Molecular Pathways and Animal Models of Truncus Arteriosus.

In normal cardiovascular development in birds and mammals, the outflow tract of the heart is divided into two distinct channels to separate the oxygenated systemic blood flow from the deoxygenated pulmonary circulation. When the process of outflow tract septation fails, a single common outflow vessel persists resulting in a serious clinical condition known as persistent truncus arteriosus or common arterial trunk. In this chapter, we will review molecular pathways and the cells that are known to play a role in the formation and development of the outflow tract and how genetic manipulation of these pathways in animal models can result in common arterial trunk.

Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease.

Pre-B cell leukemia factor 1 (PBX1) is an essential developmental transcription factor, mutations in which have recently been associated with CAKUTHED syndrome, characterized by multiple congenital defects including congenital heart disease (CHD). During analysis of a whole-exome-sequenced cohort of heterogeneous CHD patients, we identified a de novo missense variant, PBX1:c.551G>C p.R184P, in a patient with tetralogy of Fallot with absent pulmonary valve and extra-cardiac phenotypes. Functional analysis of this variant by creating a CRISPR-Cas9 gene-edited mouse model revealed multiple congenital anomalies. Congenital heart defects (persistent truncus arteriosus and ventricular septal defect), hypoplastic lungs, hypoplastic/ectopic kidneys, aplastic adrenal glands and spleen, as well as atretic trachea and palate defects were observed in the homozygous mutant embryos at multiple stages of development. We also observed developmental anomalies in a proportion of heterozygous embryos, suggestive of a dominant mode of inheritance. Analysis of gene expression and protein levels revealed that although Pbx1 transcripts are higher in homozygotes, amounts of PBX1 protein are significantly decreased. Here, we have presented the first functional model of a missense PBX1 variant and provided strong evidence that p.R184P is disease-causal. Our findings also expand the phenotypic spectrum associated with pathogenic PBX1 variants in both humans and mice.

Conditional inactivation of Foxc1 and Foxc2 in neural crest cells leads to cardiac abnormalities.

Cardiac neural crest cells (cNCCs) are required for normal heart development. cNCCs are a multipotent and migratory cell lineage that differentiates into multiple cell types. cNCCs migrate into the developing heart to contribute to the septation of the cardiac outflow tract (OFT). Foxc1 and Foxc2 are closely related members of the FOX (Forkhead box) transcription factor family and are expressed in cNCC during heart development. However, the precise role of Foxc1 and Foxc2 in cNCCs has yet to be fully described. We found that compound NCC-specific Foxc1;Foxc2 mutant embryos exhibited persistent truncus arteriosus (PTA), ventricular septal defects (VSDs), and thinning of the ventricular myocardium. Loss of Foxc1/c2 expression in cNCCs resulted in abnormal patterns of cNCC migration into the OFT without the formation of the aorticopulmonary septum. Further, loss of Foxc1 expression in cNCCs resulted in normal OFT development but abnormal ventricular septal formation. In contrast, loss of Foxc2 expression in NCCs led to no obvious cardiac abnormalities. Together, we provide evidence that Foxc1 and Foxc2 in cNCCs are cooperatively required for proper cNCC migration, the formation of the OFT septation, and the development of the ventricles. Our data also suggests that Foxc1 expression may play a larger role in ventricular development compared to Foxc2.

Bmp signaling represses Vegfa to promote outflow tract cushion development.

Congenital heart disease (CHD) is a devastating anomaly that affects ∼1% of live births. Defects of the outflow tract (OFT) make up a large percentage of human CHD. We investigated Bmp signaling in mouse OFT development by conditionally deleting both Bmp4 and Bmp7 in the second heart field (SHF). SHF Bmp4/7 deficiency resulted in defective epithelial to mesenchymal transition (EMT) and reduced cardiac neural crest ingress, with resultant persistent truncus arteriosus. Using a candidate gene approach, we found that Vegfa was upregulated in the Bmp4/7 mutant hearts. To determine if Vegfa is a downstream Bmp effector during EMT, we examined whether Vegfa is transcriptionally regulated by the Bmp receptor-regulated Smad. Our findings indicate that Smad directly binds to Vegfa chromatin and represses Vegfa transcriptional activity. We also found that Vegfa is a direct target for the miR-17-92 cluster, which is also regulated by Bmp signaling in the SHF. Deletion of miR-17-92 reveals similar phenotypes to Bmp4/7 SHF deletion. To directly address the function of Vegfa repression in Bmp-mediated EMT, we performed ex vivo explant cultures from Bmp4/7 and miR-17-92 mutant hearts. EMT was defective in explants from the Bmp4/7 double conditional knockout (dCKO; Mef2c-Cre;Bmp4/7(f/f)) and miR-17-92 null. By antagonizing Vegfa activity in explants, EMT was rescued in Bmp4/7 dCKO and miR-17-92 null culture. Moreover, overexpression of miR-17-92 partially suppressed the EMT defect in Bmp4/7 mutant embryos. Our study reveals that Vegfa levels in the OFT are tightly controlled by Smad- and microRNA-dependent pathways to modulate OFT development.

