] for 6 min at 85 C. After permeablization, nonspecific staining was blocked with blocking buffer. Slides were incubated overnight at 4 C with the Ki-67 antibody (1:200 in blocking buffer, mouse monoclonal DakoCytomation, Carpinteria, CA). Samples were incubated with the biotinylated secondary antibody (1:200 in PBS, Vectastain ABC kit, mouse IgG, Vector Laboratories, Burlingame, CA) for 2 h at room temperature, followed by incubation with the avidin and biotinylated enzyme (1:1:100 in PBS, Vectastain ABC kit, mouse IgG, Vector Laboratories) for 2 h at room temperature. Nuclei were stained with 3,3'-diaminobenzidine chromagen in substrate buffer (DakoCytomation) for 2–10 min. Cells were counterstained with 0.1% methylene blue. Samples were analyzed by counting 500 myocytes and determining the number of Ki-67-positive mononucleated myocytes; results are expressed as the percentage of Ki-67-positive mononucleated myocytes/total number of mononucleated myocytes.

Wet weight to dry weight ratios
The hearts from three control group fetuses and three cortisol treatment group fetuses were excised, blotted dry, and weighed. The hearts were desiccated in a drying oven at 40 C for 5 d and reweighed. The heart weights were expressed as wet weight to dry weight ratios for the control group and the cortisol treatment group.

Statistical analysis
Experimental data are expressed as mean ± SD. analyzed using Student’s t test for unpaired or paired measurements where appropriate to test for significant differences (30). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hemodynamic consequences of cortisol infusion
Seven fetal sheep of the cortisol treatment group were studied before and daily during the 7 d of cortisol infusion. Arterial cortisol levels increased over the duration of the infusion. Arterial plasma cortisol concentration at baseline on d 0 of the infusion was 9.2 ± 3.0 ng/ml; d 1, 17.6 ± 6.0 ng/ml; d 2, 25.0 ± 7.0 ng/ml; d 3, 27.6 ± 8.9 ng/ml; d 4, 30.8 ± 6.4 ng/ml; d 5, 27.9 ± 7.0 ng/ml; and d 6, 36.5 ± 8.8 ng/ml (Fig. 1Go). Values for arterial pH, PCO2, PO2, or O2 content on d 0 (baseline) and after 7 d of cortisol infusion are shown in Table 1Go. There was no difference in arterial pH, PCO2, PO2, or O2 content on d 6. Hemodynamic data collected on d 0 (baseline) and after 7 d of cortisol infusion into the circumflex coronary artery are shown in Table 2Go. There was no difference in mean right atrial pressure, aortic pressure, or heart rate between baseline and after 7 d of cortisol infusion. Peak systolic and diastolic pressures were also recorded from all animals, but these values are less reliable than mean pressures when measured from long polyvinyl catheters; therefore, these data are not reported. Nonetheless, neither of these pressures was altered in experimental animals, compared with their controls. Circumflex coronary artery flow velocity did not change over the period of cortisol infusion.


Figure 1
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  		FIG. 1. Plasma cortisol concentrations in fetuses that received 0.5 µg/kg·min for 7 d via the circumflex coronary artery. Day 0 is before the commencement of the infusion. Data are mean ± SD; n = 6 for all ages except at d 6 (n = 5). *, P < 0.05, compared with baseline (d 0).

 

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  		TABLE 1. Arterial pH, blood gas values, and hematocrit prior to and after 7 d of cortisol infusion (n = 7, 0.5 µg/kg·min)

 

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  		TABLE 2. Hemodynamic parameters for fetuses prior to (d 0) and after 7 d of saline (control; n = 7) or cortisol infusion (0.5 µg/kg·min, n = 7) into the circumflex coronary artery

 
Fetal cardiac mass, cardiac myocyte size, and cardiac myocyte maturational state
Heart weights and myocyte measurements for the two groups are shown in Table 3Go. Mean gestational age at postmortem was the same for controls and 7-d cortisol infusion groups. Fetal weights were also similar between groups (control: 4.6 ± 1.2 kg vs. cortisol-treated: 4.3 ± 0.6 kg). Hearts from fetuses receiving cortisol were 26% heavier (44.0 ± 8.7 g) than control hearts (34.9 ± 9.1 g, P < 0.05). Similarly, the heart weight to fetal body weight ratio was greater for the cortisol infusion fetuses, 10.3 ± 1.9 g/kg, compared with 7.7 ± 0.9 g/kg for control fetuses (P < 0.01). This increased heart weight was not due to increased water content in the cortisol-treated hearts; there was no difference in the wet weight to dry weight ratios of control (2.2 ± 1.2) and cortisol-treated (2.2 ± 0.9) hearts.


