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Journal of Physiology (2001), 535.1, pp. 231-239
© Copyright 2001 The Physiological Society

Left ventricular stroke volume in the fetal sheep is limited by extracardiac constraint and arterial pressure

Daniel A. Grant, Jean-Claude Fauchère *, Kellie J. Eede, John. V. Tyberg † and Adrian M. Walker

	ABSTRACT

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1. Extracardiac constraint and sensitivity to arterial pressure may be critical factors that limit the functional reserves of the developing fetal heart in utero. We hypothesise that extracardiac constraint is the predominant factor that limits fetal stroke volume (SV). To test this hypothesis we studied six chronically instrumented fetal sheep to determine the relative roles that extracardiac constraint and arterial pressure play in determining left ventricular (LV) function.
2. Pregnant ewes (128-131 days gestation, term = 147 days) were anaesthetised (5 mg kg^-1 Propofol I.V., then 1.5 % halothane, 50 % O₂, balance N₂O by inhalation) and instrumented using sterile surgical techniques to record LV end-diastolic pressure (P_lved), aortic pressure (P_ao), pericardial pressure (P_per), and LV SV.
3. After a minimum of 72 h recovery, LV function was assessed by altering fetal blood volume to vary P_lved. Ventricular function curves were generated using two measures of ventricular function, SV and stroke work index (SWI = SV P_ao), and two measures of ventricular filling, P_lved and LV end-diastolic transmural pressure (P_lved,tm = P_lved - P_per).
4. Although decreasing P_lved from the resting level decreased SV, increasing P_lved from the resting level did not increase SV because the ventricular function curve plateaued. This plateau was not explained solely by an increase in aortic pressure, as the plateau remained present in the SWI versus P_lved curve. When extracardiac constraint was accounted for (SV against P_lved,tm), the plateau was largely eliminated (~80 %). The remaining portion of the plateau (~20 %) was eliminated when both extracardiac constraint and arterial pressure were accounted for (SWI versus P_lved,tm).
5. Thus, the major limitation upon LV function in the near-term fetus results from extracardiac constraint limiting ventricular filling while, at the same time, a much smaller limitation arises from increasing arterial pressure.

	INTRODUCTION

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Cardiac output must increase substantially at birth to meet the increased metabolic demands of newborn life. Although the mechanisms underlying this increase are not completely understood, an increase in left ventricular (LV) filling and, as a result, an increased stroke volume (SV) are essential components (Walker, 1993; Grant, 1999). What makes this adaptation at birth remarkable is that LV filling and SV cannot easily be increased prior to birth. Although the Frank-Starling mechanism functions during fetal life (Kirkpatrick et al. 1976; Gilbert, 1980, 1982; Thornburg & Morton, 1983, 1986; Grant et al. 1992a; Grant & Walker, 1996; Grant, 1999), the effectiveness of increasing ventricular filling pressures in increasing SV beyond the normal baseline level is minimal (Thornburg & Morton, 1986; Grant et al. 1992a). Ventricular function curve analysis clearly demonstrates this limitation as an alinearity or 'plateau' in the curve when ventricular filling pressures are increased beyond control levels (Kirkpatrick et al. 1976; Gilbert, 1980, 1982; Thornburg & Morton, 1983, 1986; Grant et al. 1992a; Grant & Walker, 1996; Grant, 1999). As the fetal heart normally operates at the inflection point or 'breakpoint' of this alinearity, it has been described as lacking significant cardiac reserve.

