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INTEGRATIVE |
1 Heart Research Center, and Departments of
2 Medicine (Cardiovascular Medicine)
3 Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239-3098, USA
4 Portland VA Medical Center, Portland, OR 97207-1034, USA
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Abstract |
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145 days). Cell size, maturational state (as measured by the proportion of binucleated myocytes) and cell cycle activity (as measured by positive staining of cells for Ki-67) were determined in dissociated cardiomyocytes. UPE fetuses were hypoxaemic, but mean arterial pressures were not different from controls. UPE fetuses were lighter than control fetuses (10 days: –21%, P < 0.05; 20 days: –27%, P < 0.01) and had smaller hearts, but heart weight was appropriate for body weight. Neither lengths nor widths were different between control and UPE cardiomyocytes at either age. Ten days of UPE did not significantly alter the proportion of binucleated myocytes or cell cycle activity in either ventricle. However, 20 days of UPE reduced cell cycle activity in both ventricles by 70% (P < 0.05); the proportion of binucleated myocytes was also lower in UPE fetuses at this age (left ventricle: 31.1 ± 12.0 versus 46.0 ± 6.6%, P < 0.05; right ventricle: 29.4 ± 12.3 versus 46.3 ± 5.3%, P < 0.05). It is concluded that in the absence of fetal arterial hypertension, placental insufficiency is associated with substantially depressed growth of the heart through suppressed proliferation and maturation of cardiomyocytes.
(Received 2 October 2006;
accepted after revision 18 January 2007;
first published online 18 January 2007)
Corresponding author S. Louey: Heart Research Center, L464, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098, USA. Email: loueys{at}ohsu.edu
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Introduction |
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It is generally believed that cardiomyocytes lose their proliferative capacity during the perinatal period (Zak, 1973; Oparil et al. 1984) and have little capacity to generate new cardiomyocytes after birth. The extent to which aged cardiomyocytes replicate is a topic of current debate (Anversa et al. 2006; Murry et al. 2006). In sheep, cardiomyocyte proliferation accounts for most of the growth of the heart until mid-late gestation after which proliferating cells undergo terminal differentiation. In sheep and rodents, terminal differentiation is characterized by karyokinesis without cytokinesis resulting in a binucleated cell that can no longer divide. After terminal differentiation the heart grows predominantly via cardiomyocyte hypertrophy (Smolich et al. 1989; Lumbers et al. 2005). Rat and mouse cardiomyocytes exhibit similar changes in development. However, in these rodents, the timing of myocyte terminal differentiation occurs in the postnatal, rather than the prenatal, period (Clubb & Bishop, 1984; Soonpaa et al. 1996). Deviations from the normal programme of proliferation and maturation could have lasting effects in the adult.
There are two primary models for placental embolization that have made important contributions to our knowledge of fetal adaptations to placental insufficiency. (1) A severe placental embolization that reduces fetal oxygenation by 40–50% (from 0.82 to 0.95 gestation or until birth) leads to a mild transient fetal arterial hypertension, but does not alter heart weight relative to body weight at 0.95 gestation (Cock et al. 2001b), at 8 postnatal weeks (Louey et al. 2000) or at 2.3 years after birth (Louey et al. 2005). (2) A model with the same reduction in fetal arterial oxygenation but earlier in gestation (from 0.74 to 0.88 gestation) leads to persistent fetal arterial hypertension (+7 mmHg), and a 27% increase in heart weight (relative to body weight) (Murotsuki et al. 1997). Because it is well known that fetal cardiac myocyte maturation is sensitive to sustained loading conditions (Barbera et al. 2000), these models indicate that placental insufficiency either may or may not lead to sustained increases in cardiac load, depending upon the gestational timing and other unknown factors. Thus, it is likely that the response of the fetal cardiomyocyte to placental insufficiency depends upon the accompanying haemodynamic load.
