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. 2004 Oct 28;562(Pt 2):493–505. doi: 10.1113/jphysiol.2004.074161

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

Andrew JW Fletcher 1, Alison J Forhead 1, Abigail L Fowden 1, Will R Ford 2, Peter W Nathanielsz 3, Dino A Giussani 1
PMCID: PMC1665501  PMID: 15513943

Abstract

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−1i.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.

The Langendorff, isolated, perfused heart preparation is used widely to assess myocardial chronotropic and inotropic responsiveness to exogenous agonists in vitro (Doring, 1990). To date, the preparation has principally been employed in adult rat and rabbit hearts, with measures of heart rate and ventricular contractility being derived from the pressure pulsatility within a fluid-filled latex balloon inserted into the left ventricle (Doring, 1990). Few studies have assessed both left and right ventricular function in the Langendorff preparation in any species in either fetal or adult animals. Whilst Palmisano et al. (1995) sequentially measured left ventricular, followed by right ventricular, depression of contractility in response to halothane or isoflurane in infant rabbit hearts, only Baker et al. (1997) have simultaneously measured left and right ventricular performance using a biventricular balloon preparation. In addition, only two studies have been reported where the Langendorff preparation has been used to study fetal sheep hearts, but in both cases only left ventricular performance was assessed (Sandhu et al. 1994; Davis et al. 1995).

Since the pioneering experiments of Rudolph & Heymann (1967) in fetal sheep in utero, microspheres have been used widely to assess regional organ perfusion in a variety of species (Prinzen & Glenny, 1994). Initially, radioisotope-labelled microspheres were used (Rudolph & Heymann, 1967), but more recently, fluorescent dye-labelled microspheres have been developed that are safer and more cost-effective (Prinzen & Glenny, 1994) and that have been validated against radioisotope-labelled microspheres for use in measurement of regional organ perfusion (Glenny et al. 1993). Microspheres have been used for assessment of coronary blood flow in vivo in adult rabbits and dogs, and in fetal sheep (Fisher et al. 1982; Bassingthwaighte et al. 1987; Kowallik et al. 1991; Reller et al. 1995).

The current study combined the Langendorff and microsphere techniques to develop a novel biventricular, isolated, perfused fetal sheep heart preparation in which left and right ventricular responsiveness to exogenous agonists could be assessed independently but simultaneously, together with any associated changes in coronary perfusate distribution.

The ovine fetal heart is subject to sympathetic and parasympathetic regulation within the last third of gestation, with an increase in parasympathetic and a reduction in sympathetic nervous system activity as term approaches (Vapaavouri et al. 1973; Walker et al. 1978). 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 (Fowden et al. 1998). Currently, 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 in mediating these changes. In this study, the Langendorff preparation was used in fetal sheep hearts during late gestation to assess whether changes in myocardial responsiveness to muscarinic cholinergic and β-adrenergic stimulation occurred with fetal glucocorticoid treatment or advancing gestational age. Isoprenaline and carbachol were chosen as the pharmacological agonists used in this study because they are employed widely in isolated adult heart preparations to assess myocardial responsiveness to non-selective β-adrenergic and muscarinic cholinergic stimulation, respectively (MacLeod, 1986; Watson et al. 1988; Doring, 1990; Matsuda et al. 1993) and are known to act on ovine fetal hearts during late gestation (Anderson et al. 1990; Birk & Reimer, 1992). In the glucocorticoid treated fetuses, an intravenous infusion of cortisol for 5 days between 125 and 130 dGA was employed at a dose rate designed to elevate plasma cortisol concentrations to levels similar to those measured within the last 5 days prior to term, thereby mimicking maturational effects of the prepartum cortisol surge (Forhead et al. 2000).

Methods

Animals

A total of 16 Welsh Mountain sheep fetuses and their mothers were used in this study and were allocated to three experimental groups as described below.

Surgical preparation

All surgical procedures were performed under the UK Animals (Scientific Procedures) Act 1986, as previously described (Fletcher et al. 2000b). In brief, following induction with 20 mg kg−1 i.v. sodium thiopentone (Intraval Sodium; Rhone Mérieux, Dublin, Ireland), general anaesthesia (1.5–2.0% halothane in 50 : 50 O2–N2O) was maintained using positive-pressure ventilation. A Teflon catheter was inserted into a maternal femoral artery and advanced into the maternal caudal aorta. Following a midline laparotomy and hysterotomy, translucent polyvinylchloride catheters (Critchly Electrical Products, NSW, Australia) were inserted into a fetal carotid artery and jugular vein. Another catheter was anchored to the fetal hindlimb for measurement of amniotic cavity pressure. The abdominal incisions were closed in layers, and the catheters were filled with heparinized saline (80 i.u. heparin ml−1 in 0.9% NaCl). Catheters were exteriorized via a small incision in the maternal flank.

Postoperative care

Ewes were housed in individual pens, had free access to water and hay, were fed concentrates twice daily (100 g; Sheep Nuts no. 6; H & C Beart Ltd, Kings Lynn, UK), and generally resumed normal feeding patterns within 24 h of surgery. The ewes received 2 days of postoperative analgesia (3 g daily oral phenylbutazone; Equipalozone Paste E-pp, Arnolds Veterinary Products Ltd, Shropshire, UK) if required. Antibiotics were administered daily to the ewe (0.20–0.25 mg kg−1 i.m. Depocillin; Mycofarm, Cambridge, UK), to the fetus (150 mg kg−1 i.v. ampicillin, Penbritin; SmithKline Beecham Animal Health, Surrey, UK) and into the amniotic cavity (300 mg Penbritin) for 3 days. Daily maternal descending aortic and fetal carotid blood samples (0.4 ml) were taken for analysis of blood gas status. Vascular catheters were maintained patent by a slow continuous infusion of heparinized saline at 0.1 ml h−1. At least 5 days of postoperative recovery were allowed before experiments were performed.

Experimental procedures

The study comprised three experimental groups. Premature Control fetuses (n = 7) were surgically prepared between 117 and 120 days gestational age (dGA; term is ca 145 dGA) and were infused i.v. with heparinized saline vehicle (80 i.u. heparin ml−1 in 0.9% NaCl) prior to delivery at 130 dGA. Premature Cortisol fetuses (n = 4) were surgically prepared between 117 and 120 dGA and were delivered at 130 dGA, having been infused i.v. with cortisol (2–3 mg kg−1 day−1 (estimated weight) for 5 days; Efcortisol; Glaxo-Welcome Ltd, Ware, UK) from 125 to 130 dGA. This dose regimen for exogenous cortisol administration in the sheep fetus has been previously demonstrated to produce plasma cortisol concentrations at 125–130 dGA that are similar to those measured immediately prior to term (Forhead et al. 2000). The Term Control group comprised five non-cortisol-infused fetuses delivered at >135 dGA (range 135–143 dGA), two of which had been infused with saline vehicle.

Delivery

At the time of elective delivery, pregnant ewes and fetuses were deeply anaesthetized (20 mg kg−1 i.v. sodium pentobarbitone; Sagatal; Rhône Mérieux) and the uterus exposed via an abdominal midline incision. Fetuses were removed from the uterus via an antimesometrial incision and were weighed. Following exsanguination under anaesthesia, the fetal heart, lungs, and dorsal aorta were removed as a single unit and submerged in heparinized Krebs solution at room temperature. In all cases, organ removal was achieved within 60 s after delivery of the fetus. The ewes were killed with a lethal dose of sodium pentobarbitone (40 mg kg−1 i.v. Pentoject; Animalcare Ltd, York, UK).

Langendorff apparatus

The Langendorff apparatus consisted of two main components (Fig. 1): (1) a water-jacketed organ bath containing Krebs solution and maintained at 39.5°C by means of a heater/circulator and water bath circuit; and (2) a perfusion circuit, comprising a peristaltic pump (Masterflex L/S 7519–10; Cole-Parmer Instrument Co, UK), bubble trap and glass perfusion cannula. The peristaltic pump perfused the hearts by means of retrograde aortic perfusion (see below) with non-recirculating Krebs solution (O2-bubbled with 5% CO2 to maintain appropriate buffering; 39.5°C) at a flow rate fixed between 35 and 40 ml min−1. The perfusion flow rates selected were based upon measurements of coronary blood flow made in fetal sheep in utero using the radioisotope-labelled microsphere technique (Cohn et al. 1974; Fisher et al. 1980). A three-way tap was included in the perfusion cannula assembly for injection of agonists or microspheres into the perfusate stream. A pressure transducer (Lectromed, UK Ltd, Welwyn Garden City, UK) attached to the perfusion cannula permitted estimation of coronary perfusion pressure (Palmisano et al. 1995).

Figure 1. The Langendorff isolated, perfused fetal sheep heart preparation.

Figure 1

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A, fetal sheep heart mounted in position on the perfusion cannula. B, mounted heart immersed in water-jacketed organ bath. C, schematic representation of the perfusion apparatus. Scale bars represent 3 cm. Arrows indicate direction of fluid flow.