TGFß and Wnt in cardiac outflow tract defects in offspring of diabetic pregnancies.

BACKGROUND: Diabetes mellitus in pregnancy causes defects in infant heart, including the outflow tracts (OFTs). Development of the aorta and pulmonary artery, which are derived from the common OFT in the embryo, is regulated by the transforming growth factor ß (TGFß) and Wnt families, and can be perturbed by hyperglycemia-generated intracellular stress conditions. However, the underlying cellular and molecular mechanisms remain to be delineated. METHODS: Female mice were induced diabetic with streptozotocin. Embryonic and fetal OFTs were examined morphologically and histologically. Cell proliferation was assessed using 5'-bromo-2'-deoxyuridine incorporation assay. Oxidative and endoplasmic reticulum (ER) stress markers and TGFß factors were detected using immunohistochemistry. The expression of genes in the Wnt-signaling system was assessed using real-time reverse transcription polymerase chain reaction array. The role of activin-A in cell proliferation was addressed by treating embryos cultured in high glucose with activin-A. RESULTS: Maternal diabetes caused complex abnormalities in the OFTs, including aortic and pulmonary stenosis and persistent truncus arteriosus. The development of the endocardial cushions was suppressed, manifested with insufficient cellularization of the tissues. Cell proliferation was significantly decreased under oxidative and ER stress conditions. The expression of genes in the Wnt signaling was significantly altered. Activin-A and Smad3 were found to be expressed in the OFT. Treatment with activin-A rescued cell proliferation in the endocardial cushions. CONCLUSIONS: Maternal diabetes generates oxidative and ER stress conditions, suppresses TGFß and Wnt signaling, inhibits cell proliferation and cellularization of the endocardial cushions, leading to OFT septal defects. Activin-A plays a role in hyperglycemia-suppressed proliferation of the endocardial cells.

Long-term follow-up of persistent truncus arteriosus: Kuwait experience.

OBJECTIVE: To evaluate the long-term results of patients in Kuwait who were operated for persistent truncus arteriosus (PTA). SUBJECTS AND METHODS: The following data were collected for retrospective analysis from 24 medical records of consecutive patients with PTA in Kuwait between August 1993 and August 2009: demographics, morphology, management and outcome. Major associated abnormalities included interrupted aortic arch in 1 patient and abnormal coronary artery anatomy in 2. RESULTS: Of the 24 patients, 16 underwent total intracardiac repair. The age at operation ranged from 15 days to 5 years (mean 166.19 ± 438.63 days) and weight ranged from 2.5 to 15 kg (mean 4.3 ± 3.01 kg). The right ventricle to pulmonary artery continuity was established with aortic homograft in 11, pulmonary homograft in 4 and by implantation of a Contegra conduit in 1 patient. Four patients had moderate truncal valve regurgitation requiring concomitant truncal valve repair. After a mean follow-up period of 81.81 ± 61.58 months (range 3-166) there was no death. Eight of the 16 (50%) patients underwent redo homograft operations. One patient who had concomitant truncal valve repair subsequently underwent aortic valve replacement. CONCLUSION: The data showed that complete repair of PTA in the neonatal and early infancy period was the treatment with the best potential for survival. The homograft remained one of the conduits of choice to establish continuity between the right ventricle and the pulmonary artery in spite of the high incidence of conduit redo operations.

The quadricuspid truncal valve: Surgical management and outcomes.

OBJECTIVE: To determine the outcomes of patients with a quadricuspid truncal valve (TV) and durability of TV repair. METHOD: We reviewed 56 patients with truncus arteriosus and a quadricuspid TV who underwent complete repair between 1979 and 2018. RESULTS: TV insufficiency was present in 39 patients (mild, n = 22; moderate, n = 14; and severe, n = 3). Fourteen patients had concomitant TV surgery. Early mortality in patients who had concomitant TV surgery was 14% (2 out of 14 patients) and overall survival was 77.1% ± 11.7% at 15 years. Freedom from TV reoperation was 30.3% ± 14.6% at 15 years. Early mortality in patients who did not undergo concomitant TV surgery was 9.5% (4 out of 42 patients) and overall survival was 74.9% ± 6.9% at 15 years. Progression of TV insufficiency requiring TV surgery occurred in 16.7% (7 out of 42 patients). Freedom from TV reoperation was 77.1% ± 7.8% at 15 years. The most common method of repair was tricuspidization of the TV. Freedom from TV reoperation was 64.3% ± 21.0% at 10 years after tricuspidization and 0% at 6 years after other types of TV surgery. Overall follow-up was 97.6% (41 out of 42 patients) complete for survivors with median follow-up of 16.6 years. At last follow-up there was no TV insufficiency in 16 patients, mild insufficiency in 24 patients, and moderate insufficiency in 1 patient. CONCLUSIONS: More than one-third of patients with a quadricuspid TV require TV surgery. Tricuspidization of the quadricuspid TV appears to be a durable repair option with good long-term outcomes.

Anomalous origin of a coronary artery from the right branchiocephalic trunk associated with complex congenital heart disease.

