Fetal cardiac troponin I levels decline toward birth in sheep.

Elevated cardiac troponin I (cTnI), a myocardial damage biomarker, has been reported in cord blood of neonates delivered vaginally or by cesarean section. Although the neonatal peak likely reflects the physiological adjustment to extrauterine life, a better understanding of serial prepartum changes is required to determine physiological causes of fetal cTnI release. We longitudinally sampled eight healthy lambs (20 days before spontaneous birth to 5 days postnatal), and from three fetuses receiving intravenous IGF-1. Samples were collected into heparin, and the plasma was stored at -80°C for later determination of high-sensitivity (hs) cTnI levels (BeckmanCoulter UniCel DxI Access IA; log transformed detection limit = 0.30, quantification limit = 0.78, 99th percentile = 1.78). Positive and negative control samples were drawn from an adult ewe during a terminal experiment (myocardial ischemia) and similarly assessed. hs-cTnI data were log transformed from ng/L. Log(hs-cTnI) was 1.47 ± 0.30 (means ± SD) at 20 days before birth and declined to 1.02 ± 0.65 in fetuses 12 ± 4 h before birth (P < 0.0001, R2 = 0.7869). Birth stimulated a delayed, transient peak in hs-cTnI (P = 0.0058). Newborn (43 ± 19 min postnatal) levels were 1.39 ± 0.40 (P = 0.0650 vs. fetus on day of birth) and 2.14 ± 0.63 the day after birth (P = 0.0331 vs. newborn). The second day after birth, levels declined to 1.65 ± 0.48 (P = 0.0238 vs. day 1). IGF-1 infusion increased hs-cTnI levels 25-50% over baseline (P = 0.0252, R2 = 0.9938). Baseline adult ewe log(hs-cTnI) was below the limit of detection; 3 h following coronary artery ligation, levels were 3.21. In conclusion, we newly report that fetal hs-cTnI levels decline concomitantly with reduced proliferation of cardiomyocytes toward term.NEW & NOTEWORTHY Serial blood samples were collected from catheterized, normally developing fetal and newborn lambs and high-sensitivity cardiac troponin I (hs-cTnI) levels were assessed, providing unprecedented insight into the physiological processes leading to high levels in the perinatal period. Moderately high levels of hs-cTnI found in the normally developing fetus declined toward term. An elevation to high levels peaked the day after birth, after which hs-cTnI declined again. Stimulation of fetal cardiomyocyte proliferation with IGF-1 also elevated hs-cTnI.

Sensitivity and activation of endoplasmic reticulum stress response and apoptosis in the perinatal sheep heart.

Although the unfolded protein response (UPR) contributes to survival by removing misfolded proteins, endoplasmic reticulum (ER) stress also activates proapoptotic pathways. Changed sensitivity to normal developmental stimuli may underlie observed cardiomyocyte apoptosis in the healthy perinatal heart. We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of ER stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of eukaryotic initiation factor 2 (EIF2A) was decreased by 72% between 135 and 143 dGA (P = 0.0096) and remained low at 1 dPN (P = 0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P = 0.0380; 7.8-fold vs. 5 dPN, P = 0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P < 0.0001) and remained elevated at 1 dPN. Induced apoptosis, measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay, was highest around the time of birth (P < 0.0001). There were changes in myocardial ER stress pathway components in utero. Glucose (78 kDa)-regulated protein (GRP78) protein levels were high in the fetus and declined after birth (P < 0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P = 0.0113). In conclusion, there is dynamic regulation of ER proteostasis, ER stress, and apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for the regulation of normal maturation in the healthy perinatal heart.NEW & NOTEWORTHY Cardiomyocyte apoptosis occurs even in the healthy, normally developing perinatal myocardium. As cardiomyocyte number is a critical contributor to heart health, the sensitivity of cardiomyocytes to endoplasmic reticulum stress leading to apoptosis is an important consideration. This study suggests that the heart has less robust protective mechanisms in response to endoplasmic reticulum stress immediately before and after birth, and that more cardiomyocyte death can be induced by stress in this period.

Physiological response to fetal intravenous lipid emulsion.