The factors that limit LV stroke volume in the near-term fetus remain uncertain. Several factors are ruled out by the rapidity with which stroke volume changes at birth (Kirkpatrick et al. 1973; Anderson et al. 1982; Grant et al. 1992b). For example, the immature cellular structure (Smolich, 1995) and limited compliance of the fetal myocardium (Romero et al. 1972; Romero & Friedman, 1979) appear unimportant as LV stroke volume increases immediately at birth, long before any substantial structural changes can occur. Of the other factors that might limit SV, arterial pressure and extracardiac constraint have also been investigated. The fetal heart, particularly the right ventricle (RV), is sensitive to the increase in arterial pressure that accompanies the volume infusions routinely used to generate fetal ventricular function curves (Gilbert, 1982; Thornburg & Morton, 1983, 1986; Hawkins et al. 1989). As a result, arterial pressure and increasing afterload have been proposed to produce the plateau observed in the fetal cardiac function curve. More recently we have demonstrated that maximal SV in the fetus is largely determined by the constraining effect that the pericardium and the chest wall-lung combination (i.e. extracardiac constraint) has on ventricular filling (Grant et al. 1992b; Grant & Walker, 1996). Moreover, our work suggests that the plateau in the fetal ventricular function curve arises from the use of ventricular filling pressure as a measure of ventricular preload. When ventricular transmural pressure, a more appropriate measure of ventricular preload, is used in ventricular function curve analysis, SV is linearly related to preload (Grant et al. 1992b; Grant & Walker, 1996) and the plateau is absent. As yet, no study has sought to quantify the relative influences that ventricular constraint and arterial pressure each has upon the LV function curve in the near-term fetus in utero. In this study we sought to resolve this issue by assessing both factors in a chronically instrumented preparation in which the circulatory conditions closely match those normally existing in utero.

	METHODS

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All surgical and experimental procedures were performed in accordance with the guidelines established by the National Health and Medical Research Council of Australia, and were approved by the Monash Medical Centre Committee on Ethics in Animal Experimentation.

Six pregnant Merino-Border Leicester cross ewes (128-131 days gestation, term = 147 days) were anaesthetised (Propofol 5 mg kg^-1) and then intubated and ventilated (1.5 % halothane, 50 % oxygen, balance nitrous oxide). Each fetus was instrumented using sterile surgical techniques. A mid-line incision was made in the ewe's abdomen and the uterus was palpated to locate the fetal head. A purse string suture was made in the uterus at this location, the uterus incised and the fetal head and upper body were delivered from the uterus while taking care not to compromise the umbilical circulation.

We performed a sternotomy on the fetus and then retracted the ribcage and lungs. A small (1-2 cm) incision was made in the pericardium along the atrioventricular sulcus and an ultrasonic flow probe was positioned on the ascending aorta (probe Model 8S or 10S, flow meter Model T108, Transonic Systems Inc., Ithaca, NY, USA). To record intrapericardial pressure, we positioned a small (2 cm 2 cm internal dimensions), flat, liquid-containing balloon within the pericardial space overlying the left ventricle and secured it in place with a single suture to the myocardium. The pericardial incision was closed with interrupted sutures taking care not to reduce pericardial volume. The edges of the pericardial incision were not overlapped and no attempt was made to seal the incision since the liquid-containing balloon accurately records intrapericardial pressure under these conditions in the fetus, neonate and adult (Smiseth et al. 1985; Kingma et al. 1987; Grant et al. 1988, 1992a,b, 1994; Grant & Walker, 1996). Balloon transducers were calibrated prior to insertion and subsequently re-calibrated at the end of each study (McMahon et al. 1969).

Left ventricular pressure was measured using a pressure transducer (Model P3.5, Konigsberg Instruments Inc., Pasadena, CA, USA) positioned through the left ventricular apex and secured in position with a purse string suture. This transducer was calibrated against pressures recorded from a saline-filled catheter (1.02 mm i.d., 1.78 mm o.d.) positioned within the LV through the LV free-wall (in one fetus the Konigsberg transducer failed and left ventricular pressure was measured from the saline-filled catheter). The fetal chest was then closed and made airtight.

We also positioned a saline-filled catheter (1.02 mm i.d., 1.78 mm o.d.) non-occlusively in the ascending aorta via the carotid artery, from which aortic pressure was recorded and blood samples were withdrawn for blood gas and pH analysis (Radiometer ABL 500 blood gas analyser, Radiometer Inc., Copenhagen, Denmark). A saline-filled catheter (1.27 mm i.d., 2.29 mm o.d.) was also positioned non-occlusively in the jugular vein for blood withdrawal and infusion. Finally, a saline-filled catheter (1.27 mm i.d., 2.29 mm o.d.) with multiple side holes was secured to the surface of the fetal chest to record amniotic fluid pressure. The catheters and the flow probe lead were secured to the fetal neck and then the fetus was returned to the uterus. The uterine incision was closed and made watertight, and 500 ml of warmed saline was infused into the amniotic cavity to replace amniotic fluid that was lost during the surgery. The ewe's abdominal incision was closed in layers and the catheters and the flow probe lead were tunnelled subcutaneously to the ewe's right flank where they exited via a small incision. Antibiotics (Ilium Penstrep, Troy Laboratories Pty, Inc., Smithfield, NSW, Australia) were administered daily to the ewe throughout the period of study.

