A critical role for the chromatin remodeller CHD7 in anterior mesoderm during cardiovascular development.

CHARGE syndrome is caused by spontaneous loss-of-function mutations to the ATP-dependant chromatin remodeller chromodomain-helicase-DNA-binding protein 7 (CHD7). It is characterised by a distinct pattern of congenital anomalies, including cardiovascular malformations. Disruption to the neural crest lineage has previously been emphasised in the aetiology of this developmental disorder. We present evidence for an additional requirement for CHD7 activity in the Mesp1-expressing anterior mesoderm during heart development. Conditional ablation of Chd7 in this lineage results in major structural cardiovascular defects akin to those seen in CHARGE patients, as well as a striking loss of cardiac innervation and embryonic lethality. Genome-wide transcriptional analysis identified aberrant expression of key components of the Class 3 Semaphorin and Slit-Robo signalling pathways in Chd7(fl/fl);Mesp1-Cre mutant hearts. CHD7 localises at the Sema3c promoter in vivo, with alteration of the local chromatin structure seen following Chd7 ablation, suggestive of direct transcriptional regulation. Furthermore, we uncover a novel role for CHD7 activity upstream of critical calcium handling genes, and demonstrate an associated functional defect in the ability of cardiomyocytes to undergo excitation-contraction coupling. This work therefore reveals the importance of CHD7 in the cardiogenic mesoderm for multiple processes during cardiovascular development.

Cor triatriatum dexter versus prominent Eustachian valve in an adult congenital heart disease patient.

An eustachian valve (EV) remnant, if present, is usually noted by the presence of a thin ridge or a crescent-shaped fold of endocardium arising from the anterior rim of the inferior vena cava orifice due to the persistence of the right sinus venosus valve. Though the embryologic explanation of cor triatriatum dexter (CTD) is the same as that of the normal formation of the EV--lack of regression of the right sinus venosus valve--it is usually called CTD or divided right atrium when there are attachments on the atrial septum giving the appearance of a divided atrium. However, it's called prominent eustachian valve when the right sinus venosus valve has partly regressed, with no remaining septal attachments and without the appearance of a divided atrium. We present the case of an adult patient with an atrial septal defect with a high insertion of a giant EV, which mimics the echocardiographic appearance of divided right atrium.

Lack of endothelial diaphragms in fenestrae and caveolae of mutant Plvap-deficient mice.

Plasmalemmal vesicle-associated protein (PLVAP, PV-1) is specifically expressed in endothelial cells in which it localizes to diaphragms of fenestrae, caveolae, and transendothelial channels. To learn about its function, we generated mutant mice that lack PLVAP. In a C57BL/6N genetic background, homozygous Plvap-deficient embryos die before birth and suffer from subcutaneous edema, hemorrhages, and defects in the vascular wall of subcutaneous capillaries. In addition, hearts of Plvap(-/-) embryos show ventricular septal defects and thinner ventricular walls. In wild-type embryos, PLVAP and caveolae with a stomatal diaphragm are present in endothelial cells of subcutaneous capillaries and endocardium, while a diaphragm is missing in caveolae of Plvap(-/-) littermates. Plvap(-/-) mice in a mixed C57BL/6N/FVB-N genetic background are born and survive at the most for 4 weeks. Capillaries of exocrine and endocrine pancreas and of kidney peritubular interstitium were investigated in more detail as examples of fenestrated capillaries. In these vascular beds, Plvap(-/-) mice show a complete absence of diaphragms in fenestrae, caveolae, and transendothelial channels, findings which are associated with a substantial decrease in the number of endothelial fenestrae. The changes in the capillary phenotype correlate with a considerable retardation of postnatal growth and anemia. Plvap(-/-) mice provide an animal model to clarify the specific functional role of endothelial fenestrae and their contribution to passage of water and solutes in different organs.

The W-loop of alpha-cardiac actin is critical for heart function and endocardial cushion morphogenesis in zebrafish.

