RENAL-CARDIAC-VASCULAR
Cardiac Natriuretic Peptide Gene Expression and Plasma Concentrations During the First 72 Hours of Life in Piglets Julie Smith, Christina Christoffersen, Linn M. Nørgaard, Lisbeth H. Olsen, Niels G. Vejlstrup, Claus B. Andersen, and Jens P. Goetze Departments of Clinical Biochemistry (J.S., C.C., J.P.G.), Cardiology (N.G.V.), and Pathology (C.B.A.), Copenhagen University Hospital (Rigshospitalet), DK-2100, Copenhagen, Denmark; and Departments of Large Animal Sciences (L.M.N.) and Veterinary Disease Biology (L.H.O.), University of Copenhagen, DK-1870, Frederiksberg, Denmark
Plasma measurement of cardiac natriuretic peptides constitutes promising markers of congenital heart disease. However, concentrations change rapidly and dramatically during the first days after delivery even in healthy neonates, which complicates clinical interpretation. It is unknown whether these changes in plasma concentrations are explained by corresponding changes in the cardiac gene expression. We quantified the chamber-specific mRNA levels of ANP (A-type natriuretic peptide) and BNP (B-type natriuretic peptide) and plasma pro-ANP and BNP-32 concentrations in healthy piglets during the first 72 hours of life (from 2 litters, n ⫽ 44). Chamber-specific ANP and BNP mRNA levels reflected hemodynamic neonate changes at birth but did not correlate with circulating natriuretic peptide concentrations. However, plasma pro-ANP and creatinine concentrations were closely correlated (P ⬍ .0001; r ⫽ 0.73). Plasma pro-ANP levels were highest on the day of delivery (5580 pmol/L [4320 – 6786] decreasing to 2484 pmol/L [1602–2898] after 72 hours, P ⬍ .0001). During the 72 hours, gel chromatography suggested that the translational products in circulation and in atrial tissue were immature, ie, unprocessed pro-ANP. In contrast to pro-ANP, BNP-32 plasma concentrations were low at delivery and peaked after 48 hours (12 [10.5–20.6] vs. 88.8 [71.7–101.4] pmol/L, P ⬍ .0001). To conclude, ANP and BNP gene expression differs considerably between cardiac chambers in the first 72 hours of life in healthy piglets, resembling the transition from fetal to neonate circulation. However, the cardiac gene expression does not explain plasma concentrations. (Endocrinology 154: 1864 –1872, 2013)
ardiac natriuretic hormones and their molecular precursors are well-established plasma markers of ventricular dysfunction and congestive heart failure (CHF) (1, 2). A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are normally expressed in the cardiac atria and rapidly released upon hemodynamic and neurohumoral stimuli (3). The peptide products circulate in unprocessed and processed forms, where the processed bioactive hormones are integrally involved in fluid homeostasis via renal sodium excretion and endothelial permeability. In heart failure, however, the molecular pattern
C
shifts toward less processed forms including the intact prohormones with little or no bioactivity (4). Consequently, the propeptides and the N-terminal fragments are valuable laboratory markers but without endogenous alleviation of the congestive phenotype. In parallel with adult heart failure patients, cardiac natriuretic peptides also circulate in high concentrations in healthy neonates, which have been suggested to reflect the sudden increase in left cardiac chamber workload after birth (5). The neonate peptide heterogeneity may constitute unprocessed forms with no bioactivity as in adult
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received December 4, 2012. Accepted February 20, 2013. First Published Online March 28, 2013
Abbreviations: ANP, A-type natriuretic peptide; BNP, B-type natriuretic peptide; CV, coefficient of variation; 2D, 2-dimensional; LA, left atrium; LV, left ventricle; PDA, patent ductus arteriosus; RA, right atrium; RV, right ventricle.
