Establishment of NCHi009-A, an iPSC line from a patient with hypoplastic left heart syndrome (HLHS) carrying a heterozygous NOTCH1 mutation.

Hypoplastic left heart syndrome (HLHS) is a congenital heart malformation clinically characterized by an underdeveloped left ventricle, mitral or aortic valve stenosis or atresia, and narrowed ascending aorta. Although genetic etiology of HLHS is heterogenous, recurrent NOTCH1 variants have been associated with this defect. We report generation of an iPSC line derived from a female with HLHS with a heterozygous missense NOTCH1 (c.2058G > A; p.Gly661Ser) mutation within the conserved EGF-like repeat 17. This iPSC line exhibited typical cellular morphology, normal karyotype, high expression of pluripotent markers, and trilineage differentiation potential; and can be leveraged to dissect the complex NOTCH1-mediated HLHS disease mechanism.

Systems analysis of de novo mutations in congenital heart diseases identified a protein network in the hypoplastic left heart syndrome.

Despite a strong genetic component, only a few genes have been identified in congenital heart diseases (CHDs). We introduced systems analyses to uncover the hidden organization on biological networks of mutations in CHDs and leveraged network analysis to integrate the protein interactome, patient exomes, and single-cell transcriptomes of the developing heart. We identified a CHD network regulating heart development and observed that a sub-network also regulates fetal brain development, thereby providing mechanistic insights into the clinical comorbidities between CHDs and neurodevelopmental conditions. At a small scale, we experimentally verified uncharacterized cardiac functions of several proteins. At a global scale, our study revealed developmental dynamics of the network and observed its association with the hypoplastic left heart syndrome (HLHS), which was further supported by the dysregulation of the network in HLHS endothelial cells. Overall, our work identified previously uncharacterized CHD factors and provided a generalizable framework applicable to studying many other complex diseases. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

Novel Protein-Protein Interactions Highlighting the Crosstalk between Hypoplastic Left Heart Syndrome, Ciliopathies and Neurodevelopmental Delays.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) affecting 1 in 5000 newborns. We constructed the interactome of 74 HLHS-associated genes identified from a large-scale mouse mutagenesis screen, augmenting it with 408 novel protein-protein interactions (PPIs) using our High-Precision Protein-Protein Interaction Prediction (HiPPIP) model. The interactome is available on a webserver with advanced search capabilities. A total of 364 genes including 73 novel interactors were differentially regulated in tissue/iPSC-derived cardiomyocytes of HLHS patients. Novel PPIs facilitated the identification of TOR signaling and endoplasmic reticulum stress modules. We found that 60.5% of the interactome consisted of housekeeping genes that may harbor large-effect mutations and drive HLHS etiology but show limited transmission. Network proximity of diabetes, Alzheimer's disease, and liver carcinoma-associated genes to HLHS genes suggested a mechanistic basis for their comorbidity with HLHS. Interactome genes showed tissue-specificity for sites of extracardiac anomalies (placenta, liver and brain). The HLHS interactome shared significant overlaps with the interactomes of ciliopathy- and microcephaly-associated genes, with the shared genes enriched for genes involved in intellectual disability and/or developmental delay, and neuronal death pathways, respectively. This supported the increased burden of ciliopathy variants and prevalence of neurological abnormalities observed among HLHS patients with developmental delay and microcephaly, respectively.

Placental and fetal characteristics of the Ohia mouse line recapitulate outcomes in human hypoplastic left heart syndrome.