There remains a significant gap in our knowledge regarding the effects of placental insufficiency on cardiomyocyte growth and maturation. A paper that appeared during the review process of this study sheds new light on the cardiomyocyte responses to embolization and shows that cardiomyocyte maturation is depressed in response to embolization when hypertension is not present (Bubb et al. 2007). However, the aim of the present study was to determine the effects of placental insufficiency on cardiomyocyte growth and maturation in the last half of gestation without sustained fetal hypertension, as it related to two hormones that are known to regulate cardiac growth, angiotensin II (Sundgren et al. 2003) and cortisol (Giraud et al. 2006). We have previously shown that angiotensin II stimulates cardiomyocyte proliferation in vitro (Sundgren et al. 2003), and it has been reported that plasma renin activity is transiently increased in UPE lambs shortly after birth (Louey et al. 2000); plasma renin activity has not previously been reported in UPE fetuses. We have recently shown that infusions of subpressor doses of cortisol increase cardiomyocyte cell cycle activity in near-term fetal sheep (Giraud et al. 2006). Nevertheless, we hypothesized that cardiac myocytes from UPE fetuses would have lower proliferation rates and fewer would be terminally differentiated following either 10 or 20 days of UPE. Despite increased circulating cortisol levels in UPE fetuses (Cock et al. 2001b), we expected that the negative growth effects associated with conditions of limited oxygen and nutrient supply would override any stimulation of cardiomyocyte proliferation by increased cortisol.
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Methods |
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Time-bred mixed Western breed ewes carrying twins were purchased from a local supplier and brought to the laboratory several days before surgery for acclimatization. All procedures followed guidelines established by the Department of Comparative Medicine (Oregon Health and Sciences University) for the care and use of sheep and protocols were approved by the Institutional Animal Care and Use Committee. A total of 12 ewes underwent surgery; in 10 of the ewes, both fetuses were included for study, and in the remaining two ewes, only one fetus was included in the study. Ewes were fasted for 24 h prior to surgery, with free access to water. Anaesthesia was induced with an intravenous combination of diazepam (10 mg) and ketamine (400 mg). Ewes were then intubated and anaesthesia was maintained with 1–2% isoflurane in a 70:30 mixture of oxygen and nitrous oxide while mechanically ventilated. Sterile surgery was performed at 111 ± 1 days gestational age (d GA, term 145d GA), as previously described (Cock & Harding, 1997; Louey et al. 2000). In brief, a midline incision was made in the ewe's abdomen to expose the uterus. The fetal rump was exteriorized through a uterine incision. A catheter (1.8 mm o.d. polyvinyl; Scientific Commodities, V-8, Lake Havasu City, AZ, USA) was inserted 7 cm into the fetal femoral artery of each leg for blood sampling, haemodynamic measurements and for injecting microspheres. The tip of this catheter was placed in the abdominal aorta at a level 1–2 cm below the level of the renal arteries, and above the level of the umbilical arteries; catheter position was confirmed at autopsy. A catheter (1.8 mm o.d.) was inserted into one fetal femoral vein for the measurement of venous pressure. Before returning the fetus to the uterus, a catheter (1.8 mm o.d.) was sutured to the rump for the measurement of amniotic sac pressure. The uterus was sutured securely to minimize fluid loss. Following instrumentation of the first fetus, its twin was exposed through a second uterine incision and catheterized in the same manner. Penicillin G (1 000 000 units; Bristol-Myers Squibb, Princeton, NJ, USA) was instilled into the amniotic space of each fetus via the amniotic sac catheter before all catheters were flushed with heparinized saline. The free ends of the catheters were filled with heparinized saline and tied off, exteriorized through a small incision in the flank of the ewe, and secured to a pouch sutured to the flank. The midline incision was closed in anatomic layers. At the conclusion of the surgery, isoflurane administration was stopped and the endotracheal tube was removed once the ewe was able breathe spontaneously. Ewes were returned to a clean pen with free access to food and water for recovery. Ewes received 0.6 mg bupremorphine (S.C.) immediately following surgery and for the 2 days following surgery (twice daily) to minimize pain and distress. Animals were allowed a minimum of 3 days recovery before experiments began.