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heart failure patients (6). Plasma measurement after birth has been suggested to provide both diagnostic and prognostic information in cardiac dysfunction and congenital heart disease, particularly regarding patent ductus arteriosus (PDA) in preterm neonates, in whom PDA is associated with heart failure, bronchopulmonary dysplasia, intracranial hemorrhage, necrotizing enterocolitis, and renal failure. Prophylactic treatment has side effects and echocardiography cannot always predict a hemodynamic significant PDA. For instance, a large shunt does not always result in clinical symptoms and can even close spontaneously. Thus, there is a need for additional risk assessment and natriuretic peptide measurement has been proposed to assist echocardiography and clinical examination in predicting treatment strategies (5, 7–10). Clinical interpretation of peptide measurement in neonatal plasma is, however, complicated by the rapid changes in concentrations over the first few days of life. Moreover, there is still little information available on what these high peptide concentrations relate to and whether the dramatic changes in the biochemical phenotype primarily reflect changes in cardiac secretion due to altered local hemodynamics after birth and/or a change from a fetal to an adult elimination pattern. In the present study, we first established a porcine model for healthy neonates to elucidate chamber-specific ANP and BNP gene transcriptional expression during the first 72 hours of life and possible associations with the peptide concentrations in plasma and in cardiac tissue. In parallel with our earlier findings for pro-BNP (6), ANP translational peptide products consisted mostly of immature prohormone in both cardiac tissue and plasma from our healthy piglets. Our data suggest that cardiac tissue expression of BNP and ANP reflects the transition from fetal to postnatal circulation with a rapid decline in gene expression in the right ventricle (RV) and increase in left cardiac chambers. However, ANP and BNP transcriptional contents in the chambers cannot explain the plasma pro-ANP and BNP-32 concentrations, where in particular pro-ANP mostly reflects creatinine concentrations, a surrogate marker of renal function.
Materials and Methods Porcine neonatal model From a specific pathogen-free Danish pig farm, 2 pregnant Yorkshire/Danish Landrace sows crossed with Duroc (para III) were delivered to the University of Copenhagen. On gestation day 115 (term), the sows were sedated with midazolam (0.5 mg/kg) combined with im butorphanol (0.1 mg/kg) and iv propofol (5 mg/kg)-induced anesthesia. Anesthesia was maintained by isoflurane with minimum alveolar concentration of 1% during
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cesarean section procedures. A blood sample was taken, and the left flank was prepared for surgery, which included intracutane and im infiltration analgesia with 100 mL lidocaine mixed with noradrenaline. After uterine incision, the sows were euthanized with a lethal iv dose of pentobarbital (150 mg/kg). After delivery of the piglets, their respiration was stimulated with a drop of Dopram (20 mg/mL) placed sublingually. The piglets were placed in separate prewarmed incubators and supplied with oxygen (2–3 L/min) during the first 2 hours of life. They were allocated into 4 groups by systematic uniformly random sampling. The 4 groups varied by time of euthanasia, which was approximately 1 to 3, 24, 48, and 72 hours after delivery, and these groups will be referred to as day 0, 1, 2, and 3, respectively. Piglets not euthanized on day 0 were fitted with an orogastric feeding tube, and if previous maternal anesthesia no longer sedated the piglets, alfaxalone (0.2 mL/kg) was administered im. Two hours after delivery, the first feeding session was performed with 3 mL/kg bovine colostrum administered every third hour and raised by 3 mL/kg at every feeding session until a maximum of 15 mL/kg until euthanasia. To prevent infection, gentamicin (5 mg/kg) was given the first day together with amoxicillin (20 mg/kg); the latter was continued once daily until euthanasia. Weight, rectal temperature, and activity level were recorded at baseline and every 12th hour until euthanasia. The arterial duct was evaluated at baseline and every 12th hour by 2-dimensional (2D) echocardiography during sedation with alfaxalone (0.3– 0.35 mL/kg). Before euthanasia, the piglets were anesthetized using a zoletil mixture (0.1 mL/kg) by im injection (11). Two minutes after injection, echocardiography was completed and 10 mL of blood was collected by cardiac puncture followed by a lethal injection of 5 mL pentobarbital (200 mg/mL). All animals received humane care, and the Animal Experiments Inspectorate, Ministry of Justice, Denmark, approved all animal procedures in the research project, which complied with the institution’s guidelines.