Congenital heart defects (CHDs) are the most common birth defect worldwide. The morbidity and mortality associated with these defects is compounded by increased frequency of fetal growth abnormalities. In the Ohia mouse model of hypoplastic left heart syndrome (HLHS), the double homozygous genotype is embryonically lethal at mid-pregnancy; a time in which optimal establishment of the placenta is crucial to fetal survival. We aimed to characterize placental and fetal growth and development in the double heterozygous genotype (Sap130m/+Pcdha9m/+). There was a shift in frequency of fetuses with reduced weight near term in the Sap130m/+Pcdha9m/+ fetuses compared to wildtype, driven by lower fetal weight in male fetuses compared to female. This shift in fetal weight distribution in the Sap130m/+Pcdha9m/+ fetuses was associated with reduced labyrinth region area (P < 0.001) and reduced fetal capillary density (P < 0.001) in the placentas, the latter being significantly lower in male Sap130m/+Pcdha9m/+ placentas compared to female. mRNA expression of several nutrient transporters was also lower in placentas from males compared to placentas from females, irrespective of genotype. Overall, this data shows that whilst the double heterozygous fetuses do not carry heart defects, placental development and function is impaired, particularly in males. Such differences are similar to findings in studies of human placentas and highlights the importance of this mouse model in continuing to understand the developmental links and disruptions to the heart-placenta axis.

VarSAn: associating pathways with a set of genomic variants using network analysis.

There is a pressing need today to mechanistically interpret sets of genomic variants associated with diseases. Here we present a tool called 'VarSAn' that uses a network analysis algorithm to identify pathways relevant to a given set of variants. VarSAn analyzes a configurable network whose nodes represent variants, genes and pathways, using a Random Walk with Restarts algorithm to rank pathways for relevance to the given variants, and reports P-values for pathway relevance. It treats non-coding and coding variants differently, properly accounts for the number of pathways impacted by each variant and identifies relevant pathways even if many variants do not directly impact genes of the pathway. We use VarSAn to identify pathways relevant to variants related to cancer and several other diseases, as well as drug response variation. We find VarSAn's pathway ranking to be complementary to the standard approach of enrichment tests on genes related to the query set. We adopt a novel benchmarking strategy to quantify its advantage over this baseline approach. Finally, we use VarSAn to discover key pathways, including the VEGFA-VEGFR2 pathway, related to de novo variants in patients of Hypoplastic Left Heart Syndrome, a rare and severe congenital heart defect.

Exploration and validation of hub genes and pathways in the progression of hypoplastic left heart syndrome via weighted gene co-expression network analysis.

BACKGROUND: Despite significant progress in surgical treatment of hypoplastic left heart syndrome (HLHS), its mortality and morbidity are still high. Little is known about the molecular abnormalities of the syndrome. In this study, we aimed to probe into hub genes and key pathways in the progression of the syndrome. METHODS: Differentially expressed genes (DEGs) were identified in left ventricle (LV) or right ventricle (RV) tissues between HLHS and controls using the GSE77798 dataset. Then, weighted gene co-expression network analysis (WGCNA) was performed and key modules were constructed for HLHS. Based on the genes in the key modules, protein-protein interaction networks were conducted, and hub genes and key pathways were screened. Finally, the GSE23959 dataset was used to validate hub genes between HLHS and controls. RESULTS: We identified 88 and 41 DEGs in LV and RV tissues between HLHS and controls, respectively. DEGs in LV tissues of HLHS were distinctly involved in heart development, apoptotic signaling pathway and ECM receptor interaction. DEGs in RV tissues of HLHS were mainly enriched in BMP signaling pathway, regulation of cell development and regulation of blood pressure. A total of 16 co-expression network were constructed. Among them, black module (r = 0.79 and p value = 2e-04) and pink module (r = 0.84 and p value = 4e-05) had the most significant correlation with HLHS, indicating that the two modules could be the most relevant for HLHS progression. We identified five hub genes in the black module (including Fbn1, Itga8, Itga11, Itgb5 and Thbs2), and five hub genes (including Cblb, Ccl2, Edn1, Itgb3 and Map2k1) in the pink module for HLHS. Their abnormal expression was verified in the GSE23959 dataset. CONCLUSIONS: Our findings revealed hub genes and key pathways for HLHS through WGCNA, which could play key roles in the molecular mechanism of HLHS.

Abnormalities of placental development and function are associated with the different fetal growth patterns of hypoplastic left heart syndrome and transposition of the great arteries.

BACKGROUND: Birthweight is a critical predictor of congenital heart disease (CHD) surgical outcomes. Hypoplastic left heart syndrome (HLHS) is cyanotic CHD with known fetal growth restriction and placental abnormalities. Transposition of the great arteries (TGA) is cyanotic CHD with normal fetal growth. Comparison of the placenta in these diagnoses may provide insights on the fetal growth abnormality of CHD. METHODS: Clinical data and placental histology from placentas associated with Transposition of the Great Arteries (TGA) were analyzed for gross pathology, morphology, maturity and vascularity and compared to both control and previously analyzed HLHS placentas [1]. RNA was isolated from HLHS, TGA and control placentas and sequenced by Illumina HiSeq.Transcriptome analysis was performed using AltAnalyze. Immunohistochemistry was utilized to assess placental nutrient transporter expression in all three groups. RESULTS: Placental weight was reduced in TGA cases, and demonstrated reduced villous vasculature, immature terminal villi in the parenchyma compared to controls and reflected our previous data from HLHS placentas. However, birth weight was not reduced in TGA cases compared to controls in contrast to the HLHS cohort and birthweight:placental weight ratio was significantly increased in TGA cases but not HLHS compared to control. Transcriptomic and histologic analysis demonstrates reduced cell activity and nutrient transport capability in HLHS but not TGA placentas which appear to increase/maintain these mechanisms. CONCLUSIONS: Despite common vascular disturbances in placentas from HLHAs and TGA, these do not account for the disparities in birthweights frequently seen between these CHD subtypes, in contrast our transcriptomic and histologic analyses reveal differentially regulated mechanisms between the subtypes that may explain these disparities.

Expression of connexin-43 in the cardiac muscle of children diagnosed with hypoplastic left heart syndrome: a Western blot and confocal laser scanning microscopy study.

Hypoplastic left heart syndrome consists of several structural abnormalities in the left side of the heart and may be associated with a hereditary genetic cause, possibly related to the connexin gene GJA1; however, only a few studies have investigated it. The present study aimed to analyse the expression of connexin-43 in the cardiac muscle of hypoplastic left heart syndrome children by Western blot method and confocal laser scanning microscopy. For that, tissue samples were taken during corrective surgery to treat heart defects. Patients of control group (8) presented any type of heart defect not related to hypoplastic left heart syndrome, connexin-43, or its gene and those of hypoplastic left heart syndrome group (9) presented this disease singly, without any other associated congenital diseases. By means of confocal laser scanning microscopy, it was noticed no connexin-43 qualitative differences in positioning and location pattern between both groups. From Western blot analysis, the connexin-43 expression did not show a statistically significant difference (p = 0.0571) as well. Within the limits of this study, it is suggested that cardiomyocytes of hypoplastic left heart syndrome children are similar in connexin-43 location, distribution, and structural and conformational patterns to those of children with heart defects not related to this protein and its genes.

Hypoplastic left heart syndrome: From bedside to bench and back.

Hypoplastic Left Heart Syndrome (HLHS) is a complex Congenital Heart Disease (CHD) that was almost universally fatal until the advent of the Norwood operation in 1981. Children with HLHS who largely succumbed to the disease within the first year of life, are now surviving to adulthood. However, this survival is associated with multiple comorbidities and HLHS infants have a higher mortality rate as compared to other non-HLHS single ventricle patients. In this review we (a) discuss current clinical challenges associated in the care of HLHS patients, (b) explore the use of systems biology in understanding the molecular framework of this disease, (c) evaluate induced pluripotent stem cells as a translational model to understand molecular mechanisms and manipulate them to improve outcomes, and (d) investigate cell therapy, gene therapy, and tissue engineering as a potential tool to regenerate hypoplastic cardiac structures and improve outcomes.