Each ewe was allowed 3-5 days to recover from the surgery prior to being studied. On the day of the study (133 days gestation ± 1 day), the ewes were placed in a metabolic cart where they stood quietly throughout the study; ewes had continuous access to food and water. We connected the vascular catheters and balloon transducer to calibrated strain-gauge manometers (Transpac IV, Abbott Critical Care Systems, Abbott Ireland, Sligo, Ireland). All pressures were referenced to amniotic pressure. The strain-gauge manometers, the Konigsberg transducer, and the flow meter were connected to a signal conditioner (Cyberamp 380, Axon Instruments Inc., Foster City, CA, USA) and low pass filtered at 100 Hz. All physiological signals were recorded on a thermal chart recorder (Model 7758A, Hewlett Packard, Waltham, MA, USA), and on computer at a sampling rate of 200 Hz, using an analog-digital converting board (ADAC 4801/16 A/D board, ADAC Corp, Woburn, MA, USA) and acquisition software (CVSOFT data acquisition and analysis software, Odessa Computer Systems Ltd, Calgary, Canada).

Protocol

Each fetus was treated with atropine (0.2 mg kg^-1, supplemented at 15 min intervals) and propranolol (1.0 mg kg^-1) to block autonomic compensations of heart rate to alterations in fetal blood pressure (Thornburg & Morton, 1983). The absence of reflex heart rate changes throughout the study was indicative of an effective level of blockade. Arterial blood samples were collected for blood gas and pH analysis. Data for LV function curve analysis were collected while rapidly withdrawing fetal blood in sufficient quantities to reduce LV SV by approximately 30 % and then while reinfusing this blood. LV end-diastolic pressure was subsequently increased to a level of 20-25 mmHg with infusions of plasma substitute (Haemaccel, Hoechst Marion Roussel Australia Pty Ltd, Australia). At the completion of the study the ewes and fetuses were killed with a lethal dose of sodium pentobarbitone (150 mg kg^-1) and the pericardial balloons dissected and their calibration confirmed. In each case, the balloons maintained their calibration throughout the study period.

Assumptions

It is assumed that at end-diastole a static equilibrium exists across the LV free wall (Smiseth et al. 1985; Tyberg, 1985). At that moment, the pressure recorded within the ventricle, LV end-diastolic pressure (P_lved), equals the sum of the pressure applied across the ventricular wall (LV end-diastolic transmural pressure, P_lved,tm) and any additional forces applied to the ventricular wall by the surrounding tissues (extracardiac constraint) as recorded by the balloon transducer in the pericardial space (intrapericardial pressure, P_per). LV end-diastolic transmural pressure, calculated by subtracting P_per from P_lved, closely reflects ventricular volume and thus is indicative of ventricular preload (Smiseth et al. 1985; Tyberg, 1985).

Data analysis

LV function curves were constructed from a beat-by-beat analysis of the data recorded during alterations in fetal blood volume. Data were averaged in 1 mmHg increments of P_lved. LV stroke volume and LV stroke work index (SWI, the product of SV and mean aortic pressure) were plotted against P_lved (as an index of ventricular preload) to generate LV function curves. In addition, LV function curves were generated using P_lved,tm as the index of preload. Regression analysis was used to determine the breakpoint of each ventricular function curve. To locate the breakpoints in the curves, linear regressions of SV (or SWI) versus P_lved (or P_lved,tm) were forced through the data above and below a chosen pressure axis test point. This process was repeated for a range of test points at 1 mmHg increments. The breakpoint pressure was identified as the point on the pressure axis at which the combined sum of the residuals from the two least square regressions (total sums of squares, SS_tot) was minimal (Orr et al. 1982; Szymonowicz et al. 1990). Average ventricular function curves were subsequently generated after normalising both the index of ventricular function (SV and SWI) and P_lved to the values at the breakpoint.

