Foramen ovale blood flow and cardiac function after main pulmonary artery occlusion in fetal sheep.

NEW FINDINGS: What is the central question of this study? At near-term gestation, foramen ovale blood flow accounts for a significant proportion of fetal left ventricular output. Can the foramen ovale increase its volume blood flow when right ventricular afterload is increased by main pulmonary artery occlusion? What is the main finding and its importance? Foramen ovale volume blood flow increased during main pulmonary artery occlusion. However, this increase was attributable to an increase in fetal heart rate, because left ventricular stroke volume remained unchanged. These findings suggest that the foramen ovale has a limited capacity to increase its volume blood flow. ABSTRACT: The foramen ovale (FO) accounts for the majority of fetal left ventricular (LV) output. Increased right ventricular afterload can cause a redistribution of combined cardiac output between the ventricles. To understand the capability of the FO to increase its volume blood flow and thus LV output, we mechanically occluded the main pulmonary artery in seven chronically instrumented near-term sheep fetuses. We hypothesized that FO volume blood flow and LV output would increase during main pulmonary artery occlusion. Fetal cardiac function and haemodynamics were assessed by pulsed and tissue Doppler at baseline, 15 and 60 min after occlusion of the main pulmonary artery and 15 min after occlusion was released. Fetal ascending aorta and central venous pressures and blood gas values were monitored. Main pulmonary artery occlusion initially increased fetal heart rate (P < 0.05) from [mean (SD)] 158 (7) to 188 (23) beats min-1 and LV cardiac output (P < 0.0001) from 629 (198) to 776 (283) ml min-1 . Combined cardiac output fell (P < 0.0001) from 1524 (341) to 720 (273) ml min-1 . During main pulmonary artery occlusion, FO volume blood flow increased (P < 0.001) from 507 (181) to 776 (283) ml min-1 . This increase was related to fetal tachycardia, because LV stroke volume did not change. Fetal ascending aortic blood pressure remained stable. Central venous pressure was higher (P < 0.05) during the occlusion than after it was released. During the occlusion, fetal pH decreased and P C O 2 increased. Left ventricular systolic dysfunction developed while LV diastolic function was preserved. Right ventricular systolic and diastolic function deteriorated after the occlusion. In conclusion, the FO has a limited capacity to increase its volume blood flow at near-term gestation.

Fetal sheep central haemodynamics and cardiac function during occlusion of the ascending aorta.

NEW FINDINGS: What is the central question of this study? The fetal aortic isthmus has an important physiological role, allowing communication between the left and right ventricular outputs, which are arranged in parallel. Can the aortic isthmus provide unrestrictive communication between the left and right ventricular circulations during occlusion of the ascending aorta? What is the main finding and its importance? During occlusion of the ascending aorta, fetal carotid artery perfusion pressure fell significantly, showing that the aortic isthmus failed to redirect blood flow and pressure from the ductus arteriosus to the aortic arch. This suggests that the aortic isthmus cannot provide unrestrictive communication between left and right ventricular circulations. The fetal aortic isthmus (AoI) allows communication between left (LV) and right ventricular (RV) outputs and represents an arterial watershed between the brachiocephalic (brain) and subdiaphragmatic (placenta) circulations. To understand the capability of the AoI to maintain the balance between the upper and lower body circulations, we performed a complete occlusion of the fetal ascending aorta in nine chronically instrumented sheep at near term gestation. We hypothesized that the occlusion would significantly decrease LV output and concomitantly increase RV output in order to maintain adequate systemic cardiac output and perfusion pressure to the fetal brain circulation through retrograde filling of the AoI. Fetal cardiac function and haemodynamics were assessed by pulsed and tissue Doppler at baseline, 15 and 60 min after occlusion of the ascending aorta and 15 min after occlusion was released. Carotid artery and jugular vein pressures were monitored. Occlusion of the ascending aorta increased (P < 0.002) RV output from [mean (SD)] 684 (369) to 907 (414) ml min-1 and decreased (P < 0.0001) LV output from 440 (136) to 40 (16) ml min-1 . Combined cardiac output decreased (P < 0.02) from 1125 (494) to 946 (417) ml min-1 . During occlusion, carotid artery mean pressure decreased from 32 (7) to 12 (7) mmHg (P < 0.0001). Systemic venous pressure was unaffected. Left ventricular systolic and diastolic function deteriorated during occlusion. Right ventricular systolic function improved, while diastolic dysfunction developed. Fetal carotid artery perfusion pressure decreased significantly during occlusion of the ascending aorta, demonstrating that AoI failed to redirect blood flow and pressure from the ductus arteriosus to the aortic arch. Our finding suggests that at near term gestation the aortic AoI cannot provide unrestrictive communication between LV and RV circulations.