Plasma and cardiac tissue sampling Only at euthanasia were blood samples taken. Blood was transferred into EDTA tubes and placed on ice. All samples were centrifuged for 10 minutes at 2500g at 4°C within a half hour after collection. The plasma was stored at ⫺80°C for later analyses. Transmural biopsies of right and left atrial auricles, representing left atrium (LA) and right atrium (RA), and RV and left ventricle (LV) free wall were excised immediately after euthanasia and placed in liquid nitrogen before transfer to ⫺80°C.
Peptide measurement in plasma and cardiac tissue For plasma measurements, all piglets were included, whereas only piglets from days 0 and 3 were used for peptide tissue measurement. Frozen heart tissue was crushed using a CryoPrep (Covaris, Woburn, Massachusetts) tissue extractor, boiled in water for 20 minutes, and homogenized with an Ultra-Turrax (IKA, Staufen, Germany). After 30 minutes centrifugation at 13 000g, the supernatant was treated with trypsin (Worthington, Lakewood, New Jersey) for 30 minutes at room temperature and boiled for 10 minutes. We recently reported on this enzymatic principal elsewhere (12). The pro-ANP concentrations in tissue and in plasma were then quantitated with a porcine-specific immunoassay (13) with an intra-assay coefficient of variation (CV) ⬍15% for plasma measurements. The BNP-32 concentrations in
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plasma were measured with a commercial radioimmunoassay (Phoenix Pharmaceuticals, Karlsruhe, Germany). Intra-assay CV was ⬍15% for BNP-32 concentrations ⬎30 pmol/L.
Gel chromatography The molecular heterogeneity of pro-ANP in plasma and in cardiac atrial tissue from days 0 and 3 was examined by gel chromatography. Extracts were applied to a Sephadex G-50 Superfine column (1000 ⫻ 10 mm; Pharmacia, Uppsala, Sweden) and eluted at 4°C with a barbital buffer, pH 8.4) supplemented with 0.2M NaCl (flow rate, 4 mL/h). Void and total volumes were determined by eluting 125I-labeled albumin and 22Na-labeled NaCl, respectively.
Quantification of mRNA in cardiac tissue In total, 24 piglets were selected for ANP and BNP mRNA analyses (6 piglets from each day, 3 from each sow, with proANP plasma concentrations closest to the median). Total RNA from cardiac biopsies were purified using TRIzol (Life Technologies, Paisley, United Kingdom) according to the manufacturer’s protocol, and integrity was examined on an Agilent (Santa Clara, California) RNA 6000 Nano LabChip in 12 representative samples. First-strand cDNA was synthesized in a total volume of 20 l using 2 g RNA, M-Mulv (140 U; New England Biolabs, Ipswich, United Kingdom), and random hexamer primers (50 A260/U; Roche, Indianapolis, Indiana). Quantitative real-time PCR analysis of mRNA expression was performed with TaqMan ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, California). The PCR (20 l) contained 10 l of FAST Green I master mix, 10 pmol of each primer, cDNA synthesized from 20 ng of total RNA, and PCR-grade H2O. The relation between the time point of the log-linear increase in fluorescence signal and the relative concentration of an mRNA transcript was determined by analyzing dilutions of cDNA pool of all samples (n ⫽ 120), ie, cDNA synthesized from 200, 40, 20, 2, 0.2, and 0.02 ng total RNA. All samples were analyzed in duplicate. Sense and antisense primers for the following were used: porcine ANP (5⬘-AAGCGAGCAGAATGAGGAAG-3⬘ and 5⬘CAGTCCACTCTGTGCTCCAA-3⬘), BNP (5⬘-GTGCTCCTGCTCCTGTTCTT-3⬘ and 5⬘-TCCCAGGCTTCTGTGAGG-3⬘), and housekeeping gene -actin (5⬘-TGGACATCAGGAAGGACCTC-3⬘ and 5⬘-ACATCTGCTGGAAGGTGGAC-3⬘). The performance and validation of these mRNA assays has been reported previously (3).