Data recorded at the breakpoint P_lved pressure, 5 mmHg below this pressure, and 10 mmHg above this pressure were compared statistically using an analysis of variance for repeated measures. This range of pressures was selected to incorporate the range of P_lved common to all lambs. A Student-Newman-Keuls test was used to isolate differences detected by the analysis of variance. Student's paired t test was used when single comparisons were required. Probability (P) values of 0.05 or less were considered to be statistically significant. Data are presented as means ± S.E.M.

	RESULTS

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Blood gas and pH data recorded from the fetuses studied are consistent with reported values from this and from other laboratories (Hawkins et al. 1989; Grant & Walker, 1996; Smolich et al. 1996) and indicated a stable physiological preparation (arterial oxygen saturation, S_a,O2 = 50 ± 3 %, arterial partial pressure of oxygen, P_a,O2 = 21 ± 1 mmHg, arterial partial pressure of carbon dioxide, P_a,CO2 = 49 ± 1 mmHg, pH 7.36 ± 0.01, total haemoglobin = 8.8 ± 0.5 g dl^-1, total bicarbonate, HCO₃^- = 26 ± 1 mmol l^-1, base excess = 2 ± 1 mmol l^-1). The results observed from ewes allowed 3 days to recover from surgery did not differ from those allowed 5 days to recover.

Ventricular function curves generated by plotting either SV or SWI as a function of P_lved (Fig. 1A and B, filled circles) closely resembled the shape of those previously published from our laboratory (Grant et al. 1992b; Grant & Walker, 1996) and from others (Gilbert, 1980; Thornburg & Morton, 1986). An ascending limb, breakpoint (as indicated by a clear minimal value of the total sums of squares; Fig. 1C and D) and plateau featured prominently. As P_lved was decreased below the breakpoint, SV and SWI decreased significantly (Table 1). As P_lved was increased beyond the breakpoint SV remained unchanged from the breakpoint level (P > 0.05) while SWI continued to increase (P < 0.01), albeit at a lesser rate (Table 1 and Fig. 1A and B, filled circles). The average breakpoints of the individual SV and SWI curves occurred at P_lved of 11 ± 1 and 14 ± 1 mmHg, respectively. These breakpoint pressures did not differ significantly.

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Figure 1. Ventricular function curve and breakpoint analysis in the near-term fetal sheep Mean ventricular function curves (A and B) normalised as a percentage of the values of pressure and stroke volume (SV) or stroke work index (SWI) observed at the breakpoint. Decreasing left ventricular end-diastolic pressure (Plved) from the breakpoint value decreased LV stroke volume and stroke work index (). Increasing Plved beyond the breakpoint value produced no further increase in stroke volume and a limited increase in stroke work index as plateaus developed in the LV function curves. The plateau was largely eliminated after accounting only for the limitations of ventricular preload that result from the constraining effects of the surrounding tissues (A, ) and completely eliminated by accounting for both the limitations of ventricular preload and increasing arterial pressure (B, ). Breakpoint analysis was used to determine the breakpoint values of Plved (C and D) and LV end-diastolic transmural pressure (Plved,tm; E and F). SStot, total sums of squares of the residuals from the two linear regressions. Symbols in A and B indicate means ± S.E.M., n = 6.

When P_lved,tm pressure was used as the index of ventricular preload, the shape of the ventricular function curves changed substantially. Although an ascending limb and a plateau phase remained present in the SV-P_lved,tm curves, the plateau phase was substantially reduced in length (Fig. 1A, open circles). When SWI was plotted as a function of P_lved,tm, the breakpoint and plateau were completely absent (Fig. 1B, open circles). The absence of a breakpoint in the relationship between SWI and P_lved,tm was evidenced by the lack of a clear minimum of the total sums of squares in the breakpoint analysis (Fig. 1F).

Qualitatively, accounting for ventricular constraint largely eliminated the plateau in LV function, and accounting for arterial pressure eliminated it. To quantify the relative portions of the plateau that arose separately from extracardiac constraint and from increasing arterial pressure, we measured how much the length of the plateau (SV versus P_lved) decreased when we corrected P_lved for extracardiac constraint (SV versus P_lved,tm). We limited our assessment to the range of pressures common to all fetuses, beginning at the breakpoint and ending at a pressure equal to the breakpoint pressure plus 10 mmHg. Over this range of pressure, SV remained constant, SWI increased approximately 40 % and mean P_ao increased from 43 to 57 mmHg (Table 1). Relative to the SV-P_lved relation, the length of the plateau was shortened by 82 ± 4 % when SV was plotted as a function of P_lved,tm. The remaining 18 ± 4 % of the plateau was eliminated after accounting for increases in both extracardiac constraint and arterial pressure, as evidenced by the absence of a plateau when SWI was plotted as a function of P_lved,tm (Table 1, Fig. 1).

