Newborn Heart Research Articles

URSODEOXYCHOLIC ACID REGULATES CASPASE-9 AND ROS PRODUCTION IN PROTECTING CARDIOMYOCYTES AGAINST HYPOXIA

Ursodeoxycholic acid (UDCA) is known as a therapeutic agent in treating cholestasis and liver diseases. Recently, UDCA has been suggested as a therapeutic drug for heart related diseases. Cardioprotective effect of UDCA against the development of ischemia has been studied. Yet, the mechanism of UDCA-cardioprotection is not clearly understood. Therefore, this study aimed to elucidate the mechanisms of UDCA cardioprotection against hypoxia by investigating the expression of caspase -3/-9 and ROS generation using an in vitro hypoxic heart model. A newborn (0-2 days old) rat heart was isolated for primary cell culture of cardiomyocytes. Hypoxia was chemically induced by using CoCl2. Cardiomyocytes were then incubated with UDCA. The treated cardiomyocytes were subjected for ROS generation detection assay, QuantiGene Plex assay for caspase-3/-9 gene expression and ELISA for caspase-3/-9 protein expression. The data were analyzed by using sample paired t-test and One-way ANOVA. Our results showed that UDCA abolishes the effects on CoCl2 in ROS production and UDCA downregulates caspase-9 protein expression in CoCl2 treated cardiomyocytes. This study provides an insight of UDCA in protecting cardiomyocytes against hypoxia mediated by anti-apoptosis mechanism.

Citric Acid Cycle Metabolites Predict the Severity of Myocardial Stunning and Mortality in Newborn Pigs.

Myocardial infarction and chronic heart failure induce specific metabolic changes in the neonatal myocardium that are closely correlated to outcome. The aim of this study was to examine the metabolic responses to noninfarct heart failure and inotropic treatments in the newborn heart, which so far are undetermined. A total of 28 newborn pigs were instrumented with a microdialysis catheter in the right ventricle, and intercellular citric acid cycle intermediates and adenosine metabolite concentrations were determined at 20-minute intervals. Stunning was induced by 10 cycles of 3 minutes of ischemia, which was performed by occluding the right coronary artery, followed by 3 minutes of reperfusion. Animals were randomized for treatment with epinephrine + milrinone, dopamine + milrinone, dobutamine, or saline. University hospital animal laboratory. Ischemia-reperfusion induced right ventricular stunning and increased the concentrations of pyruvate lactate, succinate, malate, hypoxanthine, and xanthine (all, p < 0.01). During inotrope infusion, no differences in metabolite concentrations were detected between the treatment groups. In nonsurviving animals (n = 8), concentrations of succinate (p < 0.0001), malate (p = 0.009), and hypoxanthine (p = 0.04) increased compared with survivors, while contractility was significantly reduced (p = 0.03). Accumulation of citric acid cycle intermediates and adenosine metabolites reflects the presence of myocardial stunning and predicts mortality in acute noninfarct right ventricular heart failure in newborn pigs. This phenomenon occurs independently of the type of inotrope, suggesting that citric acid cycle intermediates represent potential markers of acute noninfarct heart failure.

Streptozotocin diabetes increases mRNA expression of ketogenic enzymes in the rat heart

BackgroundDiabetic cardiomyopathy develops in insulin-dependent diabetic patients who have no hypertension, cardiac hypertrophy or vascular disease. Diabetes increases cardiac fatty acid oxidation, but cardiac hypertrophy limits fatty acid oxidation. Here we examined effects of diabetes on gene expression in rat hearts. MethodsWe used oligonucleotide microarrays to examine effects of insulindependent diabetes in the rat heart. RTQ PCR confirmed results of microarrays. Specific antibodies were used to examine changes in the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). ResultsA surprising result of diabetes was increased mRNA encoding all enzymes of the ketone body synthesis pathway. Increased mRNA expression for these enzymes was confirmed by RTQ PCR. The mRNA encoding HMGCS2, the rate-controlling enzyme, was 27 times greater in diabetic hearts. Total HMGCS2 protein increased 8-fold in diabetic hearts, but no difference was found in HMGCS2 protein in control vs. diabetic liver. ConclusionsInsulin-dependent diabetes induced the enzymes of ketone body synthesis in the heart, including HMGCS2, as well as increasing enzymes of fatty acid oxidation. General significanceThe mammalian heart does not export ketone bodies to other tissues, but rather is a major consumer of ketone bodies. Induction of HMGCS2, which is normally expressed only in the fetal and newborn heart, may indicate an adaptation by the heart to combat “metabolic inflexibility” by shifting the flux of excess intramitochondrial acetyl-CoA derived from elevated fatty acid oxidation into ketone bodies, liberating free CoA to balance the acetyl-CoA/CoA ratio in favor of increased glucose oxidation through the pyruvate dehydrogenase complex.

Hemodynamics of patient-specific aorta-pulmonary shunt configurations

Optimal hemodynamics in aorta-pulmonary shunt reconstruction is essential for improved post-operative recovery of the newborn congenital heart disease patient. However, prior to in vivo execution, the prediction of post-operative hemodynamics is extremely challenging due to the interplay of multiple confounding physiological factors. It is hypothesized that the post-operative performance of the surgical shunt can be predicted through computational blood flow simulations that consider patient size, shunt configuration, cardiac output and the complex three-dimensional disease anatomy. Utilizing only the routine patient-specific pre-surgery clinical data sets, we demonstrated an intelligent decision-making process for a real patient having pulmonary artery atresia and ventricular septal defect. For this patient, a total of 12 customized candidate shunt configurations are contemplated and reconstructed virtually using a sketch-based computer-aided anatomical editing tool. Candidate shunt configurations are evaluated based on the parameters that are computed from the flow simulations, which include 3D flow complexity, outlet flow splits, shunt patency, coronary perfusion and energy loss. Our results showed that the modified Blalock-Taussig (mBT) shunt has 12% higher right pulmonary artery (RPA) and 40% lower left pulmonary artery (LPA) flow compared to the central shunt configuration. Also, the RPA flow regime is distinct from the LPA, creating an uneven flow split at the pulmonary arteries. For all three shunt sizes, right mBT innominate and central configurations cause higher pulmonary artery (PA) flow and lower coronary artery pressure than right and left mBT subclavian configurations. While there is a trade-off between energy loss, flow split and coronary artery pressure, overall, the mBT shunts provide sufficient PA perfusion with higher coronary artery pressures and could be preferred for similar patients having PA overflow risk. Central shunts would be preferred otherwise particularly for cases with very low PA overflow risk.

Three Dimensional Visualization of Human Cardiac Conduction Tissue in Whole Heart Specimens by High-Resolution Phase-Contrast CT Imaging Using Synchrotron Radiation.

The feasibility of synchrotron radiation-based phase-contrast computed tomography (PCCT) for visualization of the atrioventricular (AV) conduction axis in human whole heart specimens was tested using four postmortem structurally normal newborn hearts obtained at autopsy. A PCCT imaging system at the beamline BL20B2 in a SPring-8 synchrotron radiation facility was used. The PCCT imaging of the conduction system was performed with "virtual" slicing of the three-dimensional reconstructed images. For histological verification, specimens were cut into planes similar to the PCCT images, then cut into 5-μm serial sections and stained with Masson's trichrome. In PCCT images of all four of the whole hearts of newborns, the AV conduction axis was distinguished as a low-density structure, which was serially traceable from the compact node to the penetrating bundle within the central fibrous body, and to the branching bundle into the left and right bundle branches. This was verified by histological serial sectioning. This is the first demonstration that visualization of the AV conduction axis within human whole heart specimens is feasible with PCCT.

Comparative study of B type brain natriuretic peptide and N-terminal pro-brain natriuretic peptide in the auxiliary diagnosis of heart failure after neonatal asphyxia

Objective To observe and compare the value of B brain natriuretic peptide(BNP) and N-terminal pro-brain natriuretic peptide(NT-proBNP) in the diagnosis of heart failure after neonatal asphyxia, and to optimize early clinical diagnosis. Methods A retrospective analysis was conducted on 124 neonatal asphyxia cases from January 2013 to October 2015, who were divided into heart failure group(53 cases) and control group(71 cases) according to whether complicated with heart failure.Comparison was conducted on BNP, NT-proBNP, cardiac troponin T (cTnT), creatine kinase isoenzyme (CK-MB) through blood sampling from femoral vein puncture within 48 h. And Logistic regression analysis was introduced into explore effecting factors of heart failure, besides, correlations between BNP, NT-proBNP and left ventricular ejection fraction (LVEF) of asphyxia children were calculated, and receiver operating characteristic curve(ROC) was introduced into analyzing of BNP and NT-proBNP for diagnostic efficacy of heart failure after neonatal asphyxia. Results Heart failure group whose BNP[(835.8±154.7) pg/ml vs. (235.4±38.5) pg/ml], NT-proBNP(25 903.8 pg/ml vs. 6 974.5 pg/ml), cTnT[(0.21±0.06)ng/ml vs. (0.11±0.03) ng/ml], CK-MB[(61.3±11.7) U/L vs. (40.8±9.5) U/L] were significantly higher than those of control group (P<0.05). Logistic regression analysis indicated BNP and NT-proBNP were closely related with newborn heart failure(ORBNP=3.013, P<0.001; ORNT-proBNP=3.808, P=0.006). BNP and NT-proBNP were both significantly negatively correlated with LVEF(rBNP=-0.650, P=0.007; rNT-proBNP=-0.721, P<0.001). The ROC curve indicated the diagnostic efficacy of BNP and NT-proBNP for heart failure after neonatal asphyxia were 0.868, 0.911, with the highest diagnosis cut-off value were 268.8 pg/ml, 3 972.3 pg/ml, respectively. Conclusion BNP and NT proBNP are specific indicators reflecting heart failure after neonatal asphyxia, and NT-proBNP with higher auxiliary diagnostic efficacy comparatively. Key words: Neonatal asphyxia; Heart failure; Brain natriuretic peptide; N-terminal pro-brain natriuretic peptide; Diagnostic efficacy

Acetylation Control Contributes to Maturational Alterations in Cardiac Energy Metabolism in the Newborn Heart

Acetylation Control Contributes to Maturational Alterations in Cardiac Energy Metabolism in the Newborn Heart

Exercise during pregnancy decreases doxorubicin-induced cardiotoxic effects on neonatal hearts.

Cancer treatment with Doxorubicin (DOX) is limited due its dose-dependent cardiotoxicity, mainly related to the oxidative stress production. In experimental models of DOX treatment exercise can be used as a beneficial adjuvant therapy. This work aimed to investigate the effects of exercise during pregnancy on DOX-induced cardiotoxicity in cardiomyocytes of progeny, examining the possible intergenerational cardioprotective effects of maternal exercise. For this purpose pregnant rats were divided in control and exercise groups and pre-treated during gestational days. Hearts of newborns were used to obtain a culture of cardiomyocytes to be treated with DOX for analyses of cell viability, apoptosis and necrosis; ROS production; DNA damage; SOD and CAT activities; and Sirt6 protein expression. The results showed that exercise during pregnancy induced an increase in the viability of neonatal cardiomyocytes and a decrease in DOX-induced apoptotic and necrotic death which were correlated to the decrease in ROS production and an increase in antioxidant defenses. Exercise also protected neonatal cardiomyocytes from DOX-induced DNA damage, demonstrating a reduction in the oxidative DNA breaks. Likewise, exercise induced an increase in expression of Sirt6 in neonatal cardiomyocytes. Therefore, these results demonstrate for the first time that exercise performed by mothers protects the neonatal heart against DOX-induced toxicity. Our data demonstrate the intergenerational effect of exercise in cardiomyocytes of progeny, where the modulation of oxidative stress through antioxidant enzymes, and DNA integrity via Sirt6, were induced due to exercise in mothers, increasing the resistance of the neonatal heart against DOX toxicity.

Stem cell markers in the heart of the human newborn

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. Several recent studies now show that the different cell types that characterize the adult human heart arise from a common ancestor. Human cardiac stem cells differentiate into cardiomyocytes, and, in lesser extent, into smooth muscle and endothelial cells. The characterization of human cardiac stem cells (CSCs) has important clinical implications. In recent years, CD117 (c-kit) has been reported to mark a subtype of stem/progenitor cells in the human heart, with stem cell-like properties, including the ability to self-renewal and clonogenicity multipotentiality. Proceedings of the 2 nd International Course on Perinatal Pathology (part of the 11 th International Workshop on Neonatology · October 26 th -31 st , 2015) · Cagliari (Italy) · October 31 st , 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

Acetylation and succinylation contribute to maturational alterations in energy metabolism in the newborn heart.

Dramatic maturational changes in cardiac energy metabolism occur in the newborn period, with a shift from glycolysis to fatty acid oxidation. Acetylation and succinylation of lysyl residues are novel posttranslational modifications involved in the control of cardiac energy metabolism. We investigated the impact of changes in protein acetylation/succinylation on the maturational changes in energy metabolism of 1-, 7-, and 21-day-old rabbit hearts. Cardiac fatty acid β-oxidation rates increased in 21-day vs. 1- and 7-day-old hearts, whereas glycolysis and glucose oxidation rates decreased in 21-day-old hearts. The fatty acid oxidation enzymes, long-chain acyl-CoA dehydrogenase (LCAD) and β-hydroxyacyl-CoA dehydrogenase (β-HAD), were hyperacetylated with maturation, positively correlated with their activities and fatty acid β-oxidation rates. This alteration was associated with increased expression of the mitochondrial acetyltransferase, general control of amino acid synthesis 5 like 1 (GCN5L1), since silencing GCN5L1 mRNA in H9c2 cells significantly reduced acetylation and activity of LCAD and β-HAD. An increase in mitochondrial ATP production rates with maturation was associated with the decreased acetylation of peroxisome proliferator-activated receptor-γ coactivator-1α, a transcriptional regulator for mitochondrial biogenesis. In addition, hypoxia-inducible factor-1α, hexokinase, and phosphoglycerate mutase expression declined postbirth, whereas acetylation of these glycolytic enzymes increased. Phosphorylation rather than acetylation of pyruvate dehydrogenase (PDH) increased in 21-day-old hearts, accounting for the low glucose oxidation postbirth. A maturational increase was also observed in succinylation of PDH and LCAD. Collectively, our data are the first suggesting that acetylation and succinylation of the key metabolic enzymes in newborn hearts play a crucial role in cardiac energy metabolism with maturation.

Morphological changes of rat atrial cardiomyocytes contractile apparatus in normal conditions and under the influence of acute and chronic prenatal hypoxia. Role of α-smooth muscle actin in myofibrilogenesis.

Background. There are only a few researches considering the influence of hypoxia on myofibrils formation and cardiomyocytes differentiation. However the role of α-smooth muscle actin on sarcomyogenesis is still unclear, that leads to the further study of this question. Objective. The purpose of the work was to determine ontogenetic changes of myofibrils and cardiomyocytes differentiation in rat atrial myocardium in normal conditions and under the influence of acute and chronic prenatal hypoxia. Methods. Embryo hearts were investigated on 14th, 16th days of prenatal ontogenesis, newborn rat hearts and the hearts of rats – on 30th day of postnatal ontogenesis. Animals were subdivided into three groups: 1stexperimental group of animals was exposed to acute prenatal hypoxia, 2nd experimental group animals was exposed to chronic prenatal hypoxia and control group animals. Hypoxia modeling was conducted on pregnant females by injection of 1% sodium nitrite intraperitonealy in doses that lead to moderate hypoxia. During the work complex of histological, immunohistochemical and electron microscopy methods were used. Antibodies to α-smooth muscle actin (α-SMA) were used to study cardiomyocytes differentiation. The digital images were analyzed using a computer with NIH ImageJ program. Results and conclusion. The results show that myofibrillar differentiation is associated with reduction of α-SMA expression. Ultrastructural changes of rat atrial cardiomyocytes in 3 days after the influence of acute prenatal hypoxia manifested in disruption of myofibrillar orientation, accompanied with decreasing α-SMA expression. Chronic prenatal hypoxia from 16thday of prenatal ontogenesis to the 1stday of postnatal ontogenesis manifestated in myofibrillar defragmentation and disorientation, associated with decreased α-SMA expression compared with the control group. Conclusion. α-SMA can be used as a marker of cardiomyocytes differentiation.

Normal Newborn Heart Rate in the First Five Minutes of Life Assessed by Dry-Electrode Electrocardiography

Background: There is limited evidence regarding the heart rate (HR) during the first minutes of life. Nonetheless, resuscitative actions within the first minute are partly guided by different HR levels. The advent of an electrocardiographic (ECG) HR sensor with early HR detection has provided the opportunity to study changes immediately following delivery. Objective: The objectives were to determine immediately following delivery: (i) the time to achievement of reliable ECG signals using dry electrodes, (ii) changes in HR, and (iii) the influence of the onset of breathing and cord clamping on the HR. Methods: Healthy term neonates were randomly included between July and October 2013. The HR was recorded by the ECG sensor, placed over the abdomen immediately after birth. Results: Fifty-five newborns were included. The median time from birth to placement of the HR sensor was 3 s (quartiles: 2 and 5), and the median time to the start of breathing was 6 s (quartiles: 2 and 15). The HR was around 120 beats/min (bpm) in the first seconds of life. As determined via breakpoint analysis, the HR increased by 1 beat in the first 40 s to 149 ± 33 bpm, followed by a moderate increase until 130 s and stabilization thereafter. After the onset of breathing, the HR decreased for 10 s and then increased. Minimal HR changes were observed after cord clamping. Conclusion: A dry-electrode ECG sensor detected reliable ECG signals almost immediately after birth. The normal HR increased significantly in spontaneously breathing infants during the first minute, influenced by the onset of breathing. Delayed cord clamping had a minimal impact on the HR, likely reflecting an earlier onset of breathing.

B‐Raf is required for normal cardiomyocyte growth

ObjectivesThe postnatal heart does not retain the proliferative capacity it had during fetal life. Unlike large mammals, murine cardiomyocytes (CM) continue to divide into the first week of life before terminal differentiation and binucleation. This postnatal proliferation and maturation window offers the ability to study the mechanisms of CM terminal differentiation. We hypothesize that B‐Raf regulates ERK (extracellular‐regulated kinase) activation in newborn CM and that loss of B‐Raf suppresses cyclin levels and reduces CM proliferation. To test this, we determined whether the loss of B‐Raf disrupts the ERK cascade and impairs CM growth.MethodsCM specific knockout (KO) of B‐Raf was generated using CRE/lox (floxed B‐Raf x αMHC CRE) resulting in a truncated, unstable B‐Raf and a null phenotype. KO mice (α‐MHC‐CRE/B‐Raf lox/lox) were compared to CRE negative/B‐Raf lox/lox mice (wild type; WT). Hearts from 1d, 3d, 8d and 14d old pups were harvested for molecular analysis of B‐Raf signaling and cell cycle markers (p21, p27, p53, cyclins A1, B1, D1 and E). Mid‐wall sections of the heart were stained with Masson's trichrome to determine changes in tissue composition.ResultsHeart weight to body weight (HW/BW) ratio was less in 3d KO versus 3d WT (n=50, p&lt;0.05). HW/BW ratio became greater in 8d KO; there was no difference in 3d and 8d HW/BW in WT animals. B‐Raf and phosphorylated ERK levels were reduced in KO hearts (*p&lt;0.05). Cell cycle inhibitors p21 and p53 were increased in 3d KO hearts with decreased levels of all cyclins (Cyclin A1 and B1, p&lt;0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with decreased cyclin levels (p&lt;0.05). In 14d KO hearts, gene expression of p21, p27, and p53 are increased (p&lt;0.05), along with cyclins D1 and E. Tissue composition of cardiomyocytes decreased and collagen increased in 3d and 8d hearts (p&lt;0.05).ConclusionsERK activation was suppressed in KO hearts resulting in smaller newborn hearts but which exceeded normal HW/BW by 8d. This is may represent premature hypertrophy as the proliferative period of CM development had ended. Cell cycle analysis supports reduced CM mitosis among 8d CM. Trichrome analysis supports proliferation has ended prematurely by the reduction of the CM fraction. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.

Spontaneous Formation of Extensive Vessel-Like Structures in Murine Engineered Heart Tissue.

Engineered heart tissue (EHT) from primary heart cells contains endothelial cells (ECs), but the extent to which ECs organize into vessel-like structures or even functional vessels remains unknown and is difficult to study by conventional methods. In this study, we generated fibrin-based mini-EHTs from a transgenic mouse line (Cdh5-CreERT2 × Rosa26-LacZ), in which ECs were specifically and inducibly labeled by applying tamoxifen (EC(iLacZ)). EHTs were generated from an unpurified cell mix of newborn mouse hearts and were cultured under standard serum-containing conditions. Cre expression in 15-day-old EHTs was induced by addition of o-hydroxytamoxifen to the culture medium for 48 h, and ECs were visualized by X-gal staining. EC(iLacZ) EHTs showed a dense X-gal-positive vessel-like network with distinct tubular structures. Immunofluorescence revealed that ECs were mainly associated with cardiomyocytes within the EHT. EC(iLacZ) EHT developed spontaneous and regular contractility with forces up to 0.1 mN. Coherent contractility and the presence of an extensive vessel-like network were both dependent on the presence of animal sera in the culture medium. Contractile EC(iLacZ) EHTs successfully served as grafts in implantation studies onto the hearts of immunodeficient mice. Four weeks after implantation, EHTs showed X-gal-positive lumen-forming vessel structures connected to the host myocardium circulation as they contained erythrocytes on a regular basis. Taken together, genetic labeling of ECs revealed the extensive formation of vessel-like structures in EHTs in vitro. The EC(iLacZ) EHT model could help simultaneously study biological effects of compounds on cardiomyocyte function and tissue vascularization.

Gerald W. Dorn II: Thinker, Teacher, Tinkerer.

Cardiologist and molecular and cell biologist Gerald Dorn, MD, has built his research career crafting interesting questions and teaching himself techniques needed to find answers. Using approaches including genetic and physiological manipulation in animal models, development of mini-mouse technology to elucidate cardiac mysteries, and human genetics/genomics, Dorn is helping to unravel cellular and molecular mechanisms underlying cardiomyopathy and heart failure.1,2 Gerald W. Dorn II Dorn, 57, is founding director of the Center for Pharmacogenomics at the Washington University in St. Louis School of Medicine, where he joined the faculty in 2008. As a teenager Dorn sped through high school, starting college two years early. He earned his medical degree and trained in internal medicine, pharmacology, and clinical and interventional cardiology at the Medical University of South Carolina (MUSC) in Charleston, where two key mentors sparked in him a passion for patient care and research. After a couple of years at the University of Texas Health Science Center at San Antonio, Dorn moved to the University of Cincinnati, where an American Heart Association Established Investigator Award kick-started his research program. From 1990 to 2008 Dorn rose through the ranks at Cincinnati, capitalizing on the institution’s strength in mouse cardiac transgenesis. Dorn’s current interests include molecular mechanisms of cell death,3 microRNA regulation of cardiac genes,4 and mitochondrial dynamism5—work that has expanded to Parkinson’s disease. Building on an upbringing that valued lifelong growth, and drawing from his own broad experience, Dorn now leverages the holism of systems biology to fill in what he calls his field’s “infinitely large shades of gray.” My father was a career naval aviator. Whenever he moved, we moved. So I’m not really from anywhere. We spent a lot of time on both coasts. As a child I lived, in addition to …

Legal and Ethical Considerations in Allowing Parental Exemptions From Newborn Critical Congenital Heart Disease (CCHD) Screening

Critical congenital heart disease (CCHD) screening is rapidly becoming the standard of care in the United States after being added to the Recommended Uniform Screening Panel (RUSP) in 2011. Newborn screens typically do not require affirmative parental consent. In fact, most states allow parents to exempt their baby from receiving the required screen on the basis of religious or personally held beliefs. There are many ethical considerations implicated with allowing parents to exempt their child from newborn screening for CCHD. Considerations include the treatment of religious exemptions in our current legal system, as well as medical and ethical principles in relation to the rights of infants. Although there are significant benefits to screening newborns for CCHD, when a parent refuses for religious or personal beliefs, in the case of CCHD screening, the parental decision should stand.

Abstract 13368: Acetylation of VGLL4 Tdu Domain Modulates Its Activity to Regulate Postnatal Cardiac Growth (Best of Basic Science Abstract)

Mammalian cardiomyocytes (CM) largely exit the cell cycle shortly after birth, limiting the heart’s capacity to recover from injury. The mechanisms that enforce neonatal CM cell cycle withdrawal are largely unknown. CM proliferation is stimulated by interaction of the co-activator YAP, the terminal effector of Hippo signaling, with the transcription factor TEAD1, but YAP’s mitogenic potency declines in the adult compared to fetal or newborn heart. Here we show that VGLL4, a CM-enriched TEAD1 binding protein, inhibits CM proliferation by competing with YAP for TEAD1 binding. Moreover, VGLL4 activity is regulated by acetylation of the lysine 225 (K225) residing in its first Tondu (Tdu) domain. Acetylation at K225 antagonized its interaction with TEAD1 in the neonatal heart. Overexpression of VGLL4 with a point mutation that blocks its acetylation enhanced VGLL4-TEAD1 interaction and limited CM proliferation, resulting in lethal cardiac hypoplasia. Our study defines a novel acetylation-mediated, VGLL4-dependent switch that regulates Hippo-YAP signaling and that restrains CM proliferation. These insights may enable more effective approaches to cardiac regeneration.

Abstract 16866: Depressed Biventricular Outputs in Fetuses of Diabetic Mothers When Standardized to Estimated Fetal Weight

Background: Although the results of maternal diabetes on the newborn heart are well described, in-utero cardiovascular effects of maternal diabetes and obesity are not as well understood. Hypothesis: Echocardiographically derived measures of right and left heart flow volumes in fetuses of diabetic mothers (FDM) and fetuses of obese mothers (FOM) differ from those of normal fetuses. Methods: We prospectively enrolled 264 normal fetuses, 30 FDM and 49 FOM for prenatal research echocardiography. Estimated fetal weight (EFW) based norms for aortic and pulmonary valve velocity time integrals (AVVTI, PVVTI), right and left ventricular outflow tract diameters (LVOTD, RVOTD), aortic and pulmonary valve stroke volume (AVSV, PVSV), and cardiac output (LVCO, RVCO) were derived from the 264 control group fetuses using nonlinear regression to a Gompertz growth function. Z values for each parameter were determined, then analyzed using a non-paired two-tailed t-test to compare the three groups. Results: The mean ± SD EFW (g) for normal fetuses, FDM and FOM were 1610 ± 1201, 964 ± 694 and 1075 ± 1013; respective mean gestational ages (weeks) were 28.1±1.3, 25.3± 5, and 25 ± 6.6. The table shows mean Z value comparisons of VTI, OTD, SV and CO for the RV and LV in the three groups. Conclusions: FDM have significantly lower right and left heart stroke volumes and outputs than other fetuses matched for size, while FOM did not have demonstrable right or left heart stroke volume or cardiac output changes relative to normal fetuses. Similar to their relationship with gestational age, normal right and left ventricular stroke volumes and outputs follow a non-linear correlation with estimated fetal weight. Flow volumes in macrosomic fetuses may be better normalized to EFW rather than to gestational age. Reduced placental blood flow and ventricular compliance changes may be contributing to depressed biventricular cardiac output in FDM.

Epigenetic markers for newborn congenital heart defect (CHD)

Objective: Our objective was to determine whether there were significant differences in genome-wide DNA methylation in newborns with major congenital heart defect (CHD) compared to controls. We also evaluated methylation of cytosines in CpG motifs for the detection of these CHDs.Methods: Genome-wide DNA methylation analysis was performed on DNA from 60 newborns with various CHDs, including hypoplastic left heart syndrome, ventricular septal deficit, atrial septal defect, pulmonary stenosis, coarctation of the aorta and Tetralogy of Fallot, and 32 controls.Results: Highly significant differences in cytosine methylation were seen in a large number of genes throughout the genome for all CHD categories. Gene ontology analysis of CHD overall indicated over-represented biological processes involving cell development and differentiation, and anatomical structure morphogenesis. Methylation of individual cytosines in CpG motifs had high diagnostic accuracy for the detection of CHD. For example, for coarctation one predictive model based on levels of particular cytosine nucleotides achieved a sensitivity of 100% and specificity of 93.8% (AUC = 0.974, p < 0.00001).Conclusion: Profound differences in cytosine methylation were observed in hundreds of genes in newborns with different types of CHD. There appears to be the potential for development of accurate genetic biomarkers for CHD detection in newborns.

Ultrastructural and tissue restructuring of the rat atrial myocardium under the influence of acute and chronic prenatal hypoxia.

Background. There are relatively few researches that focused on the study of the influence of hypoxia on atrial myocardium. However the results of previous researches are contradictory and fragmentary, that leades to the further study of this item. Objective. Purpose of the work was to determine the tissue, cellular and ultrastructural changes of the rat atrial myocardium during the stages of cardiogenesis under the conditions of acute and chronic prenatal hypoxia. Methods. Embryo hearts were investigated on 14th, 16th and 18th day of prenatal ontogenesis, newborn rat hearts and the hearts of rats on the 3rd, 14th and 30th day of postnatal ontogenesis. Animals were subdivided into three groups: first experimental group animals were exposed to acute prenatal hypoxia, second experimental group animals were exposed to chronic prenatal hypoxia and control group animals. Hypoxia modeling was conducted on pregnant females by injection of 1% sodium nitrite intraperitonealy in doses that lead to moderate hypoxia. During the work complex of histological, immunohistochemical and morphometric methods was used. Results and conclusion. Effect of acute hypoxia leads to stimulation of proliferative activity of fibroblasts and endothelial cells on the right atrium, which causes to increasing of the relative volume of connective tissue and blood vessels of microvasculature of myocardium. Relative volume of connective tissue of the atrial myocardium exceeds significantly the control level of newborn rats. Ultrastructural changes of the atrial cardiomyocytes in 3 days after the influence of acute prenatal hypoxia reveals on violation of mitochondrial structure, significant inhibition of myofibrils accumulation. Until the birth of most of morphometric parameters renewal and reach control levels, however changes of the degree of myofibrils orientation on the left atrium proves stability of hypoxic damages. Chronic prenatal hypoxia causes a significant increase of relative volume of connective tissue and microvessels content of the myocardium compared with control values. Proliferative activity of fibroblasts and endothelial cells stays increased on the atrial myocardium of newborn rats. Relative volume of connective tissue observed higher significantly in 30 days after the birth. Persistent intracellular hypoxic changes of cardiomyocytes, which observed in 30 days after the rats birth, are increase numerical density of mitochondria, decrease of relative volume of myofibrils and degree of myofibrils orientation on the left atrium.