Table 1.
Plasma creatinine and carbamide were measured in all piglets on an automated platform (Roche Modular Analytics [SWA] P-module). The intra-assay CV was ⬍5%.
Echocardiography During anesthesia in lateral recumbent position, the arterial duct was evaluated in transthoracic short axis view from birth and every 12th hour by 2D echocardiography (Vivid i and 10S probe; GE Healthcare, Piscataway, New Jersey). The arterial duct was considered open if turbulence was registered in the pulmonary artery by color flow and pulse wave Doppler. At the time of euthanasia, also LV dimensions were measured with 2Dguided M-mode in short axis view, and fractional shortening was calculated for LV function. Tricuspid annular plane systolic excursion was measured for RV function. All parameters were estimated from 3 consecutive RR intervals on average. Digital recordings were evaluated with GE EchoPAC software by 1 observer blinded to the identity of the piglets.
Statistics Median (interquartile range) was calculated to represent data. Nonparametric Kruskal-Wallis was used for comparison of more than 2 groups followed by Dunn’s post hoc test. For 2-group comparisons, Mann-Whitney U test was applied and for paired data Wilcoxon’s signed rank test. Histograms, probability, and residual plots were used when simple linear regressions were applied, and if residuals were not Gaussian distributed, variables were log 10 transformed. Variables from Table 1 were tested with backward selection in multiple linear regressions for possible univariate effects on the dependent variable. Data were analyzed with SAS version 9.1 and GraphPad Prism version 5 software.
Results The two sows delivered 22 and 23 piglets, respectively. One piglet was excluded due to ventricular perforation with the feeding tube, thus day 0, 1, 2, and 3 each consisted of 11 piglets. Basic data on the piglets are shown in Table 1. Possible effects of time (ie, day), sow, gender (Table 1), and open/closed ductus (Table 2) were tested. Only time
Piglets (n ⫽ 44) During the First 3 Days of Life After Cesarean Section of 2 Sowsa
n (sow 1/sow 2) Gender (乆/么) Temperature at birth, oC Temperature at euthanasia, oC Weight at birth, g Weight at euthanasia, g a
Renal plasma markers
Day 0
Day 1
Day 2
Day 3
P
6/5 8/3 34.6 (34.1–35.2) Same as above 958 (892–1182) 938 (892–1182)
5/6 3/8 35.1 (33.9 –35.6) 37.2 (37.0 –37.8) 993 (845–1280) 967 (852–1307)
6/5 5/6 36.3 (35.6 –36.5) 37.7 (37.2–38.2) 1104 (745–1425) 1070 (707–1423)
5/6 5/6 36.1 (35.2–36.5) 38.1 (37.4 –38.9) 1116 (733–1298) 1088 (695–1235)
.0002b ⬍.0001c .9 .9
Results are listed as median (interquartile range). P values are from Kruskal-Wallis. Temperature at birth was lowest in group day 0 (Dunn’s post hoc, data not shown). Day 0 was measured shortly after delivery during the echocardiography procedure with gel, which cooled them further down. c Temperature at euthanasia. Core body temperature increases when compared with temperature at birth in the same piglets, which was as expected (32), data not shown Mann-Whitney U test for each day (not possible with day 0). b
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Table 2. Open or Closed Ductus Arteriosus in Piglets (n ⫽ 44) Evaluated With Echocardiography Ductus Arteriosus, %
At delivery (d 0) 12 h 24 h (d 1)
Open
Closed
Uncertain
80 26 3
7 61 82
13 13 15
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immunoreactivity eluted in a position corresponding to human pro-ANP 1–126 (13). No smaller fragments were detected. A smaller discrepancy was noted in the elution pattern between tissue samples from day 0 to day 3 (Figure 2B, P ⬍ .05). Weather this difference in elution represents a change in processing would be worthwhile to pursue.