The increases in P_lved that were used to generate ventricular function curves were accompanied by substantial increases in P_per (Fig. 2A) which acted to limit increases in P_lved,tm (Fig. 2B). Breakpoint analysis, applied to the relationship of P_per versus P_lved (Fig. 2C) and P_lved,tm versus P_lved (Fig. 2D), revealed that the slope of these relationships changed at a P_lved of 13 ± 2 mmHg. These breakpoint pressures were not significantly different from those observed for the relationships between SV and P_lved or SWI and P_lved. Over the range of P_lved below the breakpoint pressure, the slopes of the individual relations of P_per versus P_lved were significantly less (0.58 ± 0.08) than over the range of P_lved greater than the breakpoint pressure (0.89 ± 0.02, P = 0.012, paired t test). As a result, the slopes of the relationships between P_lved,tm and P_lved prior to the breakpoint were significantly greater (0.42 ± 0.08) than after the breakpoint (0.11 ± 0.02, P = 0.012).

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Figure 2. Pericardial and left ventricular end-diastolic transmural pressure in the near-term fetal sheep Substantial increases in pericardial pressure accompanied increases in left ventricular end-diastolic pressure (Plved, A), limiting the increase in left ventricular end-diastolic transmural pressure (Plved,tm, B). Breakpoint analysis was used to determine the breakpoint values (C and D). SStot, total sums of squares of the residuals from the two linear regressions. Symbols in A and B represent mean values ± S.E.M., n = 6.

Significant changes in arterial pressure accompanied the alterations in fetal blood volume used to generate LV function curves. Aortic pressure decreased significantly (P < 0.01) from the breakpoint value as P_lved was decreased and subsequently increased as P_lved was increased with volume infusion (P < 0.01, Fig. 3 and Table 1). Similarly, the maximum rate of pressure rise (+dP/dt_max) decreased significantly (P < 0.01) from the breakpoint value as P_lved was decreased and increased significantly as P_lved was increased with volume infusion (Fig. 3 and Table 1). Although heart rate decreased as P_lved was lowered, reflex bradycardia in response to increasing P_lved beyond the breakpoint level was not observed (Fig. 3 and Table 1).

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Figure 3. Heart rate, aortic pressure and +dP/dtmax in the near-term fetal sheep Volume loading was accompanied by only minor changes in heart rate (HR) over the entire range of left ventricular end-diastolic pressure (Plved, ) and transmural pressure (Plved,tm, ). Mean aortic pressure and +dP/dtmax both increased with volume loading. Symbols represent mean values ± S.E.M. n = 6 for HR and Pao; n = 5 for +dP/dtmax.

	DISCUSSION

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Prior to birth, the heart has a limited ability to increase the amount of blood it pumps. Until now, the cause of this limitation was uncertain, being attributed to various factors, prominent among which is the heart's response to preload and arterial pressure. Our current study reveals that both extracardiac constraint, through its effect on limiting preload, and an increase in arterial pressure, through its effect on afterload, combine to limit stroke volume and produce the plateau in the fetal LV function curve. Although both preload and afterload mechanisms exist, the major limitation in fetal LV function resides in the preload limitation placed upon the filling of the LV by the constraint of the pericardium and chest wall-lung combination.

Ventricular function curve analysis depends upon the assessment of both a measure of ventricular function, such as SV or SWI, and an accurate assessment of ventricular preload. While SV and SWI are load dependent indices of ventricular function, they nonetheless provide the opportunity to investigate the mechanisms that underlie the control of cardiac output in the unique conditions of fetal life. Although preload is conventionally defined as the stress within the ventricular wall at the end of diastole, it is more simply assessed as end-diastolic ventricular volume or as P_lved,tm. In years past, both wall stress and ventricular volume proved difficult to measure. Because ventricular filling pressure is easily measured, it has at times inappropriately been utilised as a measurement of ventricular preload. One should take care when using end-diastolic pressure to construct left ventricular function curves because this pressure does not equal preload when changes in ventricular compliance occur (Glantz & Parmley, 1978). The importance of this is exemplified in our study as the constraint placed upon the fetal ventricle by the pericardium and chest wall-lung combination limited the increase in ventricular preload (P_lved,tm) that accompanied the increase in P_lved (Fig. 2). It was this limitation that largely (80 %) accounted for the plateau in the fetal LV function curve (Fig. 1 and Table 1).