Fetal ventricular interactions and wall mechanics during ductus arteriosus occlusion in a sheep model.

We investigated the effect of fetal sheep ductus arteriosus occlusion (DO) on the distribution of cardiac output and left and right ventricular function by tissue and pulsed Doppler at baseline; after 15 and 60 min of DO induced with a vascular occluder; and 15 min after release of DO. Ductal occlusion decreased fetal pO2. Mean left ventricular output increased (p < 0.001) from 725 to 1013 mL/min, and right ventricular (1185 mL/min vs. 552 mL/min) and systemic (1757 mL/min vs. 1013 mL/min) cardiac outputs fell (p < 0.001) after 15 min of DO, compared with baseline. Pulmonary vascular impedance decreased and volume blood flow increased more than threefold during DO, whereas foramen ovale volume blood flow remained unchanged. Left ventricular systolic function was unaffected, whereas isovolumic relaxation velocity deceleration decreased. Right ventricular functional indices remained unchanged. We conclude that DO increased pulmonary volume blood flow, not foramen ovale volume blood flow. Left ventricular output increased, although not as much as right ventricular output fell, resulting in decreased systemic cardiac output. During DO, left ventricular function exhibited diminished relaxation.

Myocardial performance and its acute response to angiotensin II infusion in fetal sheep adapted to chronic anemia.

Fetal chronic anemia causes lengthening of cardiomyocytes. In adults, severe left ventricular overload may lead to irreversible ventricular dysfunction. We hypothesized that in sheep fetuses with chronic anemia, remodeled myocardium would less successfully respond to angiotensin II (AT II) infusion than in fetuses without anemia. A total of 14 ewes with twin pregnancy underwent surgery at 113 ± 1 days of gestation. After a recovery period, anemia was induced by isovolumic hemorrhage in 1 fetus of each pair. At 126 ± 1 days of gestation, longitudinal myocardial velocities of the right (RV) and left (LV) ventricles were assessed at the level of the atrioventricular valve annuli via tissue Doppler imaging. Cardiac outputs were calculated by pulsed Doppler ultrasound. All measurements were performed at baseline and during fetal AT II infusion. Fetal serum cardiac natriuretic peptide (N-terminal peptide of proatrial natriuretic peptide [NT-proANP] and B-type natriuretic peptide [BNP]) concentrations were determined. Nine ewes successfully completed the experiment. At baseline, ventricular free wall thicknesses, cardiac outputs, and NT-proANP levels were significantly greater in the anemic fetuses than in the controls. The LV isovolumic contraction velocity (IVCV) acceleration and isovolumic relaxation velocity (IVRV) deceleration were lower (P < .05) in the anemic fetuses than in the controls. In the anemic fetuses, there was a positive correlation (R = .93, P < .01) between RV IVRV deceleration and NT-proANP concentration. Angiotensin II infusion increased (P < .05) LV IVCV acceleration in the anemic fetuses. We conclude that in anemic sheep fetuses, myocardial adaptation is associated with impaired LV early contraction and relaxation. However, the LV can improve its contractility with an inotropic stimulus, even in the presence of increased afterload.

Regional myocardial function and response to acute afterload increase in chronically anemic fetal sheep: evaluation by two-dimensional strain echocardiography.