This report describes the extremely rare case of a 42-day-old boy with anomalous origin of a single coronary artery from the right branchiocephalic trunk associated with hypoplastic left ventricle, mitral atresia, truncus arteriosus, total anomalous pulmonary venous drainage, and patent foramen ovale.

Persistent truncus arteriosus with dissecting aneurysm and subsequent cardiac tamponade in a lamb.

The present case report describes the necropsy and histopathology findings of a 3-week-old lamb with persistent truncus arteriosus (PTA). This rare cardiac malformation was characterized by the presence of a common arterial trunk arising from the right ventricle and overriding a ventricular septal defect. The pulmonary arteries originated from a short common trunk from this PTA, which subsequently continued as the thoracic aorta. The death of the lamb was attributed to a rupture of the PTA with subsequent cardiac tamponade. Histologically, a dissecting aneurysm with elastic fiber fragmentation in the wall of the PTA was identified as the underlying pathologic condition. Altered hemodynamic forces with subsequent secondary vasculopathy, as well as congenital primary vasculopathies, should be considered as an underlying pathogenetic mechanism.

Common arterial trunk with thanatophoric dysplasia: a unique case.

We report a case of common arterial trunk with thanatophoric dysplasia. The skeletal dysplasia was diagnosed during fetal life. Following early induction of labour, there was a post-mortem diagnosis of type 2 common arterial trunk. This report includes the radiological and post-mortem findings. To our knowledge, thanatophoric dysplasia has never previously been reported in combination with any major cardiac defect.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development.

BACKGROUND: Congenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice. METHODS AND RESULTS: We analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos. CONCLUSION: It is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.

Myocardial wall stiffening in a mouse model of persistent truncus arteriosus.

BACKGROUND: Genetic and epigenetic programs regulate dramatic structural changes during cardiac morphogenesis. Concurrent biomechanical forces within the heart created by blood flow and pressure in turn drive downstream cellular, molecular and genetic responses. Thus, a genetic-morphogenetic-biomechanical feedback loop is continually operating to regulate heart development. During the evolution of a congenital heart defect, concomitant abnormalities in blood flow, hemodynamics, and patterns of mechanical loading would be predicted to change the output of this feedback loop, impacting not only the ultimate morphology of the defect, but potentially altering tissue-level biomechanical properties of structures that appear structurally normal. AIM: The goal of this study was to determine if abnormal hemodynamics present during outflow tract formation and remodeling in a genetically engineered mouse model of persistent truncus arteriosus (PTA) causes tissue-level biomechanical abnormalities. METHODS: The passive stiffness of surface locations on the left ventricle (LV), right ventricle (RV), and outflow tract (OFT) was measured with a pipette aspiration technique in Fgf8;Isl1Cre conditional mutant embryonic mouse hearts and controls. Control and mutant experimental results were compared by a strain energy metric based on the measured relationship between pressure and aspirated height, and also used as target behavior for finite element models of the ventricles. Model geometry was determined from 3D reconstructions of whole-mount, confocal-imaged hearts. The stress-strain relationship of the model was adjusted to achieve an optimal match between model and experimental behavior. RESULTS AND CONCLUSION: Although the OFT is the most severely affected structure in Fgf8;Isl1Cre hearts, its passive stiffness was the same as in control hearts. In contrast, both the LV and RV showed markedly increased passive stiffness, doubling in LVs and quadrupling in RVs of mutant hearts. These differences are not attributable to differences in ventricular volume, wall thickness, or trabecular density. Excellent agreement was obtained between the model and experimental results. Overall our findings show that hearts developing PTA have early changes in ventricular tissue biomechanics relevant to cardiac function and ongoing development.

Factors associated with outcomes of persistent truncus arteriosus.

OBJECTIVES: The purpose of this study was to identify trends and factors associated with outcomes of persistent truncus arteriosus (PTA). BACKGROUND: Although there have been significant improvements, PTA continues to be associated with significant morbidity and mortality. METHODS: We undertook a review of all consecutive cases of PTA (n = 205) presenting at our institution from 1953 to 1997. Data were collected regarding demographics, anatomy, management (surgical palliation and repair) and outcomes (mortality and reoperation). RESULTS: Significant trends (p < or = 0.001) related to groups defined by year of birth were as follows: number of cases (1953-1967, n = 13; 1968-1977, n = 42; 1978-1987, n = 69; 1988-1997, n = 81), median age at first assessment (8 months, 42 days, 7 days and 2 days, respectively), proportion who did not have any surgery (58%, 27%, 22% and 11%), proportion who had an initial palliative procedure (25%, 37%, 6% and 2%), proportion who underwent PTA repair (31%, 59%, 72% and 88%), median age at PTA repair (11.2 years, 1.1 years, 1.6 months and 12 days) and proportion dying before hospital discharge after repair (50%, 63%, 56% and 41%). Since 1995, mortality before hospital discharge after repair has further decreased to 2/11 (18%). Increasing time to initial conduit replacement in hospital survivors was significantly related to larger sized conduit at repair (p = 0.02) and use of pulmonary homografts (vs. aortic homografