Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects.

As part of a large-scale noninvasive fetal ultrasound screen to recover ethylnitrosourea (ENU)-induced mutations causing congenital heart defects in mice, we established a high-throughput ultrasound scanning strategy for interrogating fetal mice in utero utilizing three orthogonal imaging planes defined by the fetus' vertebral column and body axes, structures readily seen by ultrasound. This contrasts with the difficulty of acquiring clinical ultrasound imaging planes which are defined by the fetal heart. By use of the three orthogonal imaging planes for two-dimensional (2D) imaging together with color flow, spectral Doppler, and M-mode imaging, all of the major elements of the heart can be evaluated. In this manner, 10,091 ENU-mutagenized mouse fetuses were ultrasound scanned between embryonic days 12.5 and 19.5, with 324 fetuses found to die prenatally and 425 exhibiting cardiovascular defects. Further analysis by necropsy and histology showed heart defects that included conotruncal anomalies, obstructive lesions, and shunt lesions as well as other complex heart diseases. Ultrasound imaging also identified craniofacial/head defects and body wall closure defects, which necropsy revealed as encephalocele, holoprosencephaly, omphalocele, or gastroschisis. Genome scanning mapped one ENU-induced mutation associated with persistence truncus arteriosus and holoprosencephaly to mouse chromosome 2, while another mutation associated with cardiac defects and omphalocele was mapped to mouse chromosome 17. These studies show the efficacy of this novel ultrasound scanning strategy for noninvasive ultrasound phenotyping to facilitate the recovery of ENU-induced mutations causing congenital heart defects and other extracardiac anomalies.

Value of endomyocardial biopsy in infants, children and adolescents with dilated or hypertrophic cardiomyopathy and myocarditis.

Endomyocardial biopsy was performed in 20 symptomatic pediatric patients with the following clinical diagnoses: dilated cardiomyopathy in 16, hypertrophic cardiomyopathy in 2 and myocarditis in 2. Transfemoral biopsy was performed without complications in patients aged 2 months to 16 years; 6 were less than 1 year old. An average of five right ventricular specimens were obtained for histologic and ultrastructure study from each patient. In 16 patients with the clinical diagnosis of dilated cardiomyopathy, biopsy findings were compatible with the diagnosis in 8, but changed the diagnosis as follows in the other 8: myocarditis, 4; hypertrophic cardiomyopathy, 2 and carnitine deficiency syndromes, 2. In two patients with the clinical diagnosis of hypertrophic cardiomyopathy, biopsy findings confirmed the diagnosis in one and were normal in the other who had an encapsulated cardiac fibroma at operation. In two patients with the clinical diagnosis of myocarditis, biopsy findings confirmed the diagnosis in one and suggested dilated cardiomyopathy in the other. In the entire series, 25% had biopsy evidence of inflammatory disease. Biopsy findings were confirmed at subsequent autopsy in five cases.

Pulmonary atresia and intact ventricular septum. Definitive repair in the neonatal period.

Pulmonary atresia with an intact ventricular septum and a small right ventricle is associated with high mortality and lacks a consensus surgical approach. The results of operations in eight of eleven patients with either pulmonary atresia and an intact ventricular septum or critical pulmonary stenosis, hypoplastic right ventricle, and intact ventricular septum, who were operated on between 1983 and 1989, are presented. Definitive correction was performed via a right ventricular transannular patch with prolonged postoperative prostaglandin E1 infusion. Limiting conditions in using this approach were (1) severely hypoplastic right ventricle, (2) massive tricuspid regurgitation, or (3) right ventricle-dependent coronary artery blood supply. Eleven neonates had the aforementioned diagnoses; eight underwent definitive repair, five successfully. Successful outcome, up to 5 postoperative years, was achieved if the tricuspid valve diameter was greater than or equal to 0.75 cm, or if the tricuspid/mitral valve ratio was greater than or equal to 0.70. Other significant predictors of success were a tripartite right ventricle (p less than 0.006), lack of sinusoids (p less than 0.05), the ratio of the right ventricular internal and external diameters greater than or equal to 0.73 (p less than 0.05), and some contractility (p less than 0.04). Thus we choose a right ventricular transannular patch with long-term prostaglandin E1 infusion for patients with pulmonary atresia and intact ventricular septum if (1) they have a tricuspid valve diameter of 0.75 cm or 70% of the mitral valve size, (2) they have a tripartite right ventricle, (3) they exhibit some right ventricular contractility, (4) they do not have marked tricuspid valve insufficiency, and (5) the coronary arteries do not fill primarily from sinusoids.