We hypothesized that in chronic fetal anemia, remodeling of the myocardium is related to abnormalities in regional wall motion and acutely increased afterload further disturbs myocardial strain. Chronic anemia was induced in one fetus of each of seven sheep twin pregnancies. The fetuses were studied by two-dimensional (2-D) strain echocardiography at baseline and during increased afterload via angiotensin II (AT II) infusion. At baseline, the peak systolic longitudinal, radial and circumferential strains in the left ventricular lateral wall in anemic fetuses were lower than those in the controls (all p<0.05). During AT II, the circumferential strain of right ventricular free wall decreased significantly both in the control and anemic fetuses. Left ventricular free wall systolic strains were not affected by AT II. Fetal myocardial remodeling in chronic anemia decreases left ventricular systolic free wall strains. The myocardial adaptation does not change ventricular responses to acutely increased afterload.

Cardiomyocyte enlargement, proliferation and maturation during chronic fetal anaemia in sheep.

Chronic anaemia increases the workload of the growing fetal heart, leading to cardiac enlargement. To determine which cellular process increases cardiac mass, we measured cardiomyocyte sizes, binucleation as an index of terminal differentiation, and tissue volume fractions in hearts from control and anaemic fetal sheep. Fourteen chronically catheterized fetal sheep at 129 days gestation had blood withdrawn for 9 days to cause severe anaemia; 14 control fetuses were of similar age. At postmortem examination, hearts were either enzymatically dissociated or fixed for morphometric analysis. Daily isovolumetric haemorrhage reduced fetal haematocrit from a baseline value of 35% to 15% on the final day (P < 0.001). At the study conclusion, anaemic fetuses had lower arterial pressures than control fetuses (P < 0.05). Heart weights were increased by 39% in anaemic fetuses compared with control hearts (P < 0.0001), although the groups had similar body weights; the heart weight difference was not due to increased ventricular wall water content or disproportionate non-myocyte tissue expansion. Cardiomyocytes from anaemic fetuses tended to be larger than those of control fetuses. There were no statistically significant differences between groups in the cardiomyocyte cell cycle activity. The degree of terminal differentiation was greater in the right ventricle of anaemic compared with control fetuses by 8% (P < 0.05). Anaemia substantially increased heart weight in fetal sheep. The volume proportions of connective and vascular tissue were unchanged. Cardiomyocyte mass expanded by a balanced combination of cellular enlargement, increased terminal differentiation and accelerated proliferation.

Intramembranous solute and water fluxes during high intramembranous absorption rates in fetal sheep with and without lung liquid diversion.

OBJECTIVE: To examine mechanisms that mediate increased intramembranous solute and water absorption. STUDY DESIGN: Intramembranous solute and water fluxes were measured in fetal sheep under basal conditions and after intraamniotic infusion of lactated Ringer's solution of 4 L/d for 3 days with and without lung liquid diversion. RESULTS: Intramembranous sodium, potassium, chloride, calcium, glucose, and lactate fluxes increased 2.5- to 7.9-fold, were linearly related to volume fluxes (r = 0.83-0.99), and were unaffected by lung liquid. All clearance rates, except that of lactate, increased to equal the intramembranous volume absorption rate during infusion. CONCLUSION: Under basal conditions, passive diffusion makes a minor and bulk flow a major contribution to intramembranous solute absorption. During high absorption rates, the increase in solute absorption above basal levels appears to be due entirely to bulk flow and is unaffected by lung liquid. The increased bulk flow is consistent with vesicular transcytosis.

Responses of amniotic fluid volume and its four major flows to lung liquid diversion and amniotic infusion in the ovine fetus.

We designed experiments to allow direct measurement of amniotic fluid volume and continuous measurement of lung liquid production, swallowing, and urine production in fetal sheep. From these values, the rate of intramembranous absorption was calculated. Using this experimental design, the contribution of lung liquid to the control of amniotic fluid volume was examined. Fetuses were assigned to 1 of 4 protocols, each protocol lasting 3 days: control, isovolemic replacement of lung liquid, supplementation of amniotic fluid inflow by 4 L/day, and supplementation of amniotic inflow during isovolemic replacement of lung liquid. We found no effect of lung liquid replacement on any of the known flows into and out of the amniotic fluid. Although intramembranous absorption increased greatly during supplementation, the amniochorionic function curves were not altered by isovolemic lung liquid replacement. We conclude that lung liquid does not appear to contain a significant regulatory substance for amniotic fluid volume control.