Mounting of hearts in the Langendorff apparatus

The organs were rapidly transferred to the Langendorff apparatus. The glass perfusion cannula was carefully inserted into the dorsal aorta in the retrograde direction, avoiding passage into the pulmonary artery via the ductus arteriosus. The aorta was then secured to the glass cannula using cotton ligatures tied proximally to the origin of the right brachiocephalic trunk. The positioning of the tip of the cannula distal to the aortic valves was confirmed by palpation. This ensured that all the perfusate passing through the glass perfusion cannula was directed through the coronary circulation. The lungs, thymus, parietal pericardium and associated connective tissue were carefully dissected and removed, and the pulmonary artery transected to facilitate adequate right ventricular drainage. A water-filled latex balloon (size 10, 12, 14 or 16; Linton Instrumentation, Diss, Norfolk, UK) was inserted through the mitral valve into the left ventricular (LV) space via the opening created by removal of the left atrium (Fig. 1A). Similarly, another latex balloon was inserted through the tricuspid valve into the right ventricular (RV) space via the opening of the vena cava into the right atrium. Diastolic pressure was set to 10 mmHg by inflating intraventricular balloons with saline. The choice of balloon pressure was based on values used commonly in the Langendorff preparation in adult rabbit hearts (Palmisano et al. 1995), which are of similar size to the ovine fetal heart during late gestation. This balloon pressure is likely to have produced a situation in which the ventricles were operating on the plateaux of their stroke work versus end diastolic pressure function curves, as is thought to be the situation in vivo in the ovine fetus during the last third of gestation (Thornburg & Morton, 1983, 1986). The balloons were connected via rigid saline-filled catheters to separate pressure transducers for independent measurement of LV and RV balloon pressures (Fig. 1A). The heart was immersed in warm Krebs solution contained within the water-jacketed organ bath (Fig. 1B) and was left to equilibrate for 30 min (Palmisano et al. 1995).

Measurements and recording

Heart rate, coronary perfusion pressure and balloon pressures were recorded continuously using a chart recorder (Lectromed, UK Ltd). Heart rate, which was natural and not paced, was derived from balloon pressure pulsatility, and developed pressure (systolic minus diastolic balloon pressure; Carroll et al. 1997) was used as an index of ventricular inotropy (force of contraction). In three of the heart preparations (one from each group), electrocardiograms were taken under baseline conditions and these showed sinus rhythm. In all cases, visual inspection of the hearts used in this study confirmed a 1:1 regular, sequential atrial:ventricular contraction pattern which produced a correspondingly regular ventricular contraction trace on the data acquisition system output. As the peristaltic pump maintained a constant perfusion rate, changes in coronary perfusion pressure were used as an index of changes in coronary vascular resistance (Lamontagne et al. 1991).

Agonist doses

The effects of bolus doses of carbachol (CCh; carbamylcholine chloride; Sigma-Aldrich Co. Ltd, Poole, UK; range, 2 × 10−5 to 2 × 10−3 mg) and isoprenaline (Isop; (–)-isoproterenol (+)-bitartrate salt; Sigma-Aldrich Co. Ltd; range, 1.8 × 10−6 to 3.6 × 10−5 mg) on heart rate, coronary perfusion pressure and LV and RV functions were determined. The doses of isoprenaline and carbachol were selected on the basis of their effects determined in a pilot study using hearts from exsanguinated adult rabbits taken under general anaesthesia in compliance with the UK Animals (Scientific Procedures) Act 1986. Agonist doses were given sequentially but were randomised for each heart with respect to which agonist was administered first and to the dose order within each agonist. A recovery time (ranging between 5 and 15 min) was allowed between each bolus to allow heart rate and ventricular developed pressures to stabilize at baseline values before administration of the next bolus.

Distribution of coronary perfusate

The proportionate distribution of coronary perfusate to the right atrium, RV, LV and septum was determined under baseline conditions and during maximum stimulation with carbachol or isoprenaline by injection of fluorescent microspheres (red, orange, blue-green or yellow-green; 15 μm polystyrene microspheres; Fluospheres; Molecular Probes, Leiden, the Netherlands; 800 spheres per injection) into the perfusate (Hof et al. 1981; Glenny et al. 1993). The number of microspheres used per injection was determined in an initial study in two adult rabbit hearts. In these hearts, microspheres of differing colours were injected sequentially in different concentrations to determine the maximum total number of microspheres that could be injected before embolization had a measurable effect on coronary vascular resistance (3200 spheres as indexed by a rise in coronary perfusion pressure). As four sequential measurements needed to be made, each injection of microspheres was limited to 800 spheres.