Gel chromatography The elution pattern of both plasma and atrial pro-ANP were similar on day 0 and 3 (Figure 2, A and B), where the
Discordant ANP and BNP mRNA expression in cardiac chambers Cardiac ANP mRNA contents were markedly higher (70- to 100-fold) in atrial compared with ventricular tissue samples (P ⬍ .0001; Figure 3, A–D). These results were corroborated by pro-ANP peptide product concentrations in the tissue extracts, where there also was a marked difference between atria and ventricles (P ⬍ .0001; Figure 3, E–H). However, no associations between transcriptional and translational products in the tissue were observed (data not shown). Moreover, BNP mRNA contents were only up to 4-fold higher in the atria compared with ventricular tissue (P ⬍ .0001; Figure 4). In the LA and LV, ANP mRNA content was larger on day 1 compared with day 0 (P ⬍ .05) and borderline in the RA (P ⫽ .06) (Figure 3, A, C, and D). This we also observed for LA and LV BNP expression but not significantly (Figure 4, C and D). After day 1, the LV BNP mRNA content decreased (P ⬍ .05; Figure 4D). In contrast to the other chambers, the RV BNP mRNA content was highest on day 0 (P ⬍ .01; Figure 4B).
Figure 1. Neonate natriuretic peptide plasma concentrations. Plasma concentrations of pro-ANP (A) and BNP-32 (B) measured at day 0, 1, 2, and 3 after delivery. Bars represent medians. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .0001.
Discordant postpartum piglet plasma concentrations and cardiac mRNA contents ANP transcriptional contents followed a different spatial expression pattern over time as compared with proANP plasma concentrations. Measurements disclosed inverse amounts, because ANP mRNA tissue contents were lowest in piglets on day 0, whereas plasma pro-ANP concentrations were highest on this same day. Thus, no positive associations were observed between transcriptional and plasma translational products over time (LA: r ⫽ ⫺0.57, P ⫽ .003; RA: r ⫽ ⫺0.38, P ⫽ .06). This was further corroborated by a discrepancy between pro-ANP plasma and tissue concentrations from all heart chambers (data not shown). Finally, the BNP-32 plasma concentrations were not a reflection of RA BNP mRNA content. In the LA, increased BNP mRNA content seemed to parallel BNP-32 plasma concentrations; however, the after all very weak correlation (P ⫽ .03, r ⫽ 0.45) may be disputed because the peak in LA and LV BNP mRNA expression was on day 1, whereas BNP-32 plasma concentrations peaked on day 2.
had an influence on pro-ANP (P ⬍ .0001) and BNP-32 (P ⬍ .0001) plasma concentrations after backward selection. Discordant postpartum piglet pro-ANP and BNP-32 plasma concentrations Both porcine pro-ANP and BNP-32 plasma concentrations differed dramatically between day 0, 1, 2, and 3 (P ⬍ .0001; Figure 1, A and B). Overall, the pro-ANP concentrations were highest at day 0 and decreased from 5580 pmol/L (4320–6786) to 2484 pmol/L (1602–2898) on day 3. Plasma BNP-32 displayed a different concentration pattern over time, where the concentrations started at 12 pmol/L (10.5–20.6) on day 0 and peaked on day 2 (88.8 [71.7–101.4] pmol/L). In the 2 sows, mean plasma concentrations for pro-ANP and BNP-32 were 828 and 24 pmol/L, respectively.
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Figure 2. A and B, Unprocessed natriuretic peptide fragments in cardiac tissue and plasma. The high pro-ANP plasma concentrations resemble concentrations observed in cardiac heart failure patients. A and B, However, by plasma (A) and atrial tissue (B) gel chromatography of day 0 and 3, the translational product does not seem biologically active but resembles a larger unprocessed fragment like human pro-ANP 1–126 (12). Kd is the elution constant. C and D, Electron microscopy of the right (C) and left (D) atria showing secretory granules (indicated by arrow), which most likely only contain unprocessed pro-ANP as distinguished from the gel chromatography.
