J. Job Faber

Regulation of the Cardiomyocyte Population in the Developing Heart

During fetal life the myocardium expands through replication of cardiomyocytes. In sheep, cardiomyocytes begin the process of becoming terminally differentiated at about 100 gestation days out of 145 days term. In this final step of development, cardiomyocytes become binucleated and stop dividing. The number of cells at birth is important in determining the number of cardiomyocytes for life. Therefore, the regulation of cardiomyocyte growth in the womb is critical to long term disease outcome. Growth factors that stimulate proliferation of fetal cardiomyocytes include angiotensin II, cortisol and insulin-like growth factor-1. Increased ventricular wall stress leads to short term increases in proliferation but longer-term loss of cardiomyocyte generative capacity. Two normally circulating hormones have been identified that suppress proliferation: atrial natriuretic peptide (ANP) and tri-iodo-L-thyronine (T₃). Atrial natriuretic peptide signals through the NPRA receptor that serves as a guanylate cyclase and signals through cGMP. ANP powerfully suppresses mitotic activity in cardiomyocytes in the presence of angiotensin II in culture. Addition of a cGMP analog has the same effect as ANP. ANP suppresses both the extracellular receptor kinases and the phosphoinositol-3 kinase pathways. T₃ also suppresses increased mitotic activity of stimulated cardiomyocytes but does so by increasing the cell cycle suppressant, p21, and decreasing the cell cycle activator, cyclin D1.

Foetal placental blood flow in the lamb

1. Fifteen sheep foetuses of 1·5–5·2 kg body weight were prepared with indwelling arterial and venous catheters for experimentation one to six days later.

Extraplacental transfer of water in the sheep.

1. Ten pregnant ewes were operated on at 130 days of gestation. The fetal trachea was intubated with a double‐lumen tube, an inflatable occluder was placed around the umbilical cord, vascular catheters were placed in the fetal carotid artery and jugular vein and in the maternal jugular vein, and multiple catheters were placed in the amniotic and allantoic sacs. 2. At 139 days gestation, the fetus was ventilated in utero, and the umbilical cord was occluded. The extrafetal fluids were circulated by means of roller pumps. Known activities of radio‐iodinated human serum albumin, tritium‐labelled water and 14C‐labelled urea were injected into the amniotic and/or allantoic fluids. Samples were obtained at 30 min intervals for several hours. 3. Extrafetal fluid volumes were calculated from the albumin distribution volumes. The amounts of labelled water transferred to the maternal circulation were calculated from the changes in tracer concentrations in extrafetal fluids and fetal plasma. 4. No labelled albumin was detected in fetal or maternal plasma. The permeability‐surface area product of labelled water at the combined amniotic and allantoic interfaces with the ewe was 28.2 +/‐ 2.8 ml/min (mean +/‐ S.E.M.). In five preparations the values could be separately calculated for amniotic and allantoic interfaces. The two mean values (19 +/‐ 4 and 12 +/‐ 1 ml/min) were not significantly different from each other. The permeability‐surface area product at the combined interfaces with the fetus was 0.96 +/‐ 0.17 ml/min. Urea was so much less permeable than water that no reliable permeability‐surface area products could be calculated in all of the preparations. 5. We calculated that the hydraulic conductivity of the combined extraplacental pathway is more than 0.5% of that of the placenta. Because the osmotic gradient across the extraplacental pathway is one to two orders of magnitude greater than that across the placenta, extraplacental transfer of water can significantly affect intrauterine water volume.

Permeability of placenta to inulin

Inulin was administered to eight volunteer patients at term gestation over a period of 3 hours before cesarean section. Inulin concentrations were repeatedly measured in maternal plasma, in fetal plasma, and in amniotic fluid at the time of delivery. Total inulin uptake of the conceptus was taken to be the sum of the inulin in the amniotic fluid and in the newborn infant. Amniotic fluid volumes were measured by ultrasound examination, and the distribution volume of inulin in the neonate was assumed to be 180 ml/kg on the basis of animal experiments. The mean permeability was 0.15 microliter/(s.g) placenta. This value and the previously measured permeability for cyanocobalamin delimit a range of molecular weights from 1350 to 5200 daltons. In this range permeability to lipid-insoluble molecules is roughly proportional to the coefficients of free diffusion in water without further discrimination of molecular size by the placental barrier.