Tissue processing and fluorescence measurement

Following completion of the protocol, the hearts were removed from the Langendorff apparatus and weighed. The hearts were then dissected to harvest tissue samples from the right atrium, RV, LV and septum. The tissue samples were weighed, individually wrapped in aluminium foil and stored at 5°C. Fluorescence measurements for each tissue sample were performed within 4 weeks of collection as previously described (Buchwalder et al. 1998). The left atrial tissue samples, which had been removed to allow insertion of the LV balloon and therefore had not been exposed to the fluorescent microspheres, were used as zero reference for measurements of fluorescence.

Determination of regional perfusate flow

Fluorescence for each sample was corrected for background and for the signal from the left atrium (tissue control). Standard curves were then used to determine the number of microspheres present in each tissue sample, by dividing the corrected fluorescence signal by the fluorescence intensity per sphere. Proportionate distribution of the coronary perfusate to the different regions of the myocardium could then be determined by comparing the number of microspheres present in each sample (Rudolph & Heymann, 1967).

Statistical analysis

Values for all variables are expressed as mean ± s.e.m. unless otherwise indicated. Statistical significance for comparisons between doses and between treatment groups was assessed using one-way or two-way repeated measures ANOVA, as appropriate. In all cases, statistical significance was accepted when P < 0.05.

Results

Plasma cortisol concentrations

Fetal plasma cortisol concentrations on the day of delivery, and the daily concentration profile during the period of cortisol infusion are shown in Fig. 2. Plasma cortisol concentrations were significantly elevated in Term Control fetuses compared with Premature Control fetuses. Over the 5 days of cortisol infusion, plasma cortisol concentrations were elevated in Premature Cortisol fetuses from values similar to those in Premature Control fetuses to levels that were intermediate between those measured in the Premature Control and Term Control fetuses (Fig. 2).

Figure 2. Fetal plasma cortisol concentrations.

Figure 2

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Values are mean ± s.e.m. for Premature Control (○; n = 7; 125–130 dGA; saline-infused), Premature Cortisol (□; n = 4; 125–130 dGA; cortisol-infused) and Term Control fetuses (•; n = 5; > 135 dGA). Bar represents period of cortisol infusion. Significant differences (P < 0.05, one-way repeated measures ANOVA + Tukey's post hoc test): avalues during infusion versus preinfusion baseline; bPremature Cortisol or Term Control versus Premature Control fetuses; cTerm Control versus Premature Cortisol fetuses.

Fetal heart and body weights

Fetal body weights at delivery were similar between treatment groups (Table 1). Although heart weight and heart weight per kg fetal weight tended to be lower in the cortisol-infused fetuses compared with saline-infused early gestation fetuses and late gestation fetuses, these differences fell outside statistical significance (P = 0.11; Table 1).

Table 1.

Fetal biometry and baseline fetal cardiac variables in the Langendorff preparation

Premature Control Premature Cortisol Term Control
Gestational age (days; mean ±s.d.) 130 ± 1 130 ± 1 139 ± 4
Fetal weight (kg) 2.43 ± 0.17 2.51 ± 0.11 2.77 ± 0.29
Heart weight (g) 27.0 ± 3.4 17.5 ± 1.5 31.0 ± 5.1
Heart weight per kg fetal weight (g kg−1) 11.2 ± 1.6 6.9 ± 0.9 11.0 ± 1.0
Coronary perfusion pressure (mmHg) 32.9 ± 3.3 27.2 ± 2.3 23.3 ± 8.9
RV developed pressure (mmHg) 30.2 ± 3.4 32.8 ± 6.1 40.3 ± 5.5
LV developed pressure (mmHg) 30.5 ± 2.6 48.5 ± 4.6a 41.2 ± 4.7
Heart rate (beats min−1) 146.8 ± 3.7 145.8 ± 15.1 152.3 ± 8.8
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Values are means ± s.e.m. for Premature Control (n = 7; 125–130 dGA; saline-infused), Premature Cortisol (n = 4; 125–130 dGA; cortisol-infused) and Term Control hearts (n = 5; >135 dGA). Developed pressure was calculated as the difference between systolic and diastolic balloon pressures. RV, right ventricle; LV, left ventricle.

a

P < 0.05, Premature Cortisol versus Premature Control (one way ANOVA + Tukey's post hoc test).

Basal variables

Values for basal coronary perfusion pressure, RV and LV developed pressures and heart rate following 30 min of equilibration are shown in Table 1. No differences were found in basal heart rate between treatment groups or between RV and LV developed pressure within treatment groups. However, basal LV developed pressure was significantly greater in Premature Cortisol compared with Premature Control hearts (P < 0.05; Table 1).