Echocardiography The arterial duct was functionally closed 24 hours after delivery (Table 2). LV dimensions in systole measured by echocardiography increased from 8.7 (8 –10) mm on day
0 to 11 (9.7–12.3) mm on day 3 (P ⫽ .04); no difference was noted in diastole. Notably, no associations to LV geometrical or functional variables were observed with regard to local ANP and BNP gene expression, which was not
Figure 3. Neonate heart chamber-specific ANP gene expression and pro-ANP peptide in tissue. Four cardiac chambers from piglets euthanized on day 0, 1, 2, and 3 after delivery (day 0 was 1–3 hours after delivery. A–D, Regional ANP mRNA expression levels (ratio with housekeeping gene -actin to minimize sample variations. The expression data are based on ratios and thus the unit is arbitrary). E–H, Pro-ANP tissue concentrations. Bars represent medians. *P ⬍ .05.
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Figure 5. Are circulating pro-ANP concentrations affected by renal capacity? A, Creatinine in porcine plasma measured at day 0, 1, 2, and 3 after delivery. Bars represent medians. **P ⬍ .01; ***P ⬍ .0001. B, On those 4 days, a significant association existed between plasma creatinine and plasma pro-ANP. Note the scale is logarithmic.
Figure 4. Neonate heart chamber-specific BNP gene expression. A–D, Porcine regional BNP mRNA expression levels from the 4 cardiac chambers. Samples are from day 0, 1, 2, and 3 after delivery (day 0 was 1–3 hours after delivery). The ratio with housekeeping gene actin minimizes sample variation. The expression data are based on ratios, and thus, the unit is arbitrary. Bars represent medians. *P ⬍ .05; **P ⬍ .01.
found for the RV functional variable tricuspid annular plane systolic excursion or RV mRNA content (data not shown). Association between pro-ANP and creatinine plasma concentrations A close association between pro-ANP and creatinine plasma concentrations was observed; the latter also decreased during the first days after delivery (Figure 5). On the other hand, the pro-ANP concentrations were not associated to carbamide in plasma, and BNP-32 concentrations were not associated with any of the renal markers (data not shown).
Discussion In the present study, we examined the regional cardiac natriuretic peptide gene expression in neonate piglets dur-
ing the first 72 hours of life. We here show a marked difference in plasma pro-ANP and BNP-32 concentrations over time, where pro-ANP concentrations were highest at delivery, whereas BNP-32 peaked only after 48 hours of life. The rapid changes in plasma concentrations were not explained by regional cardiac mRNA content, where plasma pro-ANP concentrations mostly reflected renal function over the first days of life as assessed by creatinine. To the best of our knowledge, this is the first demonstration of a time-dependent neonate chamber-specific BNP and ANP gene expression in the myocardium. Our results also suggest that cardiac gene expression mirrors the perinatal cardiovascular physiological changes. Discordant ANP and BNP mRNA expression Cardiac natriuretic peptide mRNA expression has previously been shown to correlate to plasma concentrations of the translational products in adult heart failure and in patients with chronic myocardial hypoxia (14 –16). For pathological cardiac conditions, the correlations are usually found in the affected ventricle, whereas the atrial contribution is often not addressed, in particular for the BNP gene products (17). In the healthy adult heart, however, both ANP and BNP transcriptional expression is by far a feature of the atria with little or no ventricular expression (3). In the present study, ANP mRNA content was also negligible in healthy neonate ventricles. Thus, plasma proANP concentrations most likely represent atrial biosynthesis and secretion. Interestingly, neonate BNP mRNA content also was noted in the ventricles, which contrasts