Qualitatively, the major portion of the plateau in the fetal LV function curve was eliminated after employing P_lved,tm as the index of preload to account for extracardiac constraint. This qualitative observation was supported by our quantitative assessment in which 82 % of the plateau was associated with the effects of extracardiac constraint. One might attribute the remaining portion of the plateau to several other sources. First, it is possible, though unlikely, that our pericardial balloons underestimated pericardial pressure and thus resulted in an overestimation of P_lved,tm. While it is known that open-ended catheters underestimate pericardial pressure in many settings, including after incising the pericardium (Smiseth et al. 1985), balloon transducers have been shown to correctly measure pericardial pressure in the fetus, newborn and adult in all settings (Smiseth et al. 1985; Kingma et al. 1987; Grant et al. 1988, 1992a,b, 1994; Grant & Walker, 1996). Importantly, we confirmed the calibration of each balloon at autopsy. Second, one might attribute the plateau in the SV-P_lved,tm relation to a limitation in the Frank-Starling mechanism, i.e. beyond the breakpoint level of P_lved,tm (6 ± 1 mmHg) the preload reserve of the myocardium may be exhausted. This is also unlikely, as our previous studies demonstrate that SV can continue to increase as P_lved,tm is increased beyond 6 mmHg (Grant et al. 1992b). Finally, using SWI as a measure of cardiac function that incorporates arterial pressure in its calculation, we determined that increasing arterial pressure, and thus afterload, accounted completely for the portion of the plateau not accounted for by extracardiac constraint.

Arterial pressure has been previously shown to substantially influence fetal SV and has been credited with accounting for the entire plateau in the fetal ventricular function curve (Thornburg & Morton, 1986; Pinson et al. 1987; Hawkins et al. 1989). However, in these earlier studies the plateau may have been exaggerated by failing to adequately account for how ventricular constraint limits preload. In avoiding this difficulty, by utilising P_lved,tm as our measure of preload, we have identified that arterial pressure plays a much smaller role than does extracardiac constraint.

The presence of a plateau in the SWI-P_lved relation and its absence in the SWI-P_lved,tm relation also confirms, for the first time in the fetal LV, the speculation of Sarnoff & Berglund (1954) that a 'plot of [stroke] work against volume (or fibre length) [P_lved,tm] would be closer to a straight line' than a plot of stroke work versus P_lved. It also confirms the presence of a 'preload recruitable stroke work' relationship (Glower et al. 1985) in the fetal LV in utero. Glower et al. (1985) explained the differences between their non-linear (SW-P_lved) and linear (SW-LV end-diastolic volume) stroke work curves by the exponential relationship they observed between P_lved and LV dimensions. Similarly, the presence of the plateau in our SWI-P_lved relation and its absence in our SWI-P_lved,tm relation can be explained by the relationship that exists between P_lved and ventricular dimensions/volume in the fetus. We have previously demonstrated that the fetal LV dimension-P_lved relation is non-linear and that increases in P_lved are not accompanied by increases in dimensions (volume) because extracardiac constraint increases (Grant et al. 1992a, 1994). In the current setting, the plateau phase of the SWI-P_lved relation, and the absence of such a plateau in the SWI-P_lved,tm relation (Fig. 2), reflects the inappropriateness of using P_lved as an index of preload and indicates the importance of accounting for extracardiac constraint.