Re-evaluation of hypoplastic left heart syndrome from a developmental and morphological perspective.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) covers a spectrum of rare congenital anomalies characterised by a non-apex forming left ventricle and stenosis/atresia of the mitral and aortic valves. Despite many studies, the causes of HLHS remain unclear and there are conflicting views regarding the role of flow, valvar or myocardial abnormalities in its pathogenesis, all of which were proposed prior to the description of the second heart field. Our aim was to re-evaluate the patterns of malformation in HLHS in relation to recognised cardiac progenitor populations, with a view to providing aetiologically useful sub-groupings for genomic studies. RESULTS: We examined 78 hearts previously classified as HLHS, with subtypes based on valve patency, and re-categorised them based on their objective ventricular phenotype. Three distinct subgroups could be identified: slit-like left ventricle (24%); miniaturised left ventricle (6%); and thickened left ventricle with endocardial fibroelastosis (EFE; 70%). Slit-like ventricles were always found in combination with aortic atresia and mitral atresia. Miniaturised left ventricles all had normally formed, though smaller aortic and mitral valves. The remaining group were found to have a range of aortic valve malformations associated with thickened left ventricular walls despite being described as either atresia or stenosis. The degree of myocardial thickening was not correlated to the degree of valvar stenosis. Lineage tracing in mice to investigate the progenitor populations that form the parts of the heart disrupted by HLHS showed that whereas Nkx2-5-Cre labelled myocardial and endothelial cells within the left and right ventricles, Mef2c-AHF-Cre, which labels second heart field-derived cells only, was largely restricted to the endocardium and myocardium of the right ventricle. However, like Nkx2-5-Cre, Mef2c-AHF-Cre lineage cells made a significant contribution to the aortic and mitral valves. In contrast, Wnt1-Cre made a major contribution only to the aortic valve. This suggests that discrete cardiac progenitors might be responsible for the patterns of defects observed in the distinct ventricular sub-groups. CONCLUSIONS: Only the slit-like ventricle grouping was found to map to the current nomenclature: the combination of mitral atresia with aortic atresia. It appears that slit-like and miniature ventricles also form discrete sub-groups. Thus, reclassification of HLHS into subgroups based on ventricular phenotype, might be useful in genetic and developmental studies in investigating the aetiology of this severe malformation syndrome.

Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis.

Hypoplastic left heart syndrome (HLHS) is among the most severe forms of congenital heart disease. Although the consensus view is that reduced flow through the left heart during development is a key factor in the development of the condition, the molecular mechanisms leading to hypoplasia of left heart structures are unknown. We have generated induced pluripotent stem cells (iPSC) from five HLHS patients and two unaffected controls, differentiated these to cardiomyocytes and identified reproducible in vitro cellular and functional correlates of the HLHS phenotype. Our data indicate that HLHS-iPSC have a reduced ability to give rise to mesodermal, cardiac progenitors and mature cardiomyocytes and an enhanced ability to differentiate to smooth muscle cells. HLHS-iPSC-derived cardiomyocytes are characterised by a lower beating rate, disorganised sarcomeres and sarcoplasmic reticulum and a blunted response to isoprenaline. Whole exome sequencing of HLHS fibroblasts identified deleterious variants in NOTCH receptors and other genes involved in the NOTCH signalling pathway. Our data indicate that the expression of NOTCH receptors was significantly downregulated in HLHS-iPSC-derived cardiomyocytes alongside NOTCH target genes confirming downregulation of NOTCH signalling activity. Activation of NOTCH signalling via addition of Jagged peptide ligand during the differentiation of HLHS-iPSC restored their cardiomyocyte differentiation capacity and beating rate and suppressed the smooth muscle cell formation. Together, our data provide firm evidence for involvement of NOTCH signalling in HLHS pathogenesis, reveal novel genetic insights important for HLHS pathology and shed new insights into the role of this pathway during human cardiac development.