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  		TABLE 3. Somatic, cardiac, and ventricular myocyte growth data for control (n = 7) and cortisol-treated (n = 7, 0.5 µg/kg·min, 7 d) fetuses

 
There was no significant difference in the cardiac myocyte size measurements. The lengths of the cardiomyocytes from the left and right ventricles were similar between groups (LV: 78.3 ± 4.5 µm cortisol group vs. 74.4 ± 7.6 µm control; RV: 86.1 ± 4.9 µm cortisol group vs. 81.6 ± 6.5 µm control). Likewise, there was no significant difference in cardiac myocyte width from the LV or RV of control and cortisol-treated fetuses (LV: 10.3 ± 1.5 µm cortisol vs. 11.7 ± 1.2 µm control; RV: 12.1 ± 0.8 µm cortisol vs. 13.6 ± 1.3 µm control). There was also no significant difference in the proportion of cardiomyocytes that were binucleated from the LV or RV of control or cortisol fetuses (LV: 33 ± 8% cortisol vs. 40 ± 12% control; RV: 37 ± 8%, cortisol vs. 42 ± 8% control).

Whereas 7 d of cortisol treatment did not result in altered myocyte dimensions, cortisol infusion led to a greater number of myocytes actively in the cell cycle. Compared with age-matched controls, an increased proportion of cardiomyocytes from cortisol-treated fetuses (2 d infusion) stained positive for Ki-67 (Fig. 2Go). This increase was evident in both ventricles of cortisol-treated fetal hearts (LV: 5.5 ± 0.1 vs. 2.7 ± 0.4%, P < 0.005; RV: 4.4 ± 0.4 vs. 3.1 ± 0.7%; P < 0.05).


Figure 2
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  		FIG. 2. Percentage of Ki-67-positive mononucleated cardiomyocytes in the LVs and RVs from control (n = 4) and cortisol-infused (0.5 µg/kg·min, 2 d, n = 4) fetuses. Data are mean ± SD. *, P < 0.05, **, P < 0.005, compared with same ventricle control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We measured the direct effects of a subpressor dose of cortisol on cardiomyocyte growth and maturation in fetal sheep. The findings of this study did not support our hypothesis. Rather than seeing a suppression of proliferation and substantial increases in cardiomyocyte binucleation and cell size with cortisol treatment, we found an increase in cardiac mass without myocyte enlargement. Instead of cardiomyocyte hypertrophy, we found that the fraction of myocytes that were positive for Ki-67 had increased after only 2 d of cortisol infusion. Thus, it appears that cortisol stimulated hyperplastic growth of cardiomyocytes but not hypertrophic growth.

We believe that the infused fetuses were normal in every way. After 7 d of cortisol infusion into the circumflex coronary artery, the fetal pH, PCO2, PO2, and hematocrit values were not different from the baseline or control values. Baseline arterial pH and blood gas and blood pressure values were comparable with values from previous studies in our laboratory (24, 31, 32) and other laboratories (33, 34, 35). Fetal weights at autopsy were appropriate for the stage of gestation and were not different from that of controls. Cortisol infused fetuses did not differ significantly in weight from control group fetuses, although the mean was slightly lower in the cortisol group as shown by others. Hemodynamic measurements were also similar to those made during previous studies in our laboratory (25, 31). These observations suggest any changes observed during cortisol infusion were a direct result of the treatment and not an unusual physiological condition or hypertension in experimental fetuses.

We expected that cortisol treatment would stimulate hypertrophic growth of cardiomyocytes. The prenatal administration of dexamethasone to pregnant rats by Slotkin et al. (20) (0.2–0.8 mg/kg on E17–E19) led to a decrease in myocardial DNA per gram tissue and was interpreted by the authors as a decrease in cell density, suppression of cell proliferation, and cardiomyocyte hypertrophy. However, the DNA concentrations in the myocardium were normal on the day of birth in the Slotkin studies and decreased below controls by some 10% over the next 8 postnatal days whereupon it rose rapidly. This profound increase in DNA at d 8 is consistent with the initiation of cell binucleation, which normally occurs from postnatal d 6 to 14 (36). Thus, the Slotkin data are consistent with normal heart growth before birth and a mild suppression of proliferative growth in the heart over the first postnatal week. Furthermore, the data are difficult to interpret because the postnatal changes in DNA concentrations in the myocardium were measured at increasing times (between birth and 4 postnatal wk) after prenatal exposure to corticosteroid.