Cardiac development and perinatal care of infants with neural crest-associated conotruncal defects.

The neural crest constitutes a developmental field which is a morphogenetically reactive unit of the embryo. Disruption of this developmental field causes a constellation of anomalies to occur. Clustering of phenotypic abnormalities has allowed clinicians to recognize neural crest-associated syndromes with developmental abnormalities of the cardiovascular system, head, and neck. Basic research is beginning to unravel how these phenotypic characteristics are related to specific gene defects expressed during development. Currently, we do not know a one-to-one relationship between phenotypes and genotypes. These neonates with neural crest-associated conotruncal defects are born with recognizable complex cyanotic heart defects that are ductal-dependent. It may be difficult to judge if they have DiGeorge or velocardiofacial syndromes; thus, genetic counseling is of importance. Besides their life-threatening cardiovascular defects, these neonates frequently have either transient or persistent hypocalcemia or severe immunodeficiencies that require critical care management. This review will focus on the basic research underpinnings and currently recommended clinical care of infants with neural crest-associated conotruncal defects.

Esophageal-aortic erosion associated with double aortic arch and tracheomalacia. Experience with 2 infants.

Patients with double aortic arch may require lengthy intubation for ventilatory support. The need for endotracheal and nasogastric intubation may be prolonged in such patients because of associated tracheomalacia. Iatrogenic tracheal or esophageal erosion with subsequent aortic fistulization is an unusual but catastrophic complication that may result from such intubation. We report the cases of 2 infants with double aortic arch and tracheomalacia who developed iatrogenic esophageal-aortic erosion. This complication was successfully managed in 1 of the infants. We conclude from our experience that the important steps in preventing this complication include 1) expediting the exclusion of upper-airway compromise in intubated infants who have a presentation characteristic of bronchospastic airway disease (hyperinflation and hypercapnia) that seems unresponsive to usual therapeutic measures; and 2) expediting the diagnosis of vascular ring in order to minimize the duration of dual tracheal and esophageal intubation. Effective management of this problem, once established, requires primary closure of the esophageal perforation, removal of the nasogastric tube, interposition of thick viable tissue between the esophagus and the aorta, and decompressive gastrostomy and feeding jejunostomy. Concomitant aortopexy may be appropriate.

A novel mouse model of X-linked cardiac hypertrophy.

We recovered a novel mouse mutant exhibiting neonatal lethality associated with severe fetal cardiac hypertrophy and with some adult mice dying suddenly with left ventricular hypertrophic cardiomyopathy. Using Doppler echocardiography, we screened surviving adult mice in this mutant line for cardiac hypertrophy. Cardiac dimensions were obtained either from two-dimensional images collected using a novel ECG-gated ultra-high-frequency ultrasound system or by traditional M-mode imaging on a clinical ultrasound system. These analyses identified, among the littermates, two populations of mice: those with apparent cardiac hypertrophy with hypercontractile function characterized by ejection fraction of 75-80%, and normal littermates with ejection fraction of 53-55%. Analysis of the ECG-gated two-dimensional cines indicated that the hypertrophy was of the nonobstructive type. Further analysis of heart-to-body weight ratio confirmed the ultrasound diagnosis of left ventricular hypertrophic cardiomyopathy. Histopathology showed increased ventricular wall thickness, enlarged myocyte size, and mild myofiber disarray. Ultrastructural analysis by electron microscopy revealed mitochondria hyperproliferation and dilated sarcoplasmic reticulum. Genome scanning using microsatellite DNA markers mapped the mutation to the X chromosome. DNA sequencing showed no mutations in the coding regions of several candidate genes on the X chromosome, including several known to be associated with left ventricular hypertrophic cardiomyopathy. These findings suggest that this mouse line may harbor a mutation in a novel gene causing X-linked cardiomyopathy.

Visual understanding of cardiac development: the neural crest's contribution.

Understanding normal and abnormal cardiovascular development is of interest to basic scientists as well as to clinicians taking care of infants with heart defects. This article presents a visual overview of cardiac development. It provides a framework on which to understand how abnormal cardiac development leads to groups of cardiovascular defects requiring clinical care. Human heart development is presented schematically and is correlated with similar points in chick cardiac development. Studying both normal and abnormal cardiac development in neural crest-ablated embryos has highlighted two major themes of cardiac development: there is a mechanism of differential growth in the developing cardiovascular system that is not seen to a major extent after birth and cardiac defects can be pictured as arrested stages of normal development. At a particular stage of development, it is normal to have a certain relationship between developing structures. However, if the development is arrested and this relationship of structures is allowed to persist, it then becomes abnormal. Visualizing heart defects as arrested points in normal development is better used as a tool to categorize defects than as a causative mechanism. The exact mechanisms of how abnormal development results in cardiac defects is not well understood. Study of the neural crest model of cardiac defects suggests possible mechanisms.