Sources of amniotic fluid erythropoietin during normoxia and hypoxia in fetal sheep.

OBJECTIVE: Erythropoietin is present in human amniotic fluid and has been suggested as a marker of fetal hypoxia. The objectives of the present study were to determine whether erythropoietin is present in ovine amniotic fluid, fetal urine, and/or lung liquid and whether concentrations in these compartments change in parallel with endogenous fetal plasma erythropoietin concentration when the latter is increased experimentally. STUDY DESIGN: In late gestation chronically catheterized fetal sheep, samples of amniotic fluid and plasma, urine and plasma, lung liquid, amniotic fluid, and plasma were collected before and up to 7 days after induction of 4 types of fetal hypoxia: (1) acute anemic hypoxia that was induced by a single fetal hemorrhage, (2) progressive anemic hypoxia that was induced by daily exchange transfusion, (3) acute hypoxic hypoxia that was induced by the reduction of maternal inspired oxygen content, or (4) chronic placental insufficiency that was induced by daily umbilicoplacental embolization for 4 days. Erythropoietin concentrations were determined by radioimmunoassay. Statistical testing included analysis of variance and least squares regression. RESULTS: Under basal, nonhypoxic conditions, amniotic fluid erythropoietin concentration averaged 33.2% +/- 1.6% (SE) of fetal plasma erythropoietin concentration, and basal fetal urine and lung liquid erythropoietin concentrations ranged from low (<10% of plasma concentration) to nondetectable. Unlike the strong correlation in humans, basal amniotic fluid and plasma erythropoietin concentrations were correlated only weakly (r = 0.259; r2 = 6.7%; P = .0027; n = 132). Amniotic fluid erythropoietin concentration approximately doubled after 12 hours of severe hypoxic hypoxia or after 24 hours of embolization-induced severe hypoxia but was unchanged after 12 hours of mild-moderate hypoxic hypoxia or 24 hours of anemic hypoxia. Concomitant fetal plasma erythropoietin concentrations increased to 28.1 +/- 5.3, 12.5 +/- 2.7, 10.8 +/- 4.6, and 10.0 +/- 1.3 times basal values, respectively. During progressive fetal anemia, urinary erythropoietin concentration increased almost 10-fold (P = .0023) but remained a small fraction (3.7% +/- 0.4%) of plasma concentration; at 12 hours of hypoxic hypoxia, lung liquid erythropoietin concentration did not vary with the severity of the hypoxia and remained low relative to plasma concentration (4.2% +/- 2.1%). CONCLUSION: Erythropoietin is present in ovine amniotic fluid, urine, and lung liquid. With only 3 potential sources, the fetal membranes appear to be the primary source of amniotic fluid erythropoietin in the nonhypoxic ovine fetus because basal urine and lung liquid erythropoietin concentrations are much lower than amniotic fluid concentrations. Although unchanged during mild-to-moderate fetal hypoxia, amniotic fluid erythropoietin concentration increases modestly during severe fetal hypoxia. In sheep, fetal urinary erythropoietin may contribute to this rise in amniotic fluid erythropoietin concentration during severe hypoxia, because fetal urinary and plasma concentrations increase in parallel during anemia.

Repeated daily injections and osmotic pump infusion of isoproterenol cause similar increases in cardiac mass but have different effects on blood pressure.