Experimental groups
Four groups of fetuses were studied: fetuses that were subjected to 10 ± 1 days of UPE (n = 5; 3 females, 2 males) and their age-matched controls (n = 6; 1 female, 5 males; this group included one uninstrumented control), and fetuses that were subjected to 20 ± 0 days of UPE (n = 5; 3 females, 2 males) and their age-matched controls (n = 6; 4 females, 2 males; this group included one uninstrumented control). Studies for all fetuses commenced at 115 ± 1d GA; those in the 10 day study group were euthanized at 125 ± 1d GA and those in the 20 day study group were euthanized at 136 ± 1d GA. Fetuses were randomly assigned to each experimental group. Myocytes from fetuses at two different periods were studied to provide insight into the temporal effects of UPE on myocyte development.
Experimental protocol
Laboratory procedure. For the duration of experiments, ewes were housed in individual stanchions, with free access to food and water. An arterial blood sample was taken daily from each fetus to assess pH, blood gas status (partial pressure of carbon dioxide (Pa,CO2) and oxygen (Pa,O2), oxygen saturation (Sa,O2) and oxygen content (O2ct)) and haemoglobin (tHb) (Instrumentation Laboratories blood gas analyser, Model 1306, and Instrumentation Laboratories Co-oximeter, Model 382; Lexington, MA; values corrected to 39°C); haematocrit (Hct) was determined from samples centrifuged in microcapillary tubes. For fetuses in the UPE groups, 20–50 µm (mean ± S.D. diameter, 35 ± 9 µm) non-radioactive non-soluble microspheres (Sephadex Superfine G-25; Amersham, Sweden; 1% w/v suspended in heparinized saline with 0.02% Tween 80) were injected into the fetal femoral artery catheter to reduce Pa,O2 by approximately 8 mmHg from baseline values on study day 1 (Cock & Harding, 1997; Louey et al. 2000). UPE was performed daily as needed, as determined by an arterial blood gas sample taken each morning. If the fetus was sufficiently hypoxaemic, no UPE was performed that day. If the fetal oxygenation was higher than the target range, microspheres were injected until the level of desaturation was achieved; the number of microspheres injected was dependent on the Pa,O2 of the most recent blood gas sample. Haemodynamic measurements were made on study days 1, 4, 7, 10, 13, 16, 19 and 21. Vascular pressures (arterial and venous pressures, adjusted for amniotic sac pressures) and heart rates were measured in fetuses for a minimum of 30 min prior to UPE, during UPE and at least 2 h after fetuses had reached the target level of hypoxaemia. Pressures were measured using Transpac IV transducers (Hospira, IL, USA), calibrated against a mercury manometer, and recorded with a digital data acquisition system (Powerlab, ADInstruments, CO, USA); heart rate was digitally derived from the arterial pressure recording.
Assays. Plasma samples were collected on study days 1, 4, 7, 10, 13, 16, 19 and 21 prior to UPE for that day. Cortisol concentrations in heparinized plasma samples were measured by radioimmunoassay (Diagnostic Products Corporation, CA, USA). Plasma renin activity was determined in EDTA-treated plasma samples by the generation of angiotensin I, which was measured by radioimmunoassay (DiaSorin, MN, USA) (Faber et al. 2006).
Tissue Collection. After either 10 days or 20 days of UPE, ewes were killed with an intravenous overdose of sodium pentobarbital (Euthasol; Virbac, TX, USA). The deeply anaesthetized fetuses were exposed and 10 000 units heparin was given to the umbilical vein, followed by 10 ml of saturated KCl to euthanize the fetus and arrest the heart in diastole. The fetus was then removed from the uterus and weighed. The heart was excised, trimmed in a standardized fashion and weighed; in addition, brain and liver weights were noted.