Functional responses to carbachol and isoprenaline

Coronary perfusion pressure, RV and LV developed pressure and heart rate responses to bolus doses of carbachol and isoprenaline are shown as absolute change from baseline in Fig. 3. Within each treatment group, the RV and LV exhibited developed pressure responses that were similar in magnitude at each dose of carbachol or isoprenaline (Fig. 3). In all groups, carbachol produced a dose-dependent reduction in RV and LV developed pressures and heart rate, but had no effect on coronary perfusion pressure (Fig. 3). The magnitude of these reductions in RV and LV developed pressures and in heart rate were greater in Term Control compared with Premature Control hearts (Fig. 3).

Figure 3. Absolute change from baseline in cardiac variables during the carbachol (A) and isoprenaline (B) dose–response protocols.

Figure 3

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Values are mean ± s.e.m. of absolute changes from baseline during the carbachol and isoprenaline dose–response protocols in hearts from Premature Control (○; 130 dGA; n = 7), Premature Cortisol (▪; 130 dGA; n = 4) and Term Control fetuses (•; >135 dGA; n = 5). Significant differences (P < 0.05): adifferences by post hoc analysis indicating a significant main effect of dose compared with lowest dose; bdifferences by post hoc analysis indicating a significant main effect of treatment group compared with Premature Control (two-way repeated measures ANOVA + Tukey's post hoc test).

Whilst having no effect on coronary perfusion pressure, isoprenaline administration produced similar dose-dependent increases in RV and LV developed pressures and in heart rate in Premature Control and Premature Cortisol hearts (Fig. 3). In contrast, RV and LV developed pressure and heart rate responses to isoprenaline were significantly attenuated in Term Control compared with Premature Control hearts (P < 0.05; Fig. 3).

Regional perfusate distribution and flow

Regional perfusate distributions and flows within the fetal hearts, as assessed by the microsphere technique, are shown in Fig. 4. Microsphere measurements were made in seven hearts from Premature Control fetuses, three hearts from Premature Cortisol fetuses and three hearts from Term Control fetuses. Proportionate perfusate distribution and flow to the right atrium, septum, RV or LV were unaffected by treatment with the maximum doses of carbachol or isoprenaline (Fig. 4). The absolute perfusate flow and proportion of perfusate received by the LV and RV were similar within and between groups (Fig. 4). Whilst the absolute perfusate flow and proportion of perfusate received by the ventricles were higher than values for the right atrium in Premature Control and Premature Cortisol hearts, this trend fell outside significance in most cases for the Term Control group (Fig. 4). Furthermore, absolute perfusate flow and the proportion of perfusate received by the septum were greater than those values for the right atrium in Premature Control hearts, but not in the Premature Cortisol or Term Control hearts (Fig. 4). When absolute perfusate flow was expressed per 100 g wet tissue weight, values were found to be similar between different regions of the heart within each of the treatment groups. However, as heart weight tended to be lower in the Premature Cortisol group compared with the Premature Control group (Table 1), absolute perfusate flow per 100 g wet tissue weight tended to be greater in Premature Cortisol compared with Premature Control hearts (Fig. 4).

Figure 4. Regional perfusate distribution and flow during the carbachol and isoprenaline dose–response protocols.

Figure 4

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Values are mean ± s.e.m. for hearts from Premature Control (130 dGA; n = 7), Premature Cortisol (130 dGA; n = 3) and Term Control fetuses (>135 dGA; n = 3). RA, right atrium; RV, right ventricle; LV, left ventricle. Significant differences (P < 0.05): aLV, RV or septum versus RA; bLV or RV versus septum; cPremature Cortisol versus Premature Control (one-way ANOVA + Tukey's post hoc test).

Discussion

A novel Langendorff, biventricular, isolated, perfused preparation has been established for simultaneous measurement of LV and RV performance and regional myocardial perfusate distribution in the fetal sheep heart. This in vitro preparation has been employed to further the understanding of fetal cardiac physiology by examining fetal myocardial responsiveness to β-adrenergic and muscarinic cholinergic stimulation independently of neurohormonal influences. The model has been used to assess changes in myocardial sensitivity to β-adrenergic and muscarinic cholinergic stimulation close to term and with fetal exposure to exogenous cortisol infusion that approximated the endogenous preparturient increase in plasma cortisol concentration (Magyar et al. 1980). In this study, plasma cortisol concentrations measured in Premature Control fetuses (125–130 dGA) and Term Control fetuses (>135 dGA) were within the normal range for their respective gestational ages (Magyar et al. 1980; Fowden et al. 1998). The cortisol dosing regimen employed in this study elevated plasma cortisol concentrations, by the fifth day of infusion, to levels similar to those measured within the last 5 days prior to term (Magyar et al. 1980).