We found in mice that repeated single daily subcutaneous (s.c.) isoproterenol (ISO) injections, like constant infusions using osmotic minipumps, caused increased biventricular mass or weight relative to body weight (VW/BW). We found that 5 (1/d) s.c. injections of 2, 10, or 20 microg/g body weight caused equivalent VW/BW increases as compared with 5-d infusions at 20 microg/(g.d)). While it is often presumed that ISO elicits hypertrophy by a direct effect on the myocytes, growth may also be secondary to systemic hemodynamic effects. The 2 modes of ISO administration had different effects on mean arterial blood pressure (MABP) and heart rate. Using telemetry we observed that single injections of ISO (0, 0.5, 2, and 10 microg/g) were associated with hypotension and tachycardia with a duration but not a magnitude that was dose dependent. MABP dropped rapidly to 60 mm Hg for more than 2 h at the highest dose. Constant s.c. infusion of ISO at 20 microg/(g.d) initially lowered MABP to about 70 mm Hg for 24 h. At 48 h MABP was normal, but rose 10 mm Hg higher than baseline by day 5. Thus, different routes of administration of ISO that cause comparable increases in VW/BW had different effects on MABP. Thus when evaluating mouse models of ISO-induced cardiac hypertrophy, both repeated daily injections or infusions can cause similar increases in VW/BW, but the daily doses that are required are not the same. Furthermore, these different routes of administration have different hemodynamic sequelae and could potentially evoke different cardiac phenotypes.

Perinatal hypoxia causes ventricular enlargement associated with increased atrial natriuretic peptide (ANP) mRNA levels in newborn mice.

We sought to examine both the short-term and residual effects of perinatal hypoxia on ventricular mass and function of mice. We postulated that the magnitude of the ventricular hypertrophy would be determined by the timing of the exposure, be linked to augmented atrial natriuretic peptide (ANP) expression, and would persist to young adulthood. Furthermore, mice deficient in the ANP receptor type A (ANPRA) would have even greater hypertrophy. Newborns were placed in a 12% oxygen (O(2)) chamber either shortly after birth or at 8 days of age. Controls were raised in room air. After 8 or 16 days, pups were terminated and the right ventricle (RV) and left ventricle including the septum (LVS) were excised and weighed and total RNA was extracted. Hypoxia caused a reduction in body weight (BW) with an increase in right ventricle (RV) weight, rendering an increased RV to BW ratio and increased LVS/BW, albeit less. Hypertrophy was most pronounced in pups exposed to hypoxia in the first days of extrauterine life. A rapid postnatal decline in both RV and LVS ANP mRNA levels was observed in control animals, while the hypoxia elevated ANP mRNA. In mice missing the ANPRA, both ventricles were more massive than in wild type and hypoxia further augmented RV/BW and LVS/BW. In normal adult animals returned to room air after 16 days of hypoxia, RV but not LVS hypertrophy persisted in both sexes; there was an interaction between gender and the perinatal hypoxic stress on LVS dimension and perhaps on contractility. Thus perinatal hypoxia may "program" the adult mouse heart and vasculature.

Validation of the myocardial performance index by echocardiography in mice: a noninvasive measure of left ventricular function.

BACKGROUND: The myocardial performance index (MPI) is a Doppler-based measure of left ventricular (LV) function. It is noninvasive, independent of LV shape, and does not require dimensional measurements. However, it has never been validated in mice. METHODS: A total of 29 anesthetized mice with LV pressure catheters underwent echocardiography (2-dimensional, M-mode, and Doppler) at baseline and during manipulations of beta-adrenergic tone, temperature, preload, and afterload. The maximum derivative of LV pressure with respect to time (dP/dt(max)) was compared with MPI, fractional shortening (FS), mean velocity of circumferential fiber shortening, and the FS/MPI ratio. RESULTS: MPI (baseline 0.44 +/- 0.07) correlated strongly with dP/dt(max) (R = -.779, P <.001), as did FS and mean velocity of circumferential fiber shortening. MPI differed significantly with contractility, preload, and afterload manipulation. FS/MPI showed the best correlation with dP/dt(max). CONCLUSIONS: MPI strongly correlates with dP/dt(max) over a range of hemodynamic conditions in mice. It can be used as a noninvasive index of LV function in this species.

Fetal anemia leads to augmented contractile response to hypoxic stress in adulthood.