Dissociation of cardiomyocytes. Myocytes were dissociated by retrograde perfusion of the aorta, using established protocols in the laboratory (Giraud et al. 2006). The heart was perfused with a low-Ca2+ Tyrode buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2.6H2O, 10 mM glucose, 10 mM Hepes, no Ca2+ added; pH adjusted to 7.35 with NaOH) for 5–10 min, until the tissue was blanched. This was followed by perfusion with 160 U ml–1 Type II collagenase (Worthington Biochemicals, Lakewood, NJ, USA) and 0.78 U ml–1 Type XIV protease (Sigma) in Tyrode buffer until the heart was adequately digested (5–10 min); the collagenase/protease mixture was washed out by a minimum of 300 ml of a high-K+ buffer (74 mM glutamic acid, 30 mM KCl, 30 mM KH2PO4, 20 mM taurine, 3 mM MgSO4, 0.5 mM EGTA, 10 mM Hepes, 10 mM glucose; pH adjusted to pH 7.37 using KOH). All perfusates were prewarmed (39°C), and gassed with a mixture of 95% O2 and 5% CO2. Following perfusion, the left and right ventricular free walls were dissected from the heart and gently agitated in separate tubes containing high-K+ buffer to liberate the myocytes. The resulting cell slurry was rested at room temperature for 30–45 min before fixation in a solution of 1% paraformaldehyde.
Measurements of myocyte size and nucleation. Myocyte length and width were measured in isolated wet-mounted myocytes (lightly stained with 0.1% methylene blue in H2O), using a calibrated image analysis program (Optimas, WA, USA) at x400 magnification (Zeiss Axiophot; Bartels and Stout, WA, USA). These measurements were made on 50 mononucleated and 50 binucleated myocytes from each ventricle of each fetus. Independent of the cell size measurements, the percentage of myocytes that were binucleated (used as an indicator of the proportion of terminally differentiated myocytes) was determined in a sample of 300 myocytes from each ventricle from each fetus.
Cell cycle activity.
Cell cycle activity was determined by staining the dissociated myocytes for Ki-67, a nuclear antigen expressed in all stages of the cell cycle except G0. Myocytes were dried onto SuperFrost slides at a density of 600 cells cm–2 and then postfixed to the slides with acetone (30 min, at 4°C). Cells were permeabilized in sodium citrate (0.01 M, pH 6.0, 85°C) for 6 min. Endogenous peroxidase activity was blocked with 0.03% hydrogen peroxide for 5 min, and non-specific staining was blocked with blocking buffer (1% w/v bovine serum albumin and 0.5% v/v Triton X-100 in PBS). Cells were incubated with the Ki-67 antibody (1:200 in blocking buffer, mouse Ki-67, Clone MIB-1, DakoCytomation, CA, USA) overnight at 4°C. Slides were then incubated with biotinylated secondary antibody (1:200 in PBS, Vectastain ABC Kit, Mouse IgG; VectorLabs, CA, USA) for 1–2 h at room temperature, followed by incubation with the avidin/biotinylated enzyme complex (1:1:100 in PBS, Vectastain ABC Kit) for 1–2 h at room temperature. Ki-67-positive nuclei were visualized with 3,3'-diaminobenzidine chromagen (DAB) in substrate buffer (DakoCytomation) for 2–10 min and then cells were counterstained with 0.1% methylene blue. All incubation steps were followed by three PBS washes. The number of Ki-67-positive myocytes was counted in a sample of 500 myocytes; results are expressed as the percentage of Ki-67-positive mononucleated myocytes/total number of mononucleated myocytes.
Statistical analysis
Experimental data are expressed as means ± standard deviation (S.D.). Arterial blood parameters (pH, blood gas status, tHb, Hct, blood pressures, heart rate, plasma cortisol concentrations, plasma renin activity) in the 10 day and 20 day groups were analysed separately by two-way analysis of variance (ANOVA); significant differences were subjected to the Bonferroni post hoc test to determine significant differences between control and UPE groups. Body weight, organ weight and isolated cell data (sizes, Ki-67 staining, binucleation) were analysed by one-way ANOVA to compare all four groups; significant differences were further analysed by the Tukey Multiple Comparison Test. All analyses were performed using GraphPad Prism (Version 4.0a for Macintosh). A value of P < 0.05 was considered statistically significant.