Neural crest ablation versus sham surgical effects in a chick embryo model of defective cardiovascular development.

In chick embryos, ablation of premigratory neural crest destined for the third, fourth, and sixth pharyngeal arches results in persistent truncus arteriosus and interrupted aortic arch. Studies of cardiogenesis in these embryos have shown decreased ejection fraction and ventricular dilation with normal cardiac output. The experimental embryos that survive to d 11 of incubation have a higher cardiac output than nonsurvivors at the same earlier stage of development. We hypothesize that this survival is due to a surgically induced decrease in vascular resistance of the embryonic and vitelline vessels. Embryos from 15 opened eggs, eight sham-operated embryos, and 13 neural crest-ablated embryos were examined at stage 18. The sham-operated embryos were treated identically with the experimental embryos except that neural crest was not ablated. Ejection fraction and cardiac output were determined by cinephotography. Mean dorsal aortic and diastolic ventricular (approximates mean atrial) pressures were determined by a servo-null pressure technique and used to calculate vascular resistance. The experimental embryos had a significantly decreased ejection fraction in comparison with either sham-operated embryos or embryos from eggs that remained unopened until just before cinephotography. The experimental and sham-operated embryos had a higher cardiac output than embryos from unopened eggs. The calculated vascular resistance was also significantly lower in both the experimental and sham-operated embryos than in the embryos from unopened eggs. Neural crest ablation appears to cause a decreased ejection fraction. Sham surgery is associated with a higher cardiac output and lower vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Noninvasive assessment of cardiac output in children using impedance cardiography.

This study evaluated the effect of intracardiac shunting on the accuracy of thoracic bioimpedance-derived cardiac output determinations. Twenty-six patients, ranging in age from 3 months to 17 years, underwent cardiac catheterization during which simultaneous Fick and impedance measurements of cardiac output were obtained. The subjects were divided into three groups: 10 children with no intracardiac shunts, nine children with predominant left-to-right intracardiac shunts, and seven children with predominant right-to-left intracardiac shunts. Positive correlations between impedance and Fick-derived cardiac output determinations were obtained in the non-shunt group (r = 0.84), with lower correlations in the left-to-right shunt group (r = 0.70). In the right-to-left shunt group, the impedance derived cardiac output correlated with Fick pulmonary flow (r = 0.82), but the variability was unacceptably large. Although further study is needed, impedance cardiography appears to have validity as a methodology in pediatric critical care and cardiovascular health research.

Microcinephotography of the developing heart in neural crest-ablated chick embryos.

Microcinephotography was used to study heart development in a neural crest model of heart defects, that is, persistent truncus arteriosus, interrupted aortic arch, double outlet right ventricle, or single ventricle and tricuspid valve anomalies. These defects were created in chick embryos by ablation of premigratory neural crest destined for the aorticopulmonary and truncal septa, as well as the third and fourth aortic arch arteries. When embryogenesis reached the looped cardiac tube stage of development (Hamburger-Hamilton stage 18), 19 experimental and 15 control embryos were filmed at 100 frames per second under controlled environmental conditions. Analysis of the microcinephotography films showed the following significant distinguishing characteristics of the developing heart in the experimental embryos: altered conotruncal shape in 100%, depressed contractility and dilation of the primitive ventricle in 84%, decreased emptying of the bulbus cordis in 79%, incompetent truncal cushions in 68%, incomplete looping of the cardiac tube in 58%, and fourth right aortic arch artery without blood flow and third right aortic arch artery with increased flow in 53%. These abnormal characteristics suggested that there were functional and morphological changes in the developing heart of experimental embryos before the time when the predicted structural heart defects would be apparent. It is proposed that the primitive ventricle might attempt to compensate for depressed contractility by ventricular dilation. The incompetent truncal cushions could be secondary to the depressed contractility or secondary to the neural crest ablation that is known to cause persistent truncus arteriosus, an interrupted aortic arch, or both. The absence of blood flow in the right fourth aortic arch artery that will become the definitive aorta correlates with the expected incidence of interrupted aortic arches in this neural crest-ablation model of heart defects. It is speculated that the incomplete looping of the cardiac tube might hinder normal developmental alignment of the outflow and inflow tracts, producing a spectrum of lesions of maldevelopment of the tricuspid valve and dextroposition of the aorta.