It is well established that parasympathetic and sympathetic inputs are present and exert regulatory influences on ovine fetal cardiac function by the second half of gestation (Vapaavouri et al. 1973; Assali et al. 1977). A number of studies conducted in fetal sheep in vivo have attempted to determine how the balance in the autonomic system influences basal fetal heart rate changes with advancing gestation, but have reported conflicting results (Vapaavouri et al. 1973; Nuwayhid et al. 1975; Walker et al. 1978; Wakatsuki et al. 1992). Whilst Vapaavouri et al. (1973) reported that ovine fetal parasympathetic tone is fully developed by 120 days gestation and that sympathetic tone increases during the last third of gestation, Nuwayhid et al. (1975) found that parasympathetic tone on basal heart rate was weak prior to 130 days gestation and sympathetic tone was constant throughout gestation. However, Walker et al. (1978) reported a progressive increase in parasympathetic tone and decrease in sympathetic tone towards term in the fetal sheep. More recently, Wakatsuki et al. (1992) demonstrated that β-sympathetic and parasympathetic influences on basal ovine fetal heart rate increase with advancing gestation, regardless of fetal electrocortical state. The apparent disparity between the results of these investigations may be explained, at least in part, by the fact that these studies were carried out using β-adrenergic and cholinergic receptor agonists and antagonists in vivo in sheep fetuses in which neurohormonal reflexes were intact. Accordingly, Assali et al. (1977) have demonstrated that intravenous bolus doses of isoprenaline in the intact ovine fetus not only elicit fetal tachycardia, but also hypotension, with the magnitude of these changes increasing with advancing gestational age. Thus, the confounding influences of concomitant activation of fetal neurohormonal reflexes cannot be excluded in these studies.

In contrast, the Langendorff heart model developed in the current study allowed assessment of intrinsic myocardial responsiveness to isoprenaline and carbachol in isolation from tonic and reflex neural and endocrine inputs, and from changes in preload and afterload which affect fetal ventricular function (Thornburg & Morton, 1983, 1986). Thus, this study advances physiological understanding by demonstrating that, at the level of the myocardium itself, there is a developmental increase in ovine fetal cardiac responsiveness to muscarinic cholinergic influences but a decrease in responsiveness to β-adrenergic influences during late gestation. Furthermore, the findings of this study suggest that these changes in myocardial function are not solely regulated by preparturient increases in circulating levels of cortisol in the sheep fetus. However, these changes in fetal myocardial responsiveness provide ontogenic mechanisms that may contribute, at least in part, to the developmental reduction in basal fetal heart rate (Unno et al. 1999), the functional enhancement of the fetal cardiac baroreflex (Shinebourne et al. 1972; Segar, 1997), and the enhancement of fetal bradycardia during acute hypoxaemia (Fletcher et al. 2000a) as the fetus approaches term.

The exact mechanisms mediating these developmental changes in myocardial responsiveness to β-adrenergic and muscarinic cholinergic stimulation remain unclear. Previous studies in the fetus have reported changes in β-adrenergic and muscarinic cholinergic transmission with advancing gestational age and with glucocorticoid exposure. In rats, Bian et al. (1990, 1993) have shown a dose-dependent effect of maternal dexamethasone treatment on coupling of β-adrenoceptor activation to intracellular cyclic AMP production in the neonatal kidney and heart. In those investigations, maternal subcutaneous injections of 0.8 mg kg−1 dexamethasone on gestational days 17, 18 and 19 were found to suppress β-adrenergic signal transduction, whilst a lower dose (0.2 mg kg−1) enhanced the renal and cardiac cyclic AMP responses to isoprenaline. Although glucocorticoids enhance β-adrenergic receptor mRNA and protein expression in culture (Rodan & Rodan, 1986; Hadcock & Malbon, 1988), no change in cardiac β-adrenergic receptor numbers was found following treatment at the 0.2 mg kg−1 dose, and the enhancement of transmission was attributed to effects on adenylyl cyclase enzyme activity (Bian et al. 1992). The mechanism accounting for the suppression of transmission at the 0.8 mg kg−1 dose of dexamethasone is currently unknown, but is specific to the fetus and neonate, as adult rats treated daily with 1 mg kg−1 of dexamethasone did not exhibit suppression of cardiac sympathetic action (Lau & Slotkin, 1981). Endogenous glucocorticoids, such as cortisol or corticosterone, are also known to play an important role in regulating the coupling of β-adrenergic receptors to adenylyl cyclase, and hence to cell function (Davies & Lefkowitz, 1984), and to increasing rat myocardial muscarinic acetylcholine receptor affinity both in vivo and in vitro (Jacobsson et al. 1983; Ransnas et al. 1987).