NOTCH1-Dependent Nitric Oxide Signaling Deficiency in Hypoplastic Left Heart Syndrome Revealed Through Patient-Specific Phenotypes Detected in Bioengineered Cardiogenesis.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) attributable to multifactorial molecular underpinnings. Multiple genetic loci have been implicated to increase the risk of disease, yet genotype-phenotype relationships remain poorly defined. Whole genome sequencing complemented by cardiac phenotype from five individuals in an HLHS-affected family enabled the identification of NOTCH1 as a prioritized candidate gene linked to CHD in three individuals with mutant allele burden significantly impairing Notch signaling in the HLHS-affected proband. To better understand a mechanistic basis through which NOTCH1 contributes to heart development, human induced pluripotent stem cells (hiPSCs) were created from the HLHS-affected parent-proband triad and differentiated into cardiovascular cell lineages for molecular characterization. HLHS-affected hiPSCs exhibited a deficiency in Notch signaling pathway components and a diminished capacity to generate hiPSC-cardiomyocytes. Optimization of conditions to procure HLHS-hiPSC-cardiomyocytes led to an approach that compensated for dysregulated nitric oxide (NO)-dependent Notch signaling in the earliest specification stages. Augmentation of HLHS-hiPSCs with small molecules stimulating NO signaling in the first 4 days of differentiation provided a cardiomyocyte yield equivalent to the parental hiPSCs. No discernable differences in calcium dynamics were observed between the bioengineered cardiomyocytes derived from the proband and the parents. We conclude that in vitro modeling with HLHS-hiPSCs bearing NOTCH1 mutations facilitated the discovery of a NO-dependent signaling component essential for cardiovascular cell lineage specification. Potentiation of NO signaling with small therapeutic molecules restored cardiogenesis in vitro and may identify a potential therapeutic target for patients affected by functionally compromised NOTCH1 variants. Stem Cells 2017;35:1106-1119.

Rbfox2 function in RNA metabolism is impaired in hypoplastic left heart syndrome patient hearts.

Hypoplastic left heart syndrome (HLHS) is a fatal congenital heart disease in which the left side of the heart is underdeveloped, impairing the systemic circulation. Underdeveloped left ventricle exerts biomechanical stress on the right ventricle that can progress into heart failure. Genome-wide transcriptome changes have been identified at early stages in the right ventricle (RV) of infants with HLHS, although the molecular mechanisms remain unknown. Here, we demonstrate that the RNA binding protein Rbfox2, which is mutated in HLHS patients, is a contributor to transcriptome changes in HLHS patient RVs. Our results indicate that majority of transcripts differentially expressed in HLHS patient hearts have validated Rbfox2 binding sites. We show that Rbfox2 regulates mRNA levels of targets with 3'UTR binding sites contributing to aberrant gene expression in HLHS patients. Strikingly, the Rbfox2 nonsense mutation identified in HLHS patients truncates the protein, impairs its subcellular distribution and adversely affects its function in RNA metabolism. Overall, our findings uncover a novel role for Rbfox2 in controlling transcriptome in HLHS.

Older Age at Completion of Fontan Procedure Is Associated with Improved Percentage of Predicted Maximum Oxygen Uptake.

We tested the hypothesis that later completion of the Fontan procedure is associated with improved exercise capacity in the current period of staged single-ventricle palliation. We performed a retrospective study, in Fontan patients, of exercise stress test data from April 2003 through March 2011. Patients were included if they had received staged palliations in accordance with current surgical strategy, defined as the performance of a superior cavopulmonary connection at ≤1 year of age, followed in subsequent years by Fontan completion. Patients with a pacemaker or respiratory exchange ratio <1 were excluded. Early and late Fontan groups were created on the basis of whether Fontan completion had been performed at <4 or ≥ 4 years of age. The primary predictor variable was age at Fontan completion, and the primary marker of exercise performance was the percentage of predicted maximum oxygen consumption. During the study period, 55 patients were identified (mean age, 11.7 ± 2.8 yr). Older age at Fontan completion correlated positively with higher percentages of predicted maximum oxygen consumption (R=0.286, P=0.034). Patients in whom Fontan completion was performed at ≥4 years of age had higher percentages of predicted maximum oxygen consumption than did those in whom completion was at <4 years of age (84.4 ± 21.5 vs 72.9 ± 18.1; P=0.041). Later Fontan completion might be associated with improved exercise capacity in patients palliated in accordance with contemporary surgical strategy.

Hypoplastic left heart syndrome is associated with structural and vascular placental abnormalities and leptin dysregulation.