Relation of early hemodynamic changes to final cardiac phenotype and survival after neural crest ablation in chick embryos.

BACKGROUND: Microcinephotography was used to study a model of persistent truncus arteriosus created in chick embryos by ablation of premigratory neural crest destined for the third and fourth aortic arch arteries as well as the septum of the cardiac outflow tract. METHODS AND RESULTS: Twenty-five control embryos and 105 of 202 experimental embryos were filmed on day 3 of incubation and then reincubated. The remaining 97 experimental embryos were not filmed because of twisting of the embryos, but they were reincubated. There was no difference in either the survival rate (p greater than 0.23) from day 3 to day 11 of incubation or the incidence of persistent truncus arteriosus (p greater than 0.08) between the filmed and the nonfilmed embryos. Incomplete looping of the cardiac tube observed in experimental embryos during early cardiogenesis correlated with a right ventricular origin of the outflow vessels in the definitive heart. Hemodynamic measurements indicated that there was no difference in heart rate, ejection fraction, systolic and diastolic areas, stroke volume, and cardiac output between controls and the experimental group as a whole. However, embryos that did not survive to day 11 had decreased stroke volume (p less than 0.001) and cardiac output (p less than 0.001), whereas embryos that survived to day 11 with cardiac malformations had increased stroke volume and cardiac output in early embryogenesis. CONCLUSIONS: Increased stroke volume and cardiac output may be necessary factors for survival in embryos with cardiac dysmorphogenesis and probably are associated with dilation of the ventricular portion of the cardiac tube, which leads to malalignment of the outflow vessel or vessels.

Hemodynamic changes and compensatory mechanisms during early cardiogenesis after neural crest ablation in chick embryos.

Microcinephotography was used to study early heart development in chick embryos with ablations of the neural crest known to result in persistent truncus arteriosus with associated aortic arch anomalies. The premigratory neural crest destined for the 3rd and 4th pharyngeal arches and the aorticopulmonary septum were ablated. When the embryos reached the looped cardiac tube stage (stage 18), 15 experimental and 15 control embryos were filmed at 100 frames/s under controlled environmental conditions. End-diastolic and end-systolic dimensions were determined for the conotruncus and presumptive right ventricle that together compose the bulbus cordis. The results showed that the shortening fractions and ejection fractions were significantly depressed in the experimental embryos. The experimental embryos exhibited dilation and decreased emptying of the ventricle. There was no difference in heart rate or stroke volume between the control and experimental embryos. Thus, the calculated cardiac output was the same in the control and experimental groups. It appeared that the experimental embryos compensated for decreased contractility by ventricular dilation. These functional compensations in very early cardiac development may play an etiologic role in the subsequent development of structural heart defects.

A novel use of Amplatzer duct occluder.

This report describes the use of the Amplatzer patent ductus arteriosus occluder to close a left ventricle to descending aorta conduit. The patient was a 10-year-old male who was born with critical aortic stenosis and left ventricular outflow tract obstruction. After initial valvotomy, he underwent left ventricular to descending aorta conduit placement. At the age of 10, he had a Konno procedure to enlarge the left ventricular outflow tract and 21-mm St. Jude aortic valve placement. Closure of the conduit was not addressed because it was inaccessable from median sternotomy. Postoperatively, echocardiogram revealed significant flow through the conduit with a wide pulse pressure. Cardiac catheterization was performed with the premise to close the conduit with an Amplatzer patent ductus arteriosus occluder device.

Transient cranial hemorrhage does not cause depressed contractility in cardiac neural crest-ablated chick embryos.

Ablation of cardiac neural crest results in cardiovascular malformations. Depressed ventricular contractility has been noted in cardiac neural crest-ablated embryos several hours before the time when neural crest cells would have arrived in the cardiac outflow tract and several days before the appearance of any malformations. The reason for this depressed heart function is not known. Recently, transient cranial hemorrhages were found in chick embryos during the third day of incubation, several hours before depressed ventricular contractility can be measured. We sought to determine whether depressed ventricular contractility could be associated with these transient hemorrhages. We were also interested in defining some of the factors that influence the incidence and severity of hemorrhaging. Three groups of embryos were used: cardiac neural crest-ablated, sham-operated, and unopened controls. All groups were found to experience transient hemorrhage from a common origin in the forebrain. However, the incidence and degree of hemorrhage were higher and more severe in embryos with cardiac neural crest ablation than in the other two groups. The cardiac ejection and shortening fractions were measured at stage 18 in embryos with and without hemorrhaging, and it was found that a decrease in these parameters was associated solely with ablation of cardiac neural crest and not with hemorrhage. By altering the incubation conditions, we determined that conditions that increase the oxygen in the air space are associated with increased severity and occurrence of hemorrhage and decreased viability in the first 3 days of incubation. Our results indicate that transient cranial hemorrhages does not cause depressed contractility in cardiac neural crest-ablated embryos, and increased severity of hemorrhaging is most likely due to an increase in oxygen availability.