Advancing gestational and postnatal age may also influence cardiac adrenergic and cholinergic signal transduction. For example, during development in the chick embryo, there is functional enhancement of muscarinic acetylcholine receptor-mediated signal transduction with advancing incubation from day 4 to day 8, even though receptor density is unaltered (Halvorsen & Nathanson, 1984). In contrast, a decrease in myocardial inotropic responsiveness to β-adrenergic stimulation occurs in chick embryos after 0.8 of incubation (Higgins & Pappano, 1981; Smith & Pappano, 1985). Similarly, in neonatal rats, where a large degree of postnatal maturation of cardiac sympathetic innervation occurs (Lau & Slotkin, 1981), the myocardium is less sensitive to isoprenaline stimulation in the neonate than in the adult (Seidler & Slotkin, 1979; Tanaka & Shigenobu, 1990). Whilst data are relatively scarce in the sheep fetus, Cheng et al. (1980) have demonstrated decreasing concentrations of cardiac α-adrenoceptors with increasing gestational age in fetal sheep.

In the current study, although advancing gestational age augmented the magnitude of the myocardial responses to carbachol and attenuated responsiveness to isoprenaline, fetal treatment with cortisol failed to have any significant effect on these responses compared with the early gestation fetuses. The reasons for the disparity between these findings, as well as with the previously reported effects of fetal glucocorticoid exposure described above, are unclear but may include failure of the particular cortisol infusion regimen employed in the current study to fully induce changes that occur during late gestation with the endogenous prepartum increase in cortisol (Fowden et al. 1998). This may result from insufficient duration of fetal cortisol exposure or failure to reach a threshold level necessary to induce changes in the myocardium. Alternatively, gestation-dependent effects may be cortisol-independent or may require concomitant activity in additional endocrine systems, such as the thyroid axis (Heinsimer et al. 1984; Pracyk & Slotkin, 1991, 1992; Birk et al. 1992). It is of interest that, although in the current study fetal cortisol infusion failed to have a significant effect on chronotropic and inotropic responses to carbachol or isoprenaline, it did increase basal LV, but not RV, developed pressure. This may result from direct effects of cortisol on myocardial maturation or from effects secondary to a cortisol-induced increase in fetal arterial pressure, and therefore ventricular afterload (Rudolph et al. 1999; Forhead et al. 2000).

In this study, microspheres were employed in the Langendorff preparation to measure regional distribution of the coronary perfusate, as has been performed previously in adult rat, rabbit and cat hearts. Coronary flow is closely related to cardiac work and O2 consumption in the fetal sheep heart in vivo (Thornburg & Reller, 1999) and in the isolated, perfused working guinea-pig heart preparation, which exhibits the phenomena of flow autoregulation, and hypoxic and metabolic vasodilatation (Bardenheuer & Schrader, 1983), and in the fetal circulation, the left and right ventricles pump in parallel. Under basal conditions, RV stroke volume is greater than LV stroke volume at any given mean atrial pressure, with RV output comprising 60–67% of the combined ventricular output (Heymann et al. 1973; Anderson et al. 1981). This fetal RV dominance may be explained, at least in part, by anatomical factors, with the RV having a greater volume, greater radius:wall thickness ratio, a higher wall stress under physiological pressures and greater myocyte and myofibril density than the LV (Pinson et al. 1987; Smolich et al. 1989). Consequently, fetal RV stroke work exceeds that of the LV (Thornburg & Reller, 1999) and, accordingly, coronary flow to the free wall of the RV exceeds that delivered to the LV free wall in the fetal sheep in vivo (Fisher et al. 1982). At the transition to neonatal life, the RV dominance in work and coronary flow reverses to LV dominance by 1 h of postnatal life as the LV:RV work ratio increases (Smolich et al. 1996). In the current study, the relative distributions of perfusate to the LV and RV follow the patterns expected for fetal hearts. Under the conditions of the Langendorff preparation, both absolute perfusate flow and perfusate flow per unit tissue weight to the LV and RV free walls were similar. Values of LV and RV flow were in a similar range to those reported previously in vivo (Fisher et al. 1980; Reller et al. 1995), although under the preload conditions of the Langendorff apparatus, the RV dominance, which is observed when the hearts are working against the afterload present in vivo (Thornburg & Reller, 1999), was lost. One possible contributing factor to this difference between the situations in vivo and in vitro is a differential baseline dilatation of the coronary vasculature in the left and right ventricles under the perfusion conditions employed in the current study.