Regulatory response of intramembranous absorption of amniotic fluid to infusion of exogenous fluid in sheep

Six fetal sheep were operated on at 118 to 121 days of gestation. The pulmonary end of the trachea was connected to the gastric end of the esophagus with a section of tubing. This left urine as the only source of amniotic fluid and intramembranous absorption as sole exit. Multiple indwelling fetal vascular, intra-amniotic, allantoic, and a fetal bladder catheter were placed. Beginning 5 days after surgery, all urine was drained from the bladder and immediately reinfused into the amniotic sac to monitor urine production rate. After 4 days of urine infusion alone, the urine infusion was augmented for 6 days with an intra-amniotic infusion of Ringer solution. Amniotic and allantoic fluid volumes were measured at autopsy. During the period of Ringer infusion, intramembranous absorption of amniotic fluid increased by more than 1,191 +/- 186 (SE) ml/day (P < 0.002) and the rates of Na(+) and Cl(-) absorption increased to more than five times (P < 0.005) and eight times (P < 0.005) their initial values. Only one of six fetuses had polyhydramnios. It is concluded that intramembranous absorption of amniotic fluid makes a strong regulatory adjustment in response to an abnormal increase in inflow of exogenous fluid.Six fetal sheep were operated on at 118 to 121 days of gestation. The pulmonary end of the trachea was connected to the gastric end of the esophagus with a section of tubing. This left urine as the only source of amniotic fluid and intramembranous absorption as sole exit. Multiple indwelling fetal vascular, intra-amniotic, allantoic, and a fetal bladder catheter were placed. Beginning 5 days after surgery, all urine was drained from the bladder and immediately reinfused into the amniotic sac to monitor urine production rate. After 4 days of urine infusion alone, the urine infusion was augmented for 6 days with an intra-amniotic infusion of Ringer solution. Amniotic and allantoic fluid volumes were measured at autopsy. During the period of Ringer infusion, intramembranous absorption of amniotic fluid increased by more than 1,191 ± 186 (SE) ml/day ( P < 0.002) and the rates of Na+ and Cl- absorption increased to more than five times ( P < 0.005) and eight times ( P < 0.005) their initial values. Only one of six fetuses had polyhydramnios. It is concluded that intramembranous absorption of amniotic fluid makes a strong regulatory adjustment in response to an abnormal increase in inflow of exogenous fluid.

Filtration of water from mother to conceptus via paths independent of fetal placental circulation in sheep.

1. Four pregnant ewes were operated on at 121‐126 days of gestation. An electromagnetic flow sensor and an inflatable occluder were placed on the maternal common internal iliac artery. The ovarian arteries and veins were ligated. Indwelling catheters were placed in a maternal femoral artery and uterine vein and in the amniotic and allantoic fluids. An inflatable occluder was placed around the umbilical cord, close to the fetal abdomen. 2. Eight to nine days after surgery, the cord was occluded, the fetus killed and uterine blood flow reduced to one‐quarter of its control value. The rate of water loss from the uterine circulation was calculated from blood flow and the venoarterial difference in blood osmolality. The amniotic and allantoic fluids were made hypertonic by infusion of 2 l into each sac of a solution of 1.5 mol of mannitol per litre of saline. The rate of water loss from the maternal uterine circulation was then measured five times over the next 4.5 h. 3. The combined filtration coefficient surface area product of the interfaces between maternal blood and the amniotic and allantoic sacs, normalized per kilogram fetal body weight, was (2.8 +/‐ 0.5) x 10(‐6) cm3 s‐1 kPa‐1 kg‐1 (mean +/‐ S.E.M).

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.

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.

Review of flow limited transfer in the placenta

Summary Much of our view of the human placenta is inferred from animal experiments. Flow limited transfer is a regime of transfer in which there is complete equilibration of maternal and fetal bloods in a single pass through the placental exchange vessels. Placental exchange of non-protein bound lipid soluble materials is flow limited and the exchange of oxygen is very nearly flow limited. The relatively inefficient human placenta requires greater perfusion rates than some animal placentas do for the same rate of oxygen transfer but it offers considerable protection should one (but not both) of the placental blood flows decrease. The evolution of the placental vascular arrangement and of the difference in half saturation pressures of maternal and fetal hemoglobin point to the necessity to protect the fetus against high oxygen pressures. Wash-in and wash-out rates of non-protein bound anesthetics appear to be almost exclusively governed by the maternal and fetal placental flow rates and the distribution volume in the conceptus.

Angiotensin mediated interaction of fetal kidney and placenta in the control of fetal arterial pressure and its role in hydrops fetalis

Fetal cardiovascular control is effected by an interaction of the fetal somatic and placental circulations. Three primary regulatory mechanisms are involved: transplacental transfer of extracellular fluid, driven by a difference in hydrostatic and oncotic pressures; modulation of fetal placental and somatic vascular resistances by means of blood pressure controlled production of angiotensin; and somatic autoregulation of flow. A systems analysis incorporates these and other fetal cardiovascular functions and this analysis was modelled for computer simulation. Given physiologically plausible values for known cardiovascular parameters in the fetal sheep, the model reproduced in detail a variety of experimental protocols with known outcomes; these included the normal fetus, the fetus after bilateral nephrectomy, the nephrectomized fetus infused with angiotensin, the intact fetus infused with NaCl solutions, the fetus with lymphatic obstruction and the severely anaemic fetus. The systems analysis demonstrated that fetal cardiac failure constituted the strongest stimulus for the formation of fetal oedema of any tested pathological intervention.