Exercise electrocardiography of health black children.

Exercise electrocardiography was performed on 170 healthy black children 7 to 14 years of age in order to determine the normal childhood electrocardiographic response to exercise. R-wave amplitude decreased from 27 +/- 8 (SD) to 22 +/- 8 mm (P less than .01) and the S-wave amplitude increased from 6.9 +/- 4.4 to 7.8 +/- 5 mm (P less than .01), indicating a shift of the mean QRS vector to the right at maximum exercise. J-point depression of 1.0 mm or greater was observed in 2.3% of children at maximum exercise, using the PR isoelectric line. ST-segment slope increased from 1.5 +/- 0.7 to 4.3 +/- 1.5 mV/sec at maximum. T-wave duration decreased with exercise and T-wave amplitude initially decreased with mild exercise but exceeded resting values at maximum exercise. No dysrhythmias were observed during or after the exercise study.

Role of cardiac neural crest cells in cardiovascular development.

The discovery in the chick embryo that a specific region of the neural crest, termed the cardiac neural crest, is essential for septation of the cardiac outflow tract and for aortic arch artery development has led to the classification of a whole series of human cardiac defects as neural crest-associated. Recently, several mouse genetic models have been effectively employed to yield new insights into the relationship between cardiac neural crest and structural heart development. In all the animal models of neural crest-related heart defects, prenatal mortality is too high to be attributed to structural defects of the heart alone, and there are obvious signs of severe cardiac dysfunction. The evidence indicates that poor viability is from impaired cardiac excitation-contraction coupling and contractile function at the myocyte level. The continued study of experimental and genetically defined models with neural crest-associated heart defects will prove useful in identifying the common pathways by which the neural crest contributes to normal heart development.

Abnormalities in lymphocyte populations in infants with neural crest cardiovascular defects.

The DiGeorge syndrome has been associated with various immune deficits. Embryologically, defects of the neural crest are associated with conotruncal and aortic arch abnormalities. The objective of this study was to determine if children with neural crest congenital heart defects can have subtle but significant immunodeficiencies. Complete blood counts with differential counts and a standard lymphocyte immunophenotyping panel of selected monoclonal antibodies were performed on peripheral blood from 20 children with neural crest cardiac disease and 34 normal newborns. The children with cardiac disease were grouped as survivors and nonsurvivors. The mean total white blood cell count was similar for all groups, but the percent lymphocytes was significantly less in the nonsurvivors than in the survivors and normal newborns (p < 0. 02). The lymphocyte subsets affected were CD2, CD3, and CD4. When the cardiac patients were compared to the normal newborns, again differences in lymphocyte subsets CD2, CD3, and CD4 were seen. When comparing nonsurvivors with survivors, the mean percentages of the CD2, CD3, and CD4 T lymphocyte markers, as well as the mean lymphocyte, B cell (CD20), and natural killer cell (CD16) percentages were all lower in the nonsurvivors. It was concluded that abnormalities in specific lymphocyte populations and their subsets may be predictors of infants at greatest risk for immunodeficiency complications. Therefore children with neural crest cardiac defects should have evaluations of lymphocyte subsets at birth and be treated as if potentially immunodeficient.

HIRA, a DiGeorge syndrome candidate gene, is required for cardiac outflow tract septation.

DiGeorge syndrome (DGS) is a congenital disease characterized by defects in organs and tissues that depend on contributions by cell populations derived from neural crest for proper development. A number of candidate genes that lie within the q11 region of chromosome 22 commonly deleted in DGS patients have been identified. Orthologues of the DGS candidate gene HIRA are expressed in the neural crest and in neural crest-derived tissues in both chick and mouse embryos. By exposing a portion of the premigratory chick neural crest to phosphorothioate end-protected antisense oligonucleotides, ex ovo, followed by orthotopic backtransplantation to the untreated embryos, we have shown that the functional attenuation of cHIRA in the chick cardiac neural crest results in a significantly increased incidence of persistent truncus arteriosus, a phenotypic change characteristic of DGS, but does not affect the repatterning aortic arch arteries, the ventricular function, or the alignment of the outflow tract.