Whilst isoprenaline has been shown to increase total coronary flow in the open-chest, anaesthetized adult rabbit model (Naim et al. 1997), and carbachol produces an overall reduction in myocardial flow in isolated, perfused rat hearts (Nuutinen et al. 1985), under the constant-flow conditions of the current study, maximum doses of carbachol or isoprenaline had no effect on regional perfusate distribution in the fetal sheep hearts. This is important because any changes in perfusate distribution during stimulation with isoprenaline or carbachol may have resulted in changes in regional delivery of those agonists and may have contributed to any differences between LV and RV responses to those agonists.

In any study utilizing a novel methodology, there will be inherent limitations that cannot be addressed retrospectively following the initial development of the model. In this pioneer study, a relatively small number of hearts have been used and this reflects both the fragile nature of the study material used and the technically demanding nature of the work. Whilst there was a trend towards hearts being smaller in the cortisol-infused fetuses compared with the premature and term control groups, this fell outside statistical significance, assuming no Type II error. However, it should be acknowledged that if there was an underlying significant difference in heart sizes between the groups, then there would have been accompanying differences in ventricular chamber sizes, ventricular wall dimensions and radii of curvature of the ventricular free walls. These differences would confound interpretation of differences in ventricular developed pressure between the groups. In this study, diastolic balloon pressure was set to 10 mmHg by inflating the intraventricular balloons with saline. Whilst this imposes a relatively high filling pressure on the hearts, the choice of balloon pressure was based on values used commonly in the Langendorff preparation in adult rabbit hearts (Palmisano et al. 1995), which are of similar size to the ovine fetal heart during late gestation. Whilst ECG recordings on three of the hearts used in this study confirmed that the hearts were beating in sinus rhythm, the possibiltiy that a junctional ‘escape’ rhythm did not supervene during the maximal bradycardic responses to carbachol cannot be excluded. Furthermore, the hearts used in this study were perfused with a buffered perfusate solution rather than whole blood. Consequently, the level of oxygen carriage and myocardial oxygen delivery achieved by the perfusate would have been less than that achieved in vivo, where oxygen is largely carried bound to haemoglobin. The coronary flow rates used in this study were chosen based upon blood flow rates in ovine fetal hearts (Cohn et al. 1974; Fisher et al. 1980), which also correspond to perfusate flow rates used widely in adult rabbit Langendorff heart preparations. However, it should be noted that whilst much higher perfusate rates could, in theory, achieve oxygen delivery rates equivalent to those in vivo, this does not take account of the confounding effects of sheer stress on the coronary endothelium at very high flow rates. Another limitation of the current study is that in the assessment of coronary perfusate distribution, the number of microspheres per injection was limited to 800. This was the case because it was necessary to make four sequential measurements of perfusate distribution, and the maximum number of microspheres that could be injected before embolization had a measurable effect on coronary vascular resistance had been shown in a pilot study to be 3200 microspheres. These spheres were delivered directly into a small tissue mass, and the fluorescent signals produced in the portions of the heart harvested (septum and right and left ventricles) were significantly elevated above the control left atrium samples. Whilst it is generally accepted that 400 microspheres per tissue are required for flow determinations, this value is based upon the original measurements using radiolabelled microspheres (Buckberg et al. 1971) rather than fluorescent microspheres which have a greater range of linearity (Glenny et al. 1993).

In conclusion, a novel Langendorff, biventricular, isolated, perfused fetal sheep heart preparation has been established in which left and right ventricular responsiveness to exogenous agonists has been independently and simultaneously assessed, together with measurements of coronary perfusate distribution. Advancing gestational age, but not cortisol treatment, enhanced fetal LV and RV developed pressure and heart rate responses to carbachol whilst suppressing responses to isoprenaline. Maximum doses of either carbachol or isoprenaline had no effect on coronary perfusate distribution. The increase in myocardial negative chronotropic responsiveness to carbachol and the attenuation of the positive chronotropic responsiveness to isoprenaline in the late gestation fetal hearts provide mechanisms that contribute to the maturation of fetal cardiovascular function during late gestation.

Acknowledgments

This work was funded by the Biotechnology and Biological Sciences Research Council and Tommy's – The Baby Charity, UK. A.J.W.F. was supported by the Foster Studentship, Department of Physiology, University of Cambridge, UK. D.A.G. is a Fellow of the Lister Institute for Preventive Medicine.

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