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Results |
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UPE induced similar changes in fetal arterial pH, blood gases, tHb and Hct in the 10 and 20 day groups (Table 1) and similar to previous reports (Cock & Harding, 1997; Murotsuki et al. 1997; Louey et al. 2000; Gagnon et al. 2002). Compared with their age-matched controls, UPE fetuses were hypoxaemic, but were not hypercapnic. Fetuses in the 10 day UPE group were acidaemic at the end of the study period, while fetuses in the 20 day UPE group were not. In both UPE groups, tHb and Hct were not different from control values after 10 days of study but were significantly higher than control values after 20 days of UPE. Baseline haemodynamic measurements (made prior to daily injection of spheres) were not different between control and UPE groups at either age. Figure 1 shows Pa,O2, Sa,O2 and haemodynamic data in the 20 day control and 20 day UPE fetuses on study days 1, 4, 7, 10, 13, 16, 19 and 21 for the periods prior to daily UPE (baseline). Samples were taken 15 min after the final injection of microspheres for the day, and 2 h after the final injection of microspheres. Once the fetuses were sufficiently hypoxaemic, they remained hypoxaemic for at least 2 h after the final injection of microspheres. Although mean arterial pressure (MAP) tended to increase in response to UPE early in the study, fetuses did not remain chronically hypertensive. Circulating cortisol levels increased significantly in the 20 day group but only just prior to the end of the experimental period (Fig. 2, Table 1). Plasma renin activity was not significantly different between the two groups, except on experimental day 19 when it was significantly higher in the UPE fetuses (Fig. 2, Table 1).
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Fetal body weight was lower in both UPE groups compared with their age-matched controls; body weight was 21% lower (P < 0.05) in the 10 day UPE group and 27% lower (P < 0.01) in the 20 day UPE group (Table 2). Although there was a significant difference in body weight between the 10 day and 20 day control groups (36%, P < 0.01), body growth was slowed in the UPE group such that the body weight of fetuses in the 20 day UPE group (at 136d GA) was not significantly different to that the 10 day control group (at 125d GA). Heart weight (grams) was decreased in the UPE groups compared with controls. However, heart weight/body weight was not changed in any of the groups studied. Decreased liver weights and the relative sparing of brain growth in the 20 day UPE group compared with their age-matched controls were associated with an increased brain:liver ratio, indicative of asymmetrical growth of the body and its organs; this asymmetrical growth pattern was not apparent in the 10 day UPE group.
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Myocyte development was abnormal in the UPE groups, even though the decrease in heart weight was proportional to the decrease in body weight. The hearts of the UPE fetuses did not contain smaller myocytes; mean lengths and widths of the isolated cardiomyocytes were not different between control and UPE groups at either age (Table 3). However, the cell cycle activity of UPE cardiomyocytes from both ventricles was suppressed compared with controls (Fig. 3). After 20 days of UPE, there was a significantly lower proportion of mononucleated myocytes that were positively stained for Ki-67 compared with control cells (left ventricle: UPE 1.7 ± 1.3 versus control 6.1 ± 4.2%, P < 0.05; right ventricle: UPE 2.6 ± 2.5 versus control 7.9 ± 3.2%, P < 0.05); a similar trend was found in the 10 day UPE group; however, this was not statistically significant (left ventricle: UPE 5.6 ± 1.3 versus control 8.2 ± 4.8%; right ventricle: UPE 6.5 ± 3.1 versus control 10.0 ± 5.1%).
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Discussion |
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We reduced placental gas exchange to reach a similar level of hypoxaemia as in previous reports (Cock & Harding, 1997; Murotsuki et al. 1997; Louey et al. 2000; Gagnon et al. 2002). The reduction in fetal body weight and the effects on liver and brain growth were similar to those previously reported. Asymmetrical growth restriction typically occurs by the redistribution of blood flow during hypoxaemia to favour vital organs such as the brain at the expense of less vital organs such as the liver. This ‘brain-sparing’ pattern of growth has previously been described following 20 days of UPE (Murotsuki et al. 1997; Cock et al. 2001b). In our study, we found that this asymmetric pattern of growth restriction was evident by 20 days, but not 10 days, of UPE. Although the severity of fetal hypoxaemia was less than that in our study, one group reported asymmetrical growth after 10 days of UPE (Block et al. 1990) but another did not (Gagnon et al. 1996). Thus, it appears that a longer period of UPE rather than the severity of the hypoxaemia amplifies the abnormal organ growth patterns.
The effects of UPE on arterial pressure has differed between studies, and it is generally believed that these differences are due to the severity, duration and gestational timing of the insult (Gagnon, 2003; Louey et al. 2003). It was our intention to study the effects of placental insufficiency without sustained arterial hypertension, given that fetal arterial hypertension is well known to alter cardiomyocyte growth and maturation patterns. The haemodynamic data from our study most closely match those from the Harding laboratory (Cock & Harding, 1997; Louey et al. 2000), with transiently increased arterial pressure early in the study, but without significant changes in vascular pressures over the course of the study period. The immediate increases in arterial pressure that occur with the administration of microspheres, and the transient increase in pressure during the first 1 week of UPE may have contributed to the alterations in cardiomyocyte development in some unknown way. From previous studies, we know that sustained increases in afterload are associated with increased proliferation and terminal differentiation of cardiomyocytes. In our present study, both of these cellular processes were suppressed so that the impact of the transient increase in arterial pressure was not detectable.
We found no difference in the length or width of myocytes between UPE and control groups at either age under our protocol conditions. However, it should be noted that myocyte length and width do not increase greatly over the gestational period covered by this study.
Given that the absolute heart weight (in grams) was decreased in the UPE fetuses without a decrease in cardiomyocyte size, it appears that the smaller hearts contained fewer working myocytes. Accordingly, we found that the cardiomyocytes of the 20 days UPE (but not 10 days) fetuses had significantly reduced cell-cycle activity as determined by staining for Ki-67. We also found that the 20 day UPE group had a significantly lower proportion of terminally differentiated binucleated myocytes than controls at 136d GA; further, the percentage of binucleated myocytes present in the 20 day UPE group was the same as that of the controls 10 days earlier, suggesting that the rate of maturation of the cardiomyocytes was virtually stopped. Thus, decreases in cell cycle activity appear to be due to both decreased cell proliferation and terminal differentiation.
A trend for decreased cell cycle activity was evident at 125d GA, after 10 days of UPE (Fig. 3). An equal decrease in proliferation and terminal differentiation rates would be consistent with decreased cell cycle activity without a change to the relative percentage of binucleated myocytes in the myocardium. Fewer myocytes proliferating during the first 10 days of UPE would result in fewer mononucleated myocytes available for both proliferation and terminal differentiation during the latter 10 day phase of the experimental period. Thus the findings would explain the further decrease in cell cycle activity and greater discrepancy in the proportion of terminally differentiated myocytes between control and UPE fetuses seen in the 20 day group.
Reduced myocyte proliferation would be associated with lower DNA synthesis, as would a reduction in terminal differentiation. Our findings are consistent with a reduction in DNA synthesis rates as reported by Gagnon et al. (1995). The results from the present study suggest the presence of fewer myocytes in the myocardium of UPE fetuses; however, the number of myocytes in these hearts was not measured. Further, the contribution of apoptosis to myocyte number in this model was not determined. Thus it is possible that hypoxia increased apoptosis rates as it does in fetal rats (Bae et al. 2003). It is also not clear whether UPE altered the non-myocyte fraction of cells in the myocardium, of which myocytes only contribute to 75% of cells in the heart (Smolich et al. 1989; Barbera et al. 2000).
Few studies have reported the effects of human intrauterine growth restriction (IUGR) on cardiomyocyte development. It has been suggested by Mayhew and colleagues that myocyte maturation is delayed in IUGR fetuses with a degree of ‘catch-up’ late in gestation (Mayhew et al. 1999). However, the aetiologies of IUGR in that study were varied and the availability of tissue was understandably low. In IUGR offspring of rats fed low-protein diets (LPD) during pregnancy, heart weight/body weight of offspring was not different from controls at birth, but nevertheless, there are fewer cardiomyocyte nuclei in the hearts exposed to the maternal low-protein diet (Corstius et al. 2005). Like the sheep, terminal differentiation in rodents is associated with cardiomyocytes becoming binucleated, but unlike the sheep, this process occurs after birth (Korecky & Rakusan, 1978; Clubb & Bishop, 1984; Oparil et al. 1984; Soonpaa et al. 1996). Thus, in the low-protein rat study (Corstius et al. 2005) the proportion of binucleated myocytes was low (2–3%) in both control and LPD groups and the authors concluded that the suboptimal prenatal environment led to reduced proliferation leading to fewer cardiomyocytes. This would be consistent with the findings of the present study. Whether the hearts of UPE sheep fetuses and LPD rat pups are able to exhibit ‘catch-up’ in myocyte number in the postnatal period remains to be determined.
The terminal differentiation process occurs mostly before birth in the sheep (Smolich et al. 1989; Burrell et al. 2003). The signals for cardiomyocytes to terminally differentiate and permanently withdraw from the cell cycle are poorly understood. It is not known whether there is a predetermined number of times a myocyte must replicate before becoming terminally differentiated, or if there is a preset period of time in which a myocyte replicates before withdrawal from the cell cycle (Burton et al. 1999). The former could result in delayed terminal differentiation that might not affect the final number of myocytes in the heart, but in the latter scenario the final number of myocytes in the heart could be lower than expected.
In addition to haemodynamic influences, prenatal myocardial growth can also be modified by local and circulating factors, many of which may be altered by UPE. There is a subset of UPE fetuses in which the hypothalamo–pituitary–adrenal axis is prematurely activated, leading to birth 1 week early (Cock et al. 2001a) but some UPE fetuses do not have different cortisol levels to controls (Louey et al. 2000). In the current study, cortisol levels were not altered during the first 10 days of UPE, but were prematurely elevated toward the end of the UPE period. We have recently shown that subpressor doses of cortisol increase cell cycle activity in cardiomyocytes and lead to increases in cardiac mass (Giraud et al. 2006); in contrast, pressor doses of cortisol cause myocyte hypertrophy without a change in myocyte number (Lumbers et al. 2005). The elevated cortisol levels in the 20 day UPE fetuses did not reverse the depressed rates of proliferation of cardiomyocytes, despite similar circulating levels in UPE and cortisol-infused fetuses (Giraud et al. 2006). Further, plasma renin activity was not greatly altered by UPE over most of the 20 day period, although there was a trend for increased activities towards the end of the study; this is consistent with transiently elevated renin activity in the neonatal UPE lamb (Louey et al. 2000). While angiotensin II can stimulate proliferation in fetal sheep cardiomyocytes (Sundgren et al. 2003), the increases in plasma renin activity that were found in UPE fetuses did not reach levels that would be expected to alter myocyte growth.
Conclusions
Placental insufficiency, in the absence of persistent fetal arterial hypertension, decreases cell cycle activity and the rate of terminal differentiation in cardiac myocytes. These findings are indicative of a less mature myocardium, with potentially fewer myocytes in the heart.
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) and UPE (
) fetuses in the 20 day study group. Data are means ±
S.D., *P < 0.05, **P < 0.01 versus age-matched control; n
= 5 in all groups, except n
= 4 for UPE cortisol values.