In preterm neonates unable to obtain sufficient oral nutrition, intravenous lipid emulsion is life-saving. The contribution of post-conceptional level of maturation to pathology that some neonates experience is difficult to untangle from the global pathophysiology of premature birth. In the present study, we determined fetal physiological responses to intravenous lipid emulsion. Fetal sheep were given intravenous Intralipid 20® (n = 4 females, 7 males) or Lactated Ringer's Solution (n = 7 females, 4 males) between 125 ± 1 and 133 ± 1 d of gestation (term = 147 d). Manufacturer's recommendation for premature human infants was followed: 0.5-1 g/kg/d initial rate, increased by 0.5-1 to 3 g/kg/d. Hemodynamic parameters and arterial blood chemistry were measured, and organs were studied postmortem. Red blood cell lipidomics were analyzed by LC-MS. Intravenous Intralipid did not alter hemodynamic or most blood parameters. Compared with controls, Intralipid infusion increased final day plasma protein (P=0.004; 3.5 ± 0.3 vs. 3.9 ± 0.2 g/dL), albumin (P = 0.031; 2.2 ± 0.1 vs. 2.4 ± 0.2 g/dL), and bilirubin (P<0.001; conjugated: 0.2 ± 0.1 vs. 0.6 ± 0.2 mg/dL; unconjugated: 0.2 ± 0.1 vs. 1.1 ± 0.4 mg/dL). Circulating IGF-1 decreased following Intralipid infusion (P<0.001; 66 ± 24 vs. 46 ± 24 ng/mL). Compared with control Oil Red O liver stains (median score 0), Intralipid-infused fetuses scored 108 (P=0.0009). Lipidomic analysis revealed uptake and processing of infused lipids into red blood cells, increasing abundance of saturated fatty acids. The near-term fetal sheep tolerates intravenous lipid emulsion well, although lipid accumulates in the liver. Increased levels of unconjugated bilirubin may reflect increased red blood cell turnover or impaired placental clearance. Whether Intralipid is less well tolerated earlier in gestation remains to be determined.

Augmenting workload drives T-tubule assembly in developing cardiomyocytes.

Contraction of cardiomyocytes is initiated at subcellular elements called dyads, where L-type Ca2+ channels in t-tubules are located within close proximity to ryanodine receptors in the sarcoplasmic reticulum. While evidence from small rodents indicates that dyads are assembled gradually in the developing heart, it is unclear how this process occurs in large mammals. We presently examined dyadic formation in fetal and newborn sheep (Ovis aries), and the regulation of this process by fetal cardiac workload. By employing advanced imaging methods, we demonstrated that t-tubule growth and dyadic assembly proceed gradually during fetal sheep development, from 93 days of gestational age until birth (147 days). This process parallels progressive increases in fetal systolic blood pressure, and includes step-wise colocalization of L-type Ca2+ channels and the Na+ /Ca2+ exchanger with ryanodine receptors. These proteins are upregulated together with the dyadic anchor junctophilin-2 during development, alongside changes in the expression of amphiphysin-2 (BIN1) and its partner proteins myotubularin and dynamin-2. Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth. Conversely, reducing fetal systolic load with infusion of enalaprilat, an angiotensin converting enzyme inhibitor, blunted t-tubule formation. Interestingly, altered t-tubule densities did not relate to changes in dyadic junctions, or marked changes in the expression of dyadic regulatory proteins, indicating that distinct signals are responsible for maturation of the sarcoplasmic reticulum. In conclusion, augmenting blood pressure and workload during normal fetal development critically promotes t-tubule growth, while additional signals contribute to dyadic assembly. KEY POINTS: T-tubule growth and dyadic assembly proceed gradually in cardiomyocytes during fetal sheep development, from 93 days of gestational age until the post-natal stage. Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth and hypertrophy. In contrast, reducing fetal systolic load by enalaprilat infusion slowed t-tubule development and decreased cardiomyocyte size. Load-dependent modulation of t-tubule maturation was linked to altered expression patterns of the t-tubule regulatory proteins junctophilin-2 and amphiphysin-2 (BIN1) and its protein partners. Altered t-tubule densities did not influence dyadic formation, indicating that distinct signals are responsible for maturation of the sarcoplasmic reticulum.

Maturation of lipid metabolism in the fetal and newborn sheep heart.