Mutations in cardiac actin (ACTC) have been associated with different cardiac abnormalities in humans, including dilated cardiomyopathy and septal defects. However, it is still poorly understood how altered ACTC structure affects cardiovascular physiology and results in the development of distinct congenital disorders. A zebrafish mutant (s434 mutation) was identified that displays blood regurgitation in a dilated heart and lacks endocardial cushion (EC) formation. We identified the mutation as a single nucleotide change in the alpha-cardiac actin 1a gene (actc1a), resulting in a Y169S amino acid substitution. This mutation is located at the W-loop of actin, which has been implicated in nucleotide sensing. Consequently, s434 mutants show loss of polymerized cardiac actin. An analogous mutation in yeast actin results in rapid depolymerization of F-actin into fragments that cannot reanneal. This polymerization defect can be partially rescued by phalloidin treatment, which stabilizes F-actin. In addition, actc1a mutants show defects in cardiac contractility and altered blood flow within the heart tube. This leads to downregulation or mislocalization of EC-specific gene expression and results in the absence of EC development. Our study underscores the importance of the W-loop for actin functionality and will help us to understand the structural and physiological consequences of ACTC mutations in human congenital disorders.

ß-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos.

BACKGROUND: ß-Lapachone has antitumor and wound healing-promoting activities. To address the potential influences of various chemicals on heart development of zebrafish embryos, we previously treated zebrafish embryos with chemicals from a Sigma LOPAC1280™ library and found several chemicals including ß-lapachone that affected heart morphogenesis. In this study, we further evaluated the effects of ß-lapachone on zebrafish embryonic heart development. METHODS: Embryos were treated with ß-lapachone or dimethyl sulfoxide (DMSO) at 24 or 48 hours post fertilization (hpf) for 4 h at 28°C. Heart looping and valve development was analyzed by whole-mount in situ hybridization and histological analysis. For fractional shortening and wall shear stress analyses, AB and Tg (gata1:DsRed) embryos were recorded for their heart pumping and blood cell circulations via time-lapse fluorescence microscopy. Dextran rhodamine dye injection into the tail reticular cells was used to analyze circulation. Reactive oxygen species (ROS) was analyzed by incubating embryos in 5-(and 6-)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate (CM-H2DCFDA) and recorded using fluorescence microscopy. o-Dianisidine (ODA) staining and whole mount in situ hybridization were used to analyze erythrocytes. TUNEL assay was used to examine DNA fragmentation. RESULTS: We observed a linear arrangement of the ventricle and atrium, bradycardia arrhythmia, reduced fractional shortening, circulation with a few or no erythrocytes, and pericardial edema in ß-lapachone-treated 52-hpf embryos. Abnormal expression patterns of cmlc2, nppa, BMP4, versican, and nfatc1, and histological analyses showed defects in heart-looping and valve development of ß-lapachone-treated embryos. ROS production was observed in erythrocytes and DNA fragmentation was detected in both erythrocytes and endocardium of ß-lapachone-treated embryos. Reduction in wall shear stress was uncovered in ß-lapachone-treated embryos. Co-treatment with the NQO1 inhibitor, dicoumarol, or the calcium chelator, BAPTA-AM, rescued the erythrocyte-deficiency in circulation and heart-looping defect phenotypes in ß-lapachone-treated embryos. These results suggest that the induction of apoptosis of endocardium and erythrocytes by ß-lapachone is mediated through an NQO1- and calcium-dependent pathway. CONCLUSIONS: The novel finding of this study is that ß-lapachone affects heart morphogenesis and function through the induction of apoptosis of endocardium and erythrocytes. In addition, this study further demonstrates the importance of endocardium and hemodynamic forces on heart morphogenesis and contractile performance.

Endocardial arrhythmogenic mechanisms of torsades de pointes in patients with the congenital long QT syndrome.