INTRODUCTION: Hypoplastic left heart syndrome (HLHS) is a severe cardiovascular malformation (CVM) associated with fetal growth abnormalities. Genetic and environmental factors have been identified that contribute to pathogenesis, but the role of the placenta is unknown. The purpose of this study was to systematically examine the placenta in HLHS with and without growth abnormalities. METHODS: HLHS term singleton births were identified from a larger cohort when placenta tissue was available. Clinical data were collected from maternal and neonatal medical records, including anthropometrics and placental pathology reports. Placental tissues from cases and controls were analyzed to assess parenchymal morphology, vascular architecture and leptin signaling. RESULTS: HLHS cases (n = 16) and gestational age-matched controls (n = 18) were analyzed. Among cases, the average birth weight was 2993 g, including 31% that were small for gestational age. When compared with controls, gross pathology of HLHS cases demonstrated significantly reduced placental weight and increased fibrin deposition, while micropathology showed increased syncytial nuclear aggregates, decreased terminal villi, reduced vasculature and increased leptin expression in syncytiotrophoblast and endothelial cells. DISCUSSION: Placentas from pregnancies complicated by fetal HLHS are characterized by abnormal parenchymal morphology, suggesting immature structure may be due to vascular abnormalities. Increased leptin expression may indicate an attempt to compensate for these vascular abnormalities. Further investigation into the regulation of angiogenesis in the fetus and placenta may elucidate the causes of HLHS and associated growth abnormalities in some cases.

Aspirin Resistance in Single-Ventricle Physiology: Aspirin Prophylaxis Is Not Adequate to Inhibit Platelets in the Immediate Postoperative Period.

BACKGROUND: Incidence of thrombosis after initial stage 1 single-ventricle palliation is high. Most centers use aspirin as an antiplatelet agent to prevent thrombosis in surgically placed shunts. We hypothesize there is a significant incidence of aspirin resistance in infants after stage 1 palliation and this resistance can be overcome by an increased aspirin dose. METHODS: This is a prospective observational study of 20 patients with single-ventricle physiology who required single-ventricle palliation with a controlled source of pulmonary blood flow (Norwood/Sano, Norwood/Blalock-Taussig [BT] shunt or BT shunt alone). Aspirin resistance was determined using thromboelastography with platelet mapping (TEG) and urine thromboxane (UTX). The UTX level of less than 1,500 pg/mL and TEG value of more than 50% were used to define as adequate platelet inhibition. The UTX was measured prior to starting aspirin (20 mg/day) and TEG and UTX were obtained after 5 days of aspirin therapy A repeat UTX was measured for patients who were determined to be aspirin resistant by TEG (<50% arachidonic acid inhibition) after doubling the dose (40 mg/day). Clinical variables including patient diagnosis, age of surgery, and cardiopulmonary bypass requirement, weight, hemoglobin, and platelet count were assessed to determine their association with aspirin resistance. RESULTS: Eighty percent of patients were aspirin resistant using TEG (95% CI, 56% to 94%) and none of the patients achieved a UTX level of less than 1,500 pg/mL. Aspirin resistant patients did not respond to an increased dose of aspirin between the fifth and tenth days of therapy (p = 0.820). Patients did, however, respond to aspirin treatment when comparing the baseline UTX measurement with those recorded on the fifth day (p = 0.008) and the tenth day (p = 0.0361) of aspirin therapy. The UTX levels did not differ between those who were and those who were not aspirin resistant by TEG at any of the measurement times. The clinical variables were not associated with aspirin resistance status. CONCLUSIONS: There is a high incidence of aspirin resistance in the immediate postoperative period after single-ventricle shunt palliation. Aspirin might not be an adequate agent for shunt prophylaxis in this patient population. Further studies are needed to identify at-risk patients who might benefit from additional testing and specific anticoagulation.

Micro-RNA expression in hypoplastic left heart syndrome.