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with earlier findings in adult pigs (3). The atrial expression of ANP is activated upon physiological stimuli, whereas BNP is mainly a ventricular hormone during heart failure regulated by a constitutive mechanism. The diverse mechanisms may explain our discordant results in ANP and BNP expression even in the healthy neonate, most likely caused by change in ventricular workload after delivery. We estimated heart function with echocardiography, and apart from an LV increase in geometrical dimensions in systole from day 0 to day 3, we did not find significant changes. This is in contrast to our gene expression data. In the fetus, pulmonary vascular resistance is high and the RV workload is high, pumping blood through the arterial duct against fetal systemic pressure. After birth, pulmonary resistance decreases abruptly and causes an afterload reduction for the RV (18). This matched the lower BNP mRNA content on day 1 (neonate circuit) compared with day 0 (fetal circuit). Our first biopsies from day 0 were taken only 1 to 3 hours after birth, and we believe these expression data reflect a delayed picture of the actual fetal circulation. For left cardiac chambers and the RA, our results were opposite with low ANP and BNP mRNA content on day 0 compared with day 1. This may reflect the series of events postpartum causing the sudden increase in workload and cardiac output (milliliters per minute per kilogram), ie, closing of foramen ovale, high oxygen requirements, and removal of placenta resulting in an increase in systemic vascular resistance (18). After day 1, ventricular mRNA content seems to decrease again, perhaps caused by closure of the arterial duct. Only 1 piglet had an open arterial duct 48 hours after delivery. Interestingly, this piglet had the highest mRNA quantities of both ANP and BNP in the LA and second highest in the LV when compared with age-matched piglets, all with closed arterial ducts. However, the suggested decrease in transcriptional expression could also reflect the expected effect of ventricular adaptation to the increased pressure-volume load. This phenomenon is applied before a late switch operation in transposition of the great arteries, eg, training of the LV to systemic pressures by pulmonary artery banding resulting in LV hypertrophy and adaptation in only a few days (19). Discordant pro-ANP and BNP-32 plasma concentrations We speculated that the high concentrations of natriuretic peptides in neonate plasma could be caused by immature storage capacity. However, electron microscopy showed plenty of storage granules (Figure 2, C and D). Healthy neonates in conjunction with high concentrations of bioactive natriuretic peptides are contradictory. Our results from chromatography propose that the high cir-
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culating concentrations and the contents in storage granules are large forms and not processed to bioactive ANP. This is in accordance with our earlier observations for BNP in neonate plasma (6). Like in human neonates, we found porcine BNP-32 plasma concentrations were low at birth and with higher concentrations in the days to follow. Also resembling human neonates, porcine pro-ANP concentrations were high at delivery, which decreased in the piglets (Figure 1), contrasting with an increase in human neonates in the days after birth (20). In fetal lamb 2 weeks before birth, plasma ANP concentrations are 6 times higher than maternal plasma concentrations and even 20 times higher compared with fetal urine concentrations (21). Thus, the high pro-ANP concentrations at delivery are perhaps not only birth related but may also reflect fetal physiology (22), whereas BNP regulation starts after birth. Natriuretic peptides are suggested to regulate placental circulation, where gene expression of BNP and the related C-type natriuretic peptide (CNP) have been demonstrated in the murine placenta (23), and only in the human placenta (24). Perhaps this may help explain that plasma pro-ANP is increased in human neonates after delivery and not BNP-32, so that BNP and ANP have different endocrine roles and thus are regulated differently. The discordance may also be related to diverse hormonal processing in the fetus and neonate. In this context, it is noteworthy that the ANP and BNP peptide products in adults seem as equal plasma markers, at least in heart failure patients (25). Concentrations in neonates can be as high as in heart failure patients (5), but whether the immature prohormones have a physiological role in the neonates is unknown. It is suggested that ANP and BNP translational products have some bioactivity to retain a PDA in preterm infants (26); however, this might be the cause rather than effect. Renal elimination Cardiac ANP and BNP gene expression did not explain the fluctuating plasma concentrations of the natriuretic peptides pro-ANP and BNP-32. Because plasma concentrations represent the sum of cardiac secretion and peripheral elimination, we tested whether the profiles were associated with markers of renal function and maturation. Notably, the kidneys are well-established organs of eliminating both BNP and pro-BNP from adult plasma, which is also reflected in renal disease, where peptide concentrations in plasma increase as a function of increasing uremia (27, 28). Our data from newborn piglets corroborate this association, where a simultaneous decrease in both plasma creatinine and pro-ANP was observed (Figure 5B). Creatinine concentrations decrease after birth in neonates without renal disease, whereas plasma carbamide is not an