We injected acetylcholine (Ach) into the coronary artery to ascertain whether coronary vasospasm contributed to the syncopal events or chest oppression suffered by 3 patients with long QT syndrome (LQTS). During the test, a quadripolar electrode catheter was placed in the right ventricle and the activation-recovery interval was reanalyzed from the stored data. Intracoronary Ach transiently prolonged the QT intervals in all 3 patients without inducing coronary vasospasm. The Ach-induced QT prolongation was associated with enhanced spatial and temporal dispersion of intra-ventricular repolarization. The electrophysiological abnormalities were consistent with the putative arrhythmogenic mechanisms identified in experimental studies of LQTS.

Localization of endocardial ectopic activity by means of noninvasive endocardial surface current density reconstruction.

Localization of the source of cardiac ectopic activity has direct clinical benefits for determining the location of the corresponding ectopic focus. In this study, a recently developed current-density (CD)-based localization approach was experimentally evaluated in noninvasively localizing the origin of the cardiac ectopic activity from body-surface potential maps (BSPMs) in a well-controlled experimental setting. The cardiac ectopic activities were induced in four well-controlled intact pigs by single-site pacing at various sites within the left ventricle (LV). In each pacing study, the origin of the induced ectopic activity was localized by reconstructing the CD distribution on the endocardial surface of the LV from the measured BSPMs and compared with the estimated single moving dipole (SMD) solution and precise pacing site (PS). Over the 60 analyzed beats corresponding to ten pacing sites (six for each), the mean and standard deviation of the distance between the locations of maximum CD value and the corresponding PSs were 16.9 mm and 4.6 mm, respectively. In comparison, the averaged distance between the SMD locations and the corresponding PSs was slightly larger (18.4 ± 3.4 mm). The obtained CD distribution of activated sources extending from the stimulus site also showed high consistency with the endocardial potential maps estimated by a minimally invasive endocardial mapping system. The present experimental results suggest that the CD method is able to locate the approximate site of the origin of a cardiac ectopic activity, and that the distribution of the CD can portray the propagation of early activation of an ectopic beat.

End-stage cardiac failure caused by isolated ventricular non-compaction: cardiac transplantation in a 5-year-old boy.

Isolated ventricular non-compaction (IVNC) is a rare congenital cardiomyopathy characterized by arrest in endomyocardial morphogenesis. Presenting features in children include systolic and diastolic ventricular dysfunction, cardiac arrhythmias, and thromboembolism. End-stage cardiac failure necessitating cardiac transplantation is described in adults. We report the unique case of a 5-year-old boy with IVNC and end-stage heart failure who underwent cardiac transplantation. Diagnostic echocardiographic features, gross cardiac morphology, and histopathologic findings are illustrated.

Clinical features of isolated left ventricular noncompaction in children.

BACKGROUND: Ventricular noncompaction is a rare unclassified cardiomyopathy due to intrauterin arrest of compaction of the loose interwoven meshwork with limited data regarding diagnosis and outcome in children. METHODS: In this study we describe clinical features of isolated left ventricular noncompaction (IVNC) in children and compare our findings with those previously reported. A diagnosis of ventricular noncompaction was made according to the characteristic echocardiographic appearance of two-layered myocardial wall consisting of a thin compacted epicardial and a thick noncompacted endocardial layer with numerous, prominent trabeculations and deep intertrabecular recesses communicating with left ventricular cavity. RESULTS: Nine children, eight male and one female, with ages ranging from 10 days to 12 years and follow-up as long as 5 years were included into the study. The primary diagnosis of IVNC had been missed in four of the patients. Clinical manifestations were heart failure in five patients, cardiac murmur in two, dizziness in one, and palpitation in one patient. All patients, except two asymptomatic ones, had electrocardiographic abnormalities. Four patients required hospital admission for decompansated heart failure. Two patients died during follow-up while waiting for heart transplantation. Familial occurrence, ventricular tachycardia and thromboembolic events were not observed in any of the patients. CONCLUSIONS: Although IVNC is present at birth, it may become clinically overt at any time from infancy through adolescence. Physicians and echocardiographers should be familiar with the diagnostic pattern of ventricular noncompaction in order to prevent any delay in diagnosis. Since associated morbidity and mortality rates are high, these patients require regular follow-up.