At birth, the fetus experiences a dramatic change in environment that is accompanied by a shift in myocardial fuel preference from lactate and glucose in fetal life to fatty acid oxidation after birth. We hypothesized that fatty acid metabolic machinery would mature during fetal life in preparation for this extreme metabolic transformation at birth. We quantified the pre- (94-day and 135-day gestation, term ∼147 days) and postnatal (5 ± 4 days postnatal) gene expression and protein levels for fatty acid transporters and enzymes in hearts from a precocial species, the sheep. Gene expression of fatty acid translocase (CD36), acyl-CoA synthetase long-chain 1 (ACSL1), carnitine palmitoyltransferase 1 (CPT1), hydroxy-acyl dehydrogenase (HADH), acetyl-CoA acetyltransferase (ACAT1), isocitrate dehydrogenase (IDH), and glycerol phosphate acyltransferase (GPAT) progressively increased through the perinatal period, whereas several genes [fatty acid transport protein 6 (FATP6), acyl-CoA synthetase long chain 3 (ACSL3), long-chain acyl-CoA dehydrogenase (LCAD), very long-chain acyl-CoA dehydrogenase (VLCAD), pyruvate dehydrogenase kinase (PDK4), phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT)] were stable in fetal hearts and had high expression after birth. Protein expression of CD36 and ACSL1 progressively increased throughout the perinatal period, whereas protein expression of carnitine palmitoyltransferase 1a (fetal isoform) (CPT1a) decreased and carnitine palmitoyltransferase 1b (adult isoform) (CPT1b) remained constitutively expressed. Using fluorescent-tagged long-chain fatty acids (BODIPY-C12), we demonstrated that fetal (125 ± 1 days gestation) cardiomyocytes produce 59% larger lipid droplets (P < 0.05) compared with newborn (8 ± 1 day) cardiomyocytes. These results provide novel insights into the perinatal maturation of cardiac fatty acid metabolism in a precocial species.NEW & NOTEWORTHY This study characterized the previously unknown expression patterns of genes that regulate the metabolism of free fatty acids in the perinatal sheep myocardium. This study shows that the prenatal myocardium prepares for the dramatic switch from carbohydrate metabolism to near complete reliance on free fatty acids postnatally. Fetal and neonatal cardiomyocytes also demonstrate differing lipid storage mechanisms where fetal cardiomyocytes form larger lipid droplets compared with newborn cardiomyocytes.

Changes in microvascular perfusion of heart and skeletal muscle in sheep around the time of birth.

NEW FINDINGS: What is the central question of this study? How does the microvascular perfusion of striated muscle change during the dynamic developmental period between the late gestation fetus and early neonate? What is the main finding and its importance? In both myocardium and skeletal muscle, perfusion of striated muscle is significantly reduced in the neonate compared to the late term fetus, but flow reserve is unchanged. The results suggest striated muscle capillary networks grow more slowly relative to the myofibres they nourish during the perinatal period. ABSTRACT: Microvascular perfusion of striated muscle is an important determinant of health throughout life. Birth is a transition with profound effects on the growth and function of striated muscle, but the regulation of microvascular perfusion around this transition is poorly understood. We used contrast-enhanced ultrasound perfusion imaging (CEUS) to study the perfusion of left ventricular myocardium and hindlimb biceps femoris, which are populations of muscle with different degrees of change in pre- to postnatal workloads and different capacities for postnatal proliferative growth. We studied separate groups of lambs in late gestation (135 days' gestational age; 92% of term) and shortly after birth (5 days' postnatal age). We used CEUS to quantify baseline perfusion, perfusion during hyperaemia induced by adenosine infusion (myocardium) or electrically stimulated unloaded exercise (skeletal muscle), flow reserve and oxygen delivery. We found heart-to-body weight ratio was greater in neonates than fetuses. Microvascular volume and overall perfusion were lower in neonates than fetuses in both muscle groups at baseline and with hyperaemia. Flux rate differed with muscle group, with myocardial flux being faster in neonates than fetuses, but skeletal muscle flux being slower. Oxygen delivery to skeletal muscle at baseline was lower in neonates than fetuses, but was not significantly different in myocardium. Flow reserve was not different between ages. Given the significant somatic growth, and the transition from hyperplastic to hypertrophic myocyte growth occurring in the perinatal period, we postulate that the primary driver of lower neonatal striated muscle perfusion is faster growth of myofibres than their associated capillary networks.

Intrauterine growth restriction elevates circulating acylcarnitines and suppresses fatty acid metabolism genes in the fetal sheep heart.

At birth, the mammalian myocardium switches from using carbohydrates as the primary energy substrate to free fatty acids as the primary fuel. Thus, a compromised switch could jeopardize normal heart function in the neonate. Placental embolization in sheep is a reliable model of intrauterine growth restriction (IUGR). It leads to suppression of both proliferation and terminal differentiation of cardiomyocytes. We hypothesized that the expression of genes regulating cardiac fatty acid metabolism would be similarly suppressed in IUGR, leading to compromised processing of lipids. Following 10 days of umbilicoplacental embolization in fetal sheep, IUGR fetuses had elevated circulating long-chain fatty acylcarnitines compared with controls (C14: CTRL 0.012 ± 0.005 nmol/ml vs. IUGR 0.018 ± 0.005 nmol/ml, P < 0.05; C18: CTRL 0.027 ± 0.009 nmol/mol vs. IUGR 0.043 ± 0.024 nmol/mol, P < 0.05, n = 12 control, n = 12 IUGR) indicative of impaired fatty acid metabolism. Uptake studies using fluorescently tagged BODIPY-C12-saturated free fatty acid in live, isolated cardiomyocytes showed lipid droplet area and number were not different between control and IUGR cells. mRNA levels of sarcolemmal fatty acid transporters (CD36, FATP6), acylation enzymes (ACSL1, ACSL3), mitochondrial transporter (CPT1), ß-oxidation enzymes (LCAD, HADH, ACAT1), tricarboxylic acid cycle enzyme (IDH), esterification enzymes (PAP, DGAT) and regulator of the lipid droplet formation (BSCL2) gene were all suppressed in IUGR myocardium (P < 0.05). However, protein levels for these regulatory genes were not different between groups. This discordance between mRNA and protein levels in the stressed myocardium suggests an adaptive protection of key myocardial enzymes under conditions of placental insufficiency. KEY POINTS: The fetal heart relies on carbohydrates in utero and must be prepared to metabolize fatty acids after birth but the effects of compromised fetal growth on the maturation of this metabolic system are unknown. Plasma fatty acylcarnitines are elevated in intrauterine growth-restricted (IUGR) fetuses compared with control fetuses, indicative of impaired fatty acid metabolism in fetal organs. Fatty acid uptake and storage are not different in IUGR cardiomyocytes compared with controls. mRNA levels of genes regulating fatty acid transporter and metabolic enzymes are suppressed in the IUGR myocardium compared with controls, while protein levels remain unchanged. Mismatches in gene and protein expression, and increased circulating fatty acylcarnitines may have long-term implications for offspring heart metabolism and adult health in IUGR individuals. This requires further investigation.

Coronary vascular growth matches IGF-1-stimulated cardiac growth in fetal sheep.

As loss of contractile function in heart disease could often be mitigated by increased cardiomyocyte number, expansion of cardiomyocyte endowment paired with increased vascular supply is a desirable therapeutic goal. Insulin-like growth factor 1 (IGF-1) administration increases fetal cardiomyocyte proliferation and heart mass, but how fetal IGF-1 treatment affects coronary growth and function is unknown. Near-term fetal sheep underwent surgical instrumentation and were studied from 127 to 134 d gestation (term = 147 d), receiving either IGF-1 LR3 or vehicle. Coronary growth and function were interrogated using pressure-flow relationships, an episode of acute hypoxia with progressive blockade of adenosine receptors and nitric oxide synthase, and by modeling the determinants of coronary flow. The main findings were that coronary conductance was preserved on a per-gram basis following IGF-1 treatment, adenosine and nitric oxide contributed to hypoxia-mediated coronary vasodilation similarly in IGF-1-treated and Control fetuses, and the relationships between coronary flow and blood oxygen contents were similar between groups. We conclude that IGF-1-stimulated fetal myocardial growth is accompanied by appropriate expansion and function of the coronary vasculature. These findings support IGF-1 as a potential strategy to increase cardiac myocyte and coronary vascular endowment at birth.

Thyroid hormone receptor function in maturing ovine cardiomyocytes.

KEY POINTS: Plasma thyroid hormone (tri-iodo-l-thyronine; T3 ) concentrations rise near the end of gestation and is known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for the action of thyroid hormone in fetal cardiomyocytes. Loss of thyroid hormone receptor (TR)α1 abolishes T3 signalling via extracellular signal-related kinase and Akt in fetal cardiomyocytes. The expression of TRα1 and TRß1 in ovine fetal myocardium increases with age, although TRα1 levels always remain higher than those of TRß1. Near term fetal cardiac myocytes are more sensitive than younger myocytes to thyroid receptor blockade by antagonist, NH3, and to the effects of TRα1/α2 short interfering RNA. Although T3 is known to abrogate ovine cardiomyocyte proliferation stimulated by insulin-like growth factor 1, this effect is mediated via the genomic action of thyroid hormone receptors, with little evidence for non-genomic mechanisms. ABSTRACT: We have previously shown that the late-term rise in tri-iodo-l-thyronine (T3 ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T3 -induced changes are mediated via thyroid hormone receptors (TRs) or by non-genomic mechanisms. Fetal cardiomyocytes were isolated from 102 ± 3 and 135 ± 1 days of gestational age (dGA) sheep (n = 7 per age; term ∼145 dGA). Cells were treated with T3 (1.5 nm), insulin-like growth factor (IGF)-1 (1 µg mL-1 ) or a combination in the presence of TR antagonist NH3 (100 nm) or following short interfering RNA (siRNA) knockdown of TRα1/α2. Proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) uptake (10 µm). Western blots measured protein levels of extracellular signal-related kinase (ERK), Akt, TRα1/ß1 and p21. Age specific levels of TRα1/ß1 were measured in normal hearts from fetuses [95 dGA (n = 8), 135 dGA (n = 7)], neonates (n = 8) and adult ewes (n = 7). TRα1 protein levels were consistently >50% more than TRß1 at each gestational age (P < 0.05). T3 reduced IGF-1 stimulated proliferation by ∼50% in 100 dGA and by ∼75% in 135 dGA cardiomyocytes (P < 0.05). NH3 blocked the T3  + IGF-1 reduction of BrdU uptake without altering the phosphorylation of ERK or Akt at both ages. NH3 did not suppress T3 -induced p21 expression in 100 dGA cardiomyocytes in 135 dGA cardiomyocytes, NH3 alone reduced BrdU uptake (-28%, P < 0.05), as well as T3 -induced p21 (-75%, P < 0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T3  + IGF-1 reduction of BrdU uptake and dramatically reduced ERK and Akt signalling in 135 dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T3 signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.

Cardiac myocyte proliferation and maturation near term is inhibited by early gestation maternal testosterone exposure.

Polycystic ovary syndrome is a complex and common disorder in women, and those affected experience an increased burden of cardiovascular disease. It is an intergenerational syndrome, as affected women with high androgen levels during pregnancy "program" fetal development, leading to a similar phenotype in their female offspring. The effect of excess maternal testosterone exposure on fetal cardiomyocyte growth and maturation is unknown. Pregnant ewes received biweekly injections of vehicle (control) or 100 mg testosterone propionate between 30 and 59 days of gestation (early T) or between 60 and 90 days of gestation (late T). Fetuses were delivered at ~135 days of gestation, and their hearts were enzymatically dissociated to measure cardiomyocyte growth (dimensional measurements), maturation (proportion binucleate), and proliferation (nuclear Ki-67 protein). Early T depressed serum insulin-like growth factor 1 and caused intrauterine growth restriction (IUGR; P < 0.0005). Hearts were smaller with early T ( P < 0.001) due to reduced cardiac myocyte maturation ( P < 0.0005) and proliferation ( P = 0.017). Maturation was also lower in male than female fetuses ( P = 0.004) independent of treatment. Late T did not affect cardiac growth. Early excess maternal testosterone exposure depresses circulating insulin-like growth factor 1 near term and causes IUGR in both female and male offspring. These fetuses have small, immature hearts with reduced proliferation, which may reduce cardiac myocyte endowment and predispose to adverse cardiac growth in postnatal life. While excess maternal testosterone exposure leads to polycystic ovary syndrome and cardiovascular disease in female offspring, it may also predispose to complications of IUGR and cardiovascular disease in male offspring. NEW & NOTEWORTHY Using measurements of cardiac myocyte growth and maturation in an ovine model of polycystic ovary syndrome, this study demonstrates that early gestation excess maternal testosterone exposure reduces near-term cardiomyocyte proliferation and maturation in intrauterine growth-restricted female and male fetuses. The effect of testosterone is restricted to exposure during a specific period early in pregnancy, and the effects appear mediated through reduced insulin-like growth factor 1 signaling. Furthermore, male fetuses, regardless of treatment, had fewer mature cardiomyocytes than female fetuses.

Systemic arterial hypertension but not IGF-I treatment stimulates cardiomyocyte enlargement in neonatal lambs.

Although cardiomyocyte terminal differentiation is nearly complete at birth in sheep, as in humans, very limited postnatal expansion of myocyte number may occur. The capacity of newborn cardiomyocytes to respond to growth stimulation by proliferation is poorly understood. Our objective was to test this growth response in newborn lambs with two stimuli shown to be potent inducers of cardiomyocyte growth in fetuses and adults: increased systolic load (Load) and insulin-like growth factor I (IGF-I). Vascular catheters and an inflatable aortic occluder were implanted in lambs. Hearts were collected for analysis at 18 days of age after a 7-day experiment and compared with control hearts. Load hearts, but not IGF-I hearts, were heavier ( P = 0.001) because of increased mass of the left ventricle (LV), septum, and left atrium (40-50%, P = 0.004). Terminal differentiation and cell cycle activity were not different between groups. Myocyte length was 7% greater in Load lamb hearts ( P < 0.05), and binucleated myocytes, which comprise ~90% of LV cells, were 25% larger in volume ( P = 0.03). Myocyte number per gram of myocardium was decreased in all ventricles of Load lambs ( P = 0.01). Cells from the IGF-I group were not different by any comparison. These results suggest that the newborn sheep LV responds to systolic stress with cardiomyocyte hypertrophy, not proliferation. Furthermore, IGF-I is ineffective at stimulating cardiomyocyte proliferation at this age (despite effectiveness when administered before birth). Thus, to expand cardiomyocyte number in the newborn heart, therapies other than systolic pressure load and IGF-I treatment need to be developed.

Role of adenosine signaling in coordinating cardiomyocyte function and coronary vascular growth in chronic fetal anemia.

Fetal anemia causes rapid and profound changes in cardiac structure and function, stimulating proliferation of the cardiac myocytes, expansion of the coronary vascular tree, and impairing early contraction and relaxation. Although hypoxia-inducible factor-1α is sure to play a role, adenosine, a metabolic byproduct that increases coronary flow and growth, is implicated as a major stimulus for these adaptations. We hypothesized that genes involved in myocardial adenosine signaling would be upregulated in chronically anemic fetuses and that calcium-handling genes would be downregulated. After sterile surgical instrumentation under anesthesia, gestationally timed fetal sheep were made anemic by isovolumetric hemorrhage for 1 wk (16% vs. 35% hematocrit). At 87% of gestation, necropsy was performed to collect heart tissue for PCR and immunohistochemical analysis. Anemia increased mRNA expression levels of adenosine receptors ADORA 1, ADORA2A, and ADORA2B in the left and right ventricles (adenosine receptor ADORA3 was unchanged). In both ventricles, anemia also increased expression of ectonucleoside triphosphate diphosphohydrolase 1 and ecto-5'-nucleotidase. The genes for both equilibrative nucleoside transporters 1 and 2 were expressed more abundantly in the anemic right ventricle but were not different in the left ventricle. Neither adenosine deaminase nor adenosine kinase cardiac levels were significantly changed by chronic fetal anemia. Chronic fetal anemia did not significantly change cardiac mRNA expression levels of the voltage-dependent L-type calcium channel, ryanodine receptor 1, sodium-calcium exchanger, sarcoplasmic/endoplasmic reticulum calcium transporting ATPase 2, phospholamban, or cardiac calsequestrin. These data support local metabolic integration of vascular and myocyte function through adenosine signaling in the anemic fetal heart.

Functional adaptations of the coronary microcirculation to anaemia in fetal sheep.

KEY POINTS: In fetuses, chronic anaemia stimulates cardiac growth; simultaneously, blood flow to the heart muscle itself is increased, and reserve blood flow capacity of the coronary vascular bed is preserved. Here we examined functional adaptations of the capillaries and small blood vessels responsible for delivering oxygen to the anaemic fetal heart muscle using contrast-enhanced echocardiography. We demonstrate that coronary microvascular flux rate doubled in anaemic fetuses compared to control fetuses, both at rest and during maximal flow, suggesting reduced microvascular resistance consistent with capillary widening. Cardiac fractional microvascular blood volume was not greater in anaemic fetuses, suggesting that growth of new microvascular vessels does not contribute to the increased flow per volume of myocardium. These unusual changes in microvascular function during anaemia may indicate novel adaptive strategies in the fetal heart. ABSTRACT: Fetal anaemia causes cardiac adaptations that have immediate and life-long repercussions on heart function and health. It is known that resting and maximal coronary conductance both increase during chronic fetal anaemia, but the coronary microvascular changes responsible for the adaptive response are unknown. Until recently, technical limitations have prevented quantifying functional capillary-level adaptations in the in vivo fetal heart. Our objective was to characterise functional microvascular adaptations in chronically anaemic fetal sheep. Chronically instrumented fetuses were randomized to a control group (n = 11) or were made anaemic by isovolumetric haemorrhage (n = 12) for 1 week prior to myocardial contrast echocardiography at 85% of gestation. Anaemia augmented cardiac mass by 23% without changing body weight. In anaemic fetuses, microvascular blood flow per volume of myocardium was twice that of control fetuses at rest, during vasodilatory hyperaemia, and during hyperaemia plus increased aortic pressure. The elevated blood flow was attributable almost entirely to an increase in microvascular blood flux rate whereas microvascular blood volumes were not different between groups at baseline, during hyperaemia, or with hyperaemia plus increased aortic pressure. Increased coronary microvascular flux rate in response to chronic fetal anaemia is consistent with expected reductions in capillary resistance from capillary diameter widening detected in earlier histological studies.

Timing of cardiomyocyte growth, maturation, and attrition in perinatal sheep.

Studies in altricial rodents attribute dramatic changes in perinatal cardiomyocyte growth, maturation, and attrition to stimuli associated with birth. Our purpose was to determine whether birth is a critical trigger controlling perinatal cardiomyocyte growth, maturation and attrition in a precocial large mammal, sheep (Ovis aries). Hearts from 0-61 d postnatal lambs were dissected or enzymatically dissociated. Cardiomyocytes were measured by micromorphometry, cell cycle activity assessed by immunohistochemistry, and nuclear number counted after DNA staining. Integration of this new data with published fetal data from our laboratory demonstrate that a newly appreciated >30% decrease in myocyte number occurred in the last 10 d of gestation (P < 0.0005) concomitant with an increase in cleaved poly (ADP-ribose) polymerase 1 (P < 0.05), indicative of apoptosis. Bisegmental linear regressions show that most changes in myocyte growth kinetics occur before birth (median = 15.2 d; P < 0.05). Right ventricular but not left ventricular cell number increases in the neonate, by 68% between birth and 60 d postnatal (P = 0.028). We conclude that in sheep few developmental changes in cardiomyocytes result from birth, excepting the different postnatal degrees of free wall hypertrophy between the ventricles. Furthermore, myocyte number is reduced in both ventricles immediately before term, but proliferation increases myocyte number in the neonatal right ventricle.