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accurate marker for glomerular filtration (29), especially after milk substitute (30). Clearly, the decisive test for renal peptide extraction is determining the arteriovenous gradient in piglets, which would be interesting to pursue if the markers are to be implemented in future neonatal diagnosis and risk assessment. BNP-32 in plasma increased over the first 3 days, which does not reflect renal maturation in itself. However, LA and LV BNP gene expression and renal immaturity in combination might best explain the rapidly increasing concentrations. For pro-ANP in plasma, postpartum cardiac secretion is possibly concealed in the existing high concentrations from the fetal contribution (21, 22). In the present report, we note that the concentrations of pro-ANP are associated with creatinine in plasma. However, creatinine is a weak surrogate measure for actual neonate renal function. Indeed, assessing renal function in neonates is a challenge. Creatinine and carbamide also reflect muscle to fat mass and feeding (in neonates, the muscle mass is relatively low). Interpretation of the markers is even more complex, because they both cross the placenta. Thus, creatinine in plasma at birth largely represents the maternal concentration, and the first measurement should therefore be interpreted with caution. Inulin clearance is not used in human neonates, because this measure is imprecise. Renal function in neonates is generally low with clearance rates of 20 mL/min/1.73 m2 at 1 month of age in term and preterm neonates (31). For now, we speculate that pro-ANP and BNP-32 plasma measurement may not be optimal markers for cardiac events during the first 72 hours of life, because the renal impact on plasma concentrations seems to override cardiac changes. This is in accord with observations in healthy neonates and neonates with congenital heart disease where the diagnostic accuracy with BNP plasma measurement improves after the third or fourth day of life (5). Studies on neonate animals and children with renal disturbances should therefore be pursued to elucidate the potential diagnostic pitfalls. Our model could be further used to study the impact of a PDA on cardiovascular endocrinology. The PDA could be set up by prostaglandin treatment or, to reduce a hormonal effect, with deployment of a stent in ductus arteriosus. Conclusion Our data show that cardiac natriuretic peptide gene expression reflects local hemodynamic changes after birth in neonate piglets. However, the chamber-specific cardiac transcriptional expression does not explain the rapidly changing and high natriuretic peptide concentrations in circulation after birth. We suggest that this discrepancy may be related to renal function in the neonate, and plasma measurement should be interpreted with great care
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if applied as biomarkers for cardiac disease in the first 72 hours of life.
Acknowledgments We thank Lina Christel Monica Anklew, Mandy Johanne Greig, Christina Tirsdal Kjempff, Anne Nordhagen, Mette Helga Schmidt, and Andreas Vegge for animal handling; Line Bisgaard, Rikke Krøncke, Alice Lieth, and Dijana Terzic for expert assistance with laboratory analyses; and personal assistant Connie Bundgaard for help with formatting the manuscript. Address all correspondence and requests for reprints to: Jens Peter Goetze, Professor, MD, DMSc, Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. E-mail:
[email protected]. This work was supported by Copenhagen University Hospital Research Council, Biotek, Kurt Bønnelycke and Grethe Bønnelycke’s Foundation, Torben and Alice Frimodt’s Foundation, Augustinus Foundation, Sophus and Astrid Jacobsen’s Foundation, and the Danish Council of Independent Research. Disclosure Summary: The authors have nothing to disclose
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