Although the fetal LV was unable to increase SV beyond the breakpoint value of P_lved, it was able to maintain the plateau level of SV constant in the face of increasing arterial pressure; over the range of P_lved from the breakpoint to 10 mmHg beyond the breakpoint, SV was unchanged while P_ao increased approximately 33 %. To maintain SV constant throughout the plateau the LV increased SWI by approximately 40 % (Table 1). This increase in SWI is unlikely to represent homeometric autoregulation of the fetal heart (Sarnoff et al. 1960; Sarnoff & Mitchell, 1962; Klautz et al. 1995) since this increase, and the increase in contractility as assessed by +dP/dt_max, were linearly related to increases in ventricular preload (P_lved,tm, Fig. 3, open symbols), and thus reflect the volume-dependent nature of these measures of contractility (Parmley et al. 1975; Grant & Walker, 1996).

Although our study demonstrates that extracardiac constraint is the predominant mechanism by which fetal LV function is limited, it is uncertain whether or not the same can be said of the fetal RV. In acute studies, we have shown that a limitation of preload as a result of extracardiac constraint limits RV stroke volume (Grant & Walker, 1996). However, the fetal RV is known to be more sensitive to afterload than the fetal LV (Thornburg & Morton, 1983, 1986; Pinson et al. 1987; Reller et al. 1987; Thornburg et al. 1987) and thus afterload may play a larger role in determining RV function than LV function. Moreover, the unique circulatory pathways of the fetus, including the presence of the ductus arteriosus and the highly constricted pulmonary vasculature (Walker, 1993), represent a setting in which the interaction between the RV and the pulmonary vasculature (ventricular-vascular interaction) has a significant impact upon blood flow from the RV (Grant et al. 1999). As ventricular-vascular interactions may have a more significant impact upon RV function than LV function, future studies should address the relative roles that preload and arterial pressure play in determining the limits of RV SV.

We did not measure the relative magnitude of the constraint applied to the LV by each of the tissues that surround the fetal heart in this study. However, based upon our earlier studies, and based upon observations made by Kirkpatrick et al. (1973), it is likely that the pericardium and the fetal chest wall-lung are the major contributors. As our studies were conducted with the fetus in utero, the maternal tissues may also have contributed. We took care not to introduce any artificial constraint into our preparations. Pericardial incisions were small and the volume of instrumentation within the pericardial space was minimal. All animals recovered well from the surgery, as indicated by normal arterial blood gas and pH data and by the normal levels of LV stroke volume. Moreover, at autopsy the fetal heart appeared normal and there were very few intrathoracic adhesions.

It is uncertain why we did not observe a significant afterload effect in our previous acute studies of fetal cardiac function (Grant et al. 1992b). It seems unclear how the presence of anaesthesia in our early studies would have acted to make the LV less sensitive to increasing arterial pressure. A more likely explanation may lie in the different ages of the fetuses studied. The fetuses in our current study (131-135 days gestation), and those in much of the earlier work (Thornburg & Morton, 1983, 1986) were studied earlier in gestation that those in our initial study (140-144 days). In the development of the fetal sheep, the last 10-15 days of gestation is a period of rapid cardiorespiratory development that may have a substantial impact on the heart's response to arterial pressure. Moreover, this is also a period when a marked surge in plasma cortisol occurs, which has been shown to have a direct effect on myocardial growth (Rudolph et al. 1999). Whatever the reason, both our earlier results and our current results point clearly to a limitation in preload, not arterial pressure, as playing the major role in determining ventricular function in utero. The ability to utilise preload recruitable stroke work may represent an important compensatory mechanism by which the fetus maintains LV output in the face of increased arterial pressure such as might accompany periods of hypoxaemia (Cohn et al. 1974). Moreover, it may be teleologically advantageous for the LV to become less sensitive to increases in arterial pressure in preparation for the increase in arterial pressure that occurs at birth (Walker, 1993).

In summary, our study suggests that extracardiac constraint and its effect of limiting ventricular preload is the major limitation on the fetal LV and is largely responsible for producing the plateau in the fetal LV function curve.

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Top Abstract Introduction Methods Results Discussion References

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Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia (D.A.G. and A.M.W.) J.-C.F was supported by an Overseas Postgraduate Research Scholarship (DEETYA, Australia); Monash Graduate Scholarship (Monash University); and by the Department of Education, Canton Zurich Switzerland.

Corresponding author

D. A. Grant: Ritchie Centre for Baby Health Research, Monash Institute of Reproduction and Development, Locked Bag 29, Clayton, Victoria 3168, Australia.

Email: daniel.grant{at}med.monash.edu.au

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