In response to chronic fetal anemia, coronary blood flow, maximal coronary conductance, and coronary reserve increase. We sought to determine whether chronic fetal anemia alters left ventricular (LV) function in adulthood. We studied adult sheep that had been made anemic for 20 days in utero by phlebotomy. They were transfused just before birth. At 7 mo of age, LV function was measured by pressure-volume loops at rest and during hypoxic stress. The in utero anemia group (n = 8) did not differ from controls (n = 5) with respect to hematocrit, heart and body weight, or baseline hemodynamic parameters. However, the effect of hypoxia (relative to baseline) on multiple indexes of systolic function was different between the two groups. End-systolic elastance increased in the in utero anemia group (baseline to hypoxia) by 4.15 +/- 3.47 mmHg/ml (mean +/- SD) but changed little in controls (0.24 +/- 0.45), which shows that the response to hypoxia was significantly different (P < 0.01) between groups. Similarly, the maximum derivative of LV pressure with respect to time increased in the in utero anemia group (486 +/- 340 mmHg/s,) but on average fell in the controls (-503 +/- 211 mmHg/s) with the response again being significantly different (P < 0.03). We conclude that in sheep, perinatal anemia can alter cardiac responses to hypoxic stress in the adult long after restoration of normocythemia.

Renal and placental secretion of erythropoietin during anemia or hypoxia in the ovine fetus.

OBJECTIVE: The source of the erythropoietin (EPO) that circulates in the fetus is unknown although it is known that EPO does not cross the placenta and that fetal kidneys, liver, and placenta express the EPO gene. This study tested to what extent in vivo EPO secretion by the fetal kidneys and placenta can be demonstrated under normoxic and hypoxic conditions. STUDY DESIGN: Renal arterial and venous EPO concentrations were determined in eight late-gestation chronically catheterized fetal sheep made progressively anemic by exchange transfusion with saline solution over 5 to 8 days. In a separate additional series of experiments, umbilical arterial and venous EPO concentrations were determined in nine normoxic fetuses and in nine fetuses subjected to 12 hours of hypoxia induced by lowering maternal-inspired oxygen content. Organ secretion rates were calculated as the product of plasma flow rate and the arteriovenous concentration differences. RESULTS: Renal vein plasma EPO concentration was higher than the arterial concentration in 36 of 40 paired samples (P<.0001) by 16.3%+/-2.7% (mean+/-SE). This difference was concentration independent over a range of 12 to 4100 mU/mL. Renal EPO secretion rates were variable and averaged 155+/-105 mU/min when hematocrit was 31.3%+/-1.6% (n=5) and 1124+/-300 mU/min post-exchange transfusion when hematocrit was 15.6%+/-0.8% (n=12). In contrast, umbilical venous and arterial EPO concentrations (range 9-35 mU/mL), although highly correlated (r=0.94), were not different during normoxia (Po(2)=21.6+/-0.5 mm Hg, n=9). Under hypoxic conditions (Po(2)=15.6+/-0.4 mm Hg, n=9), umbilical vein EPO concentration (range 151-1245 mU/mL) was higher than arterial concentration (range 140-951 mU/mL) in eight of nine paired samples by 13.6%+/-3.3% (P<.01). Under these conditions, estimated umbilical EPO secretion rate was 27,900+/-11,500 mU/min. CONCLUSION: Under nonanemic, normoxic basal conditions, the kidneys secreted EPO into the fetal circulation, whereas secretion by the placenta was not demonstrated. In the phlebotomy-induced fetal anemia experiments, the kidney demonstrated marked, progressive increases in the rate of EPO production. Similarly, in the fetal hypoxemia experiments, the placenta demonstrated progressive increases--albeit an order of magnitude greater than the kidneys--in EPO production rate. As an extension of these findings, we speculate that the hypoproliferative neonatal anemia that invariably occurs in the early weeks after birth is in part the result of loss of EPO production by the placenta.

The role of nerve cell density in the regulation of bud production in hydra.

The role of nerve cell density in the regulation of bud production in hydra was examined. Animals with different rates of bud production were produced by altering the temperature, population density and illumination of their cultures. When the distribution of cell types was examined in animals with different rates of bud production, the density of nerve cells in those animals was found to be correlated with their rate of bud production. Transfer of animals from one environment to another resulted in immediate changes in the rate of differentiation of large interstitial cells into nerve cells. This suggests that the density of nerve cells may play a role in regulating the rate of bud production in hydra.