OBJECTIVE: Micro-RNAs (miRNAs) are important regulators of gene expression through interaction with the 3'UTR of target messenger RNAs (mRNAs). The role of miRNAs has been extensively studied in adult human and nonhuman animal models of heart disease. Hypoplastic left heart syndrome (HLHS) is the most common form of severe congenital heart disease and is an important cause of morbidity and mortality in infants and children. The objective of this work was to analyze the miRNA profile in HLHS patients. METHODS AND RESULTS: miRNA profile was determined in the right ventricle with the use of miRNA array, and expression was validated with the use of reverse-transcription polymerase chain reaction (RT-PCR). Based on bioinformatics analysis, targets were selected and their expression analyzed with the use of RT-PCR.We found that the miRNA profile of HLHS is novel, with few similarities between pediatric and adult idiopathic dilated cardiomyopathy. Moreover, our analysis identified putative targets for these miRNAs that are known to be important for cardiac development and disease, and that miRNAs and their putative targets are antithetically regulated. We also found that miRNA expression changes with stage of surgery, suggesting that volume unloading of the ventricle has important consequences for gene expression. CONCLUSIONS: Our data suggest a unique miRNA profile for HLHS that may be associated with defects in cardiac development and disease.

Oxygen supply to the fetal cerebral circulation in hypoplastic left heart syndrome: a simulation study based on the theoretical models of fetal circulation.

Hypoxia due to congenital heart diseases (CHDs) adversely affects brain development during the fetal period. Head circumference at birth is closely associated with neuropsychiatric development, and it is considerably smaller in newborns with hypoplastic left heart syndrome (HLHS) than in normal newborns. We performed simulation studies on newborns with CHD to evaluate the cerebral circulation during the fetal period. The oxygen saturation of cerebral blood flow in newborns with CHD was simulated according to a model for normal fetal circulation in late pregnancy. We compared the oxygen saturation of cerebral blood flow between newborns with tricuspid atresia (TA; a disease showing univentricular circulation and hypoplasia of the right ventricle), those with transposition of the great arteries (TGA; a disease showing abnormal mixing of arterial and venous blood), and those with HLHS. The oxygen saturation of cerebral blood flow in newborns with normal circulation was 75.7 %, whereas it was low (49.5 %) in both newborns with HLHS and those with TA. Although the oxygen level is affected by the blood flow through the foramen ovale, the oxygen saturation in newborns with TGA was even lower (43.2 %). These data, together with previous reports, suggest that the cerebral blood flow rate is decreased in newborns with HLHS, and the main cause was strongly suspected to be retrograde cerebral perfusion through a patent ductus arteriosus. This study provides important information about the neurodevelopmental prognosis of newborns with HLHS and suggests the need to identify strategies to resolve this unfavorable cerebral circulatory state in utero.

Calcium signaling regulates ventricular hypertrophy during development independent of contraction or blood flow.

In utero interventions aimed at restoring left ventricular hemodynamic forces in fetuses with prenatally diagnosed hypoplastic left heart syndrome failed to stimulate ventricular myocardial growth during gestation, suggesting chamber growth during development may not rely upon fluid forces. We therefore hypothesized that ventricular hypertrophy during development may depend upon fundamental Ca(2+)-dependent growth pathways that function independent of hemodynamic forces. To test this hypothesis, zebrafish embryos were treated with inhibitors or activators of Ca(2+) signaling in the presence or absence of contraction during the period of chamber development. Abolishment of contractile function alone in the setting of preserved Ca(2+) signaling did not impair ventricular hypertrophy. In contrast, inhibition of L-type voltage-gated Ca(2+) influx abolished contraction and led to reduced ventricular hypertrophy, whereas increasing L-type voltage-gated Ca(2+) influx led to enhanced ventricular hypertrophy in either the presence or absence of contraction. Similarly, inhibition of the downstream Ca(2+)-sensitive phosphatase calcineurin, a known regulator of adult cardiac hypertrophy, led to reduced ventricular hypertrophy in the presence or absence of contraction, whereas hypertrophy was rescued in the absence of L-type voltage-gated Ca(2+) influx and contraction by expression of a constitutively active calcineurin. These data suggest that ventricular cardiomyocyte hypertrophy during chamber formation is dependent upon Ca(2+) signaling pathways that are unaffected by heart function or hemodynamic forces. Disruption of Ca(2+)-dependent hypertrophy during heart development may therefore represent one mechanism for impaired chamber formation that is not related to impaired blood flow.