Torres et al. (21) administered dexamethasone to pregnant rats by pellet (~48 µg/d) beginning on the 17th day of pregnancy until term to approximate clinical doses for promoting fetal lung maturation in premature labor. They found that dexamethasone exposure produced smaller pups with increased heart/body weight ratios. Importantly, the proliferative index of cardiomyocytes was greatly increased, suggesting a glucocorticoid-stimulated proliferation in the fetal rat ventricular myocardium. Our data fit best with these findings.

Rudolph et al. (22) infused cortisol into the left coronary artery of the near term sheep fetus for 72–80 h. They found decreases in LV DNA concentration, compared with controls and increases in the protein to DNA ratios in the LV but not RV. Although they infused doses of cortisol similar to those in our study (Rudolph et al.: 1.7 mg/d; our study: 2.16 mg/d), they did not directly measure the effects of cortisol on cardiomyocyte growth, as we did, nor did they measure arterial pressure. Although in most tissues an increase in protein to DNA ratios is indicative of cellular hypertrophy, the ratio is complicated in fetal cardiac tissue because of unknown rates of proliferation, binucleation, and hypertrophy. Nevertheless, previous studies do not paint a clear picture of the role of cortisol during the maturation phase in myocardium.

The Lumbers group (6) infused high levels of cortisol (72 mg/d) into the near-term sheep fetus for about 60 h. In the cortisol-treated fetuses, plasma levels reached 200 ng/ml within an hour and more than 370 ng/ml by the conclusion of the study, compared with the approximately 3 ng/ml of the controls. These levels would be higher than found ordinarily in a sheep fetus, even at birth. The cortisol treatment did not lead to increases in cardiomyocyte binucleation. However, LV but not RV mono- and binucleated cells became larger than their control counterparts. Thus, these data superficially appear to support the conclusion of the Rudolph study that cellular hypertrophy caused the protein to DNA ratio to decrease.

However, these two sets of fetal sheep data are not easily interpreted. In the Lumbers’ paper, the cortisol infusion caused fetal hypertension; mean systolic pressure was 22 mm Hg higher in the cortisol-treated fetuses than in control fetuses (78.5 ± 2.4 mm Hg vs. 57.2 ± 4.5 mm Hg). We and others have shown that pressure increases of this magnitude cause proliferation, hypertrophy, and binucleation in the RV (17). Whereas the Lumbers’ study was important for showing the effect that might be expected from a large maternal course of glucocorticoid, it raises several questions regarding the specific actions of cortisol on the myocyte: 1) why did not RV cardiomyocytes increase in size with the increased pressure load that accompanied the cortisol infusion when the RV is known to be most sensitive to increased fetal arterial pressure? 2) was the increase in LV myocyte size the result of cortisol acting directly on the myocyte or was it due primarily to the increase in LV systolic load or both?

The different findings generated from these outstanding research groups are not easily explained. However, there are at least four features of perinatal myocardial growth that complicate the interpretation of protein to DNA ratios: 1) cardiomyocyte binucleation and terminal differentiation occur prenatally in the sheep and postnatally in the rat, and this process temporarily increases the concentration of myocardial DNA; 2) immature cardiomyocytes have relatively less contractile protein than mature cells and thus able to increase protein content per cell without significant cell enlargement (37, 38); 3) under conditions of systolic load, cardiomyocytes are able to increase their volume without increasing amounts of contractile protein (17); and 4) the effects of load and hormonal action are very sensitive to the stage of development. These features need to be considered when interpreting developmental patterns in immature hearts.

In this study, the increase in heart weight of cortisol infusion fetuses could not be accounted for by an increase in heart water content. There was no difference in wet weight to dry weight ratio between the cortisol-treated hearts and the control hearts. Rudolph et al. (22) came to the same conclusion in their study. The cortisol-treated hearts had an increased number of cardiomyocytes in the cell cycle. We directly measured cardiac myocyte size to assess the effects of cortisol on cardiac myocyte hypertrophy and found no hypertrophy. These findings taken together are consistent with the finding that cortisol augmented hyperplastic growth.

To keep animal use to a minimum, we used controls from three sources. Some were sham controls with or without saline infusion and some were uninstrumented controls. Because we found no differences in hemodynamic values or blood gas tensions between saline vehicle controls and treatment animals or between cortisol pre- and posttreatment conditions, we were statistically justified in using normal age matched uninstrumented animals as untreated controls as well.

The MR and the GR are both expressed in cardiac tissue of rat (39, 40), human (41, 42), and fetal sheep (6, 7). Binding of the receptors leads to nuclear translocation in which the ligand-receptor complexes bind DNA (8). Rapid nongenomic actions of glucocorticoids have also been described. MR has a higher affinity for both gluco- and mineralocorticosteroid ligands, compared with GR (43), and thus, MR stimulation is dependent on the relative local concentrations of ligand (40). Because plasma levels of the most abundant active glucocorticoid, cortisol, is found in much higher concentrations than the most important mineralocorticoid, aldosterone, it is believed that in most cells, the local concentrations of active cortisol must be inactivated by the enzyme, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) 2, if aldosterone is to be effective in binding MR (42). However, 11ß-HSD2 mRNA is not detectable in neonatal rat myocardium, whereas mRNA for 11ß-HSD1, which enables the conversion of inactive cortisone to active cortisol, is in relatively high abundance (40). Because the HSD1 isoform catalyzes the formation of active glucocorticoid, it is likely that corticosterone (the active glucocorticoid in rat) binds both MR and GR.

Unfortunately, very little is known about the signaling of cortisol in the developing sheep heart. It is likely under the conditions of our study, in which cortisol levels were elevated severalfold, that the effect of cortisol was due to both MR and GR binding, if the hydroxysteroid isoform levels are similar to those in the rat. It is not clear, however, what signaling pathways might have been important in eliciting the proliferation effects of cortisol in the present experiments. Potential targets include the cyclin-dependent kinases and their inhibitors (44, 45) and/or the phosphorylation cascades stimulated by growth factors (46). The relative expression patterns of hydroxysteroid dehydrogenases underlie tissue-specific responses to glucocorticoid stimulation that are unique to each fetal tissue and these can change in a developmental fashion (45).

Cortisol may stimulate proliferation in cardiomyocytes indirectly. When cortisol is infused into late gestation fetal sheep and the angiotensin II type 1 receptors are antagonized, the normal cortisol-induced hypertension is abolished (47). Other investigators (15) have shown that exogenous cortisol increases plasma angiotensin II levels and components of the renin angiotensin system in the myocardium in near term fetal sheep (6, 48) when given at pressor doses. Hegarty et al. (49) infused a low dose of cortisol (3 mg/d, 3–5 d), resulting in a small increase in plasma cortisol (to ~10 ng/ml), a small increase in arterial pressure (+4 mm Hg) and no effect on total angiotensin II receptor density or receptor affinity for ligands in the RV. We have shown that angiotensin II stimulates the proliferation of fetal sheep cardiomyocytes in vitro but does not stimulate hypertrophy (29). In addition to angiotensin II, thyroid hormone (50, 51, 52, 53) and the IGFs (54) are potential secondary-effect regulators of the fetal myocardium under the influence of cortisol. Whereas we have shown that IGF-I stimulates proliferation of fetal sheep cardiomyocytes in culture (55), neither thyroid hormone nor IGF-I has been studied sufficiently to make a strong case for their actions under the conditions of this study.

Conclusions
Subpressor levels of cortisol infused into the circumflex coronary artery result in: 1) an increase in heart weight and an increase in heart weight to body weight ratio, 2) no effect on LV or RV myocyte size, and 3) no effect on LV or RV myocyte maturational state. After a 2-d cortisol infusion, the number of cardiac myocytes staining positive for Ki-67 is increased, consistent with more cardiac myocytes in the cell cycle during cortisol infusion. These findings taken together are consistent with accelerated hyperplastic growth under the influence of cortisol. We cannot yet explain the different findings of previous studies in the same and other species. It is likely that species variation in the timing of terminal differentiation and maturation of cortisol signaling pathways are important.


   Acknowledgments
 
The authors thank Robert Webber and Patricia Renwick for technical assistance.


   Footnotes
 
This work was supported by the National Institute of Child Health and Human Development Grant P01HD34430; the M. Lowell Edwards Endowment; and a predoctoral fellowship (0110242Z) (to S.J.) and a postdoctoral fellowship (0525929Z) (to S.L.) from the American Heart Association.

Disclosure statement: all authors have nothing to declare.

First Published Online May 11, 2006

Abbreviations: E, Embryonic day; GR, glucocorticoid receptor; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; KB, potassium buffer; LV, left ventricle; MR, mineralocorticoid receptor; RV, right ventricle.

Received January 17, 2006.

Accepted for publication May 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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