Increased systolic load causes adverse remodeling of fetal aortic and mitral valves.

While abnormal hemodynamic forces alter fetal myocardial growth, little is known about whether such insults affect fetal cardiac valve development. We hypothesized that chronically elevated systolic load would detrimentally alter fetal valve growth. Chronically instrumented fetal sheep received either a continuous infusion of adult sheep plasma to increase fetal blood pressure, or a lactated Ringer's infusion as a volume control beginning on day 126 ± 4 of gestation. After 8 days, mean arterial pressure was higher in the plasma infusion group (63.0 mmHg vs. 41.8 mmHg, P < 0.05). Mitral annular septal-lateral diameter (11.9 mm vs. 9.1 mm, P < 0.05), anterior leaflet length (7.7 mm vs. 6.4 mm, P < 0.05), and posterior leaflet length (P2; 4.0 mm vs. 3.0 mm, P < 0.05) were greater in the elevated load group. mRNA levels of Notch-1, TGF-ß2, Wnt-2b, BMP-1, and versican were suppressed in aortic and mitral valve leaflets; elastin and α1 type I collagen mRNA levels were suppressed in the aortic valves only. We conclude that sustained elevated arterial pressure load on the fetal heart valve leads to anatomic remodeling and, surprisingly, suppression of signaling and extracellular matrix genes that are important to valve development. These novel findings have important implications on the developmental origins of valve disease and may have long-term consequences on valve function and durability.

Regulation of intramembranous absorption and amniotic fluid volume by constituents in fetal sheep urine.

Our objective was to test the hypothesis that fetal urine contains a substance(s) that regulates amniotic fluid volume by altering the rate of intramembranous absorption of amniotic fluid. In late gestation ovine fetuses, amniotic fluid volumes, urine, and lung liquid production rates, swallowed volumes and intramembranous volume and solute absorption rates were measured over 2-day periods under control conditions and when urine was removed and continuously replaced at an equal rate with exogenous fluid. Intramembranous volume absorption rate decreased by 40% when urine was replaced with lactated Ringer solution or lactated Ringer solution diluted 50% with water. Amniotic fluid volume doubled under both conditions. Analysis of the intramembranous sodium and chloride fluxes suggests that the active but not passive component of intramembranous volume absorption was altered by urine replacement, whereas both active and passive components of solute fluxes were altered. We conclude that fetal urine contains an unidentified substance(s) that stimulates active intramembranous transport of amniotic fluid across the amnion into the underlying fetal vasculature and thereby functions as a regulator of amniotic fluid volume.

Thyroid hormone drives fetal cardiomyocyte maturation.

Tri-iodo-l-thyronine (T(3)) suppresses the proliferation of near-term serum-stimulated fetal ovine cardiomyocytes in vitro. Thus, we hypothesized that T(3) is a major stimulant of cardiomyocyte maturation in vivo. We studied 3 groups of sheep fetuses on gestational days 125-130 (term ∼145 d): a T(3)-infusion group, to mimic fetal term levels (plasma T(3) levels increased from ∼0.1 to ∼1.0 ng/ml; t(1/2)∼24 h); a thyroidectomized group, to produce low thyroid hormone levels; and a vehicle-infusion group, to serve as intact controls. At 130 d of gestation, sections of left ventricular freewall were harvested, and the remaining myocardium was enzymatically dissociated. Proteins involved in cell cycle regulation (p21, cyclin D1), proliferation (ERK), and hypertrophy (mTOR) were measured in left ventricular tissue. Evidence that elevated T(3) augmented the maturation rate of cardiomyocytes included 14% increased width, 31% increase in binucleation, 39% reduction in proliferation, 150% reduction in cyclin D1 protein, and 500% increase in p21 protein. Increased expression of phospho-mTOR, ANP, and SERCA2a also suggests that T(3) promotes maturation and hypertrophy of fetal cardiomyocytes. Thyroidectomized fetuses had reduced cell cycle activity and binucleation. These findings support the hypothesis that T(3) is a prime driver of prenatal cardiomyocyte maturation.

Thyroid hormone increases fatty acid use in fetal ovine cardiac myocytes.