The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Endodermal heterotopia of the atrioventricular node associated with transposition of the great arteries.

A 13-year-old girl with surgically corrected complete transposition of the great arteries died suddenly while walking to school. Postmortem examination revealed endodermal heterotopia of the atrioventricular node, a condition not previously associated with transposition of the great arteries. New immunohistochemical observations are described, and the embryogenesis of the condition and its association with complete transposition are discussed.

Double-outlet right ventricle and overriding tricuspid valve reflect disturbances of looping, myocardialization, endocardial cushion differentiation, and apoptosis in TGF-beta(2)-knockout mice.

BACKGROUND: Transforming growth factor-beta(2) (TGF-beta(2)) is a member of a family of growth factors with the potential to modify multiple processes. Mice deficient in the TGF-beta(2) gene die around birth and show a variety of defects of different organs, including the heart. METHODS AND RESULTS: We studied the hearts of TGF-beta(2)-null mouse embryos from 11.5 to 18.5 days of gestation to analyze the types of defects and determine which processes of cardiac morphogenesis are affected by the absence of TGF-beta(2). Analysis of serial sections revealed malformations of the outflow tract (typically a double-outlet right ventricle) in 87.5%. There was 1 case of common arterial trunk. Abnormal thickening of the semilunar valves was seen in 4.2%. Associated malformations of the atrioventricular (AV) canal were found in 62.5% and were composed of perimembranous inlet ventricular septal defects (37.5%), AV valve thickening (33.3%), overriding tricuspid valve (25.0%), and complete AV septal defects (4.2%). Anomalies of the aorta and its branches were seen in 33.3%. Immunohistochemical staining showed failure of myocardialization of the mesenchyme of the atrial septum and the ventricular outflow tract as well as deficient valve differentiation. Morphometry documented this to be associated with absence of the normal decrease of total endocardial cushion volume in the older stages. Apoptosis in TGF-beta(2)-knockout mice was increased, although regional distribution was normal. CONCLUSIONS: TGF-beta(2)-knockout mice exhibited characteristic cardiovascular anomalies comparable to malformations seen in the human population.

Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo.

As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.

Hypoplasia of cushion ridges in the proximal outflow tract elicits formation of a right ventricle-to-aortic route in retinoic acid-induced complete transposition of the great arteries in the mouse: scanning electron microscopic observations of corrosion cast models.

BACKGROUND: The major morphologic change associated with retinoic acid (RA)-induced complete transposition of the great arteries (TGA), a congenital malformation of the heart, was investigated in a mouse model in which TGA was found in 80% of surviving fetuses. METHODS: Corrosion casts of embryonic hearts with or without prior exposure to retinoic acid were observed under a scanning electron microscope. RESULTS: In control hearts, indentations caused by expanded parietal and septal ridges in the outflow tract established right ventricle-to-left ventral pulmonic and left ventricle-to-right dorsal aortic routes before the aorticopulmonary septum completion. In RA-treated hearts, indentations of proximal regions of the parietal and septal ridges were small in the proximal outflow tract, whereas those in the distal regions developed well. These morphological features in the RA-treated hearts elicited right ventricle-to-right ventral aortic and left ventricle-to-left dorsal pulmonic routes in the TGA morphology. CONCLUSIONS: Hypoplasticity of the proximal regions of parietal and septal ridges in the outflow tract is one of the primary morphological abnormalities of the RA-induced TGA.

Dysplastic cardiac development presenting as cardiomyopathy.

Multiple manifestations of disordered or arrested cardiac muscle development are reported in a 14-year-old boy with clinical evidence of heart disease. These include persistent noncommunicating intramyocardial sinusoids, anomalous right ventricular muscle bands, muscular deficiency of ventricular septum, and papillary muscle underdevelopment. To our knowledge, this complex of findings has not been previously described.