Cardiac metabolic substrate preference shifts at parturition from carbohydrates to fatty acids. We hypothesized that thyroid hormone (T3 ) and palmitic acid (PA) stimulate fetal cardiomyocyte oxidative metabolism capacity. T3 was infused into fetal sheep to a target of 1.5 nM. Dispersed cardiomyocytes were assessed for lipid uptake and droplet formation with BODIPY-labeled fatty acids. Myocardial expression levels were assessed PCR. Cardiomyocytes from naïve fetuses were exposed to T3 and PA, and oxygen consumption was measured with the Seahorse Bioanalyzer. Cardiomyocytes (130-day gestational age) exposed to elevated T3 in utero accumulated 42% more long-chain fatty acid droplets than did cells from vehicle-infused fetuses. In utero T3 increased myocardial mRNA levels of CD36, CPT1A, CPT1B, LCAD, VLCAD, HADH, IDH, PDK4, and caspase 9. In vitro exposure to T3 increased maximal oxygen consumption rate in cultured cardiomyocytes in the absence of fatty acids, and when PA was provided as an acute (30 min) supply of cellular energy. Longer-term exposure (24 and 48 h) to PA abrogated increased oxygen consumption rates stimulated by elevated levels of T3 in cultured cardiomyocytes. T3 contributes to metabolic maturation of fetal cardiomyocytes. Prolonged exposure of fetal cardiomyocytes to PA, however, may impair oxidative capacity.

Chronic in utero mitral inflow obstruction unloads left ventricular volume in a novel late gestation fetal lamb model.

Objective: The in utero no flow/no grow hypothesis postulates that reduced inflow of blood into the left ventricle due to a stenotic mitral valve could lead to ventricular hypoplasia and hypoplastic left heart syndrome. This has been demonstrated in chick embryos, but less so in large animals. We investigated the impact of mitral obstruction on left and right ventricular growth in fetal lambs. Methods: Twelve pregnant ewes, most bearing twins, were instrumented at 119 ± 1 days gestational age. Carotid artery and jugular vein catheters, an ascending aorta flow probe, and a left atrial deflated balloon catheter were implanted into 1 fetus (left atrial balloon group), and the twin remained an uninstrumented control. The balloon was inflated gradually over 8 days until net antegrade aortic flow was eliminated. Fetal transesophageal echocardiography was performed at the time of surgery and just before termination in both groups. Results: Terminal fetal body weights were comparable between groups. Terminal heart/body weight ratio was higher in left atrial balloon group fetuses (6.9 ± 0.8 g/kg) compared with controls (5.9 ± 0.6 g, P = .0126). The left ventricular/right ventricular weight ratio was 24% (P = .0077) lower in left atrial balloon group fetuses than in controls. Left ventricular/heart weight (0.24 ± 0.04 g/g vs 0.30 ± 0.04 g/g, P = .0009), left ventricular end-diastolic volume (2.3 ± 0.7 mL vs 7.1 ± 0.8 mL; P = .0012), and left ventricular end-systolic volume (1.01 mL [0.95-1.95 mL] vs 3.38 mL [3.28-3.57 mL], P = .0042) were lower in left atrial balloon group fetuses compared with controls. Right ventricular weight (g/kg), right ventricular end-diastolic volume, and right ventricular end-systolic volume were similar between groups. Conclusions: In this late-gestation fetal lamb model, in utero obstruction of mitral inflow slowed left ventricular growth and caused right ventricular remodeling.

Development of a novel Guinea Pig model producing transgenerational endothelial transcriptional changes driven by maternal food restriction and a second metabolic insult of high fat diet.

Developmental programming of chronic adverse cardiovascular health outcomes has been studied both using numerous human populations and an array of animal models. However, the mechanisms that produce transgenerational effects have been difficult to study due to a lack of developmentally relevant models. As such, how increased disease risk is carried to the second generation has been poorly studied. We hypothesized that the endothelium which mediates many acute and chronic vascular inflammatory responses is a key player in these effects, and epidemiological studies implicate transgenerational nutritional effects on endothelial health. To study the mutigenerational effects of maternal undernutrition on offspring endothelial health, we developed a model of transgenerational nutritional stress in guinea pigs, a translationally relevant precocial species with a relatively short lifespan. First- and second-generation offspring were subjected to a high fat diet in adolescence to exacerbate negative cardiovascular health. To assess transcriptional changes, we performed bulk RNA-sequencing in carotid artery endothelial cells, with groups stratified as prenatal control or food restricted, and postnatal control or high fat diet. We detected statistically significant gene alterations for each dietary permutation, some of which were unique to treatments and other transcriptional signatures shared by multiple or all conditions. These findings highlight a core group of genes altered by high fat diet that is shared by all cohorts and a divergence of transgenerational effects between the prenatal ad libitum and dietary restriction groups. This study establishes the groundwork for this model to be used to better understand the interplay of prenatal stress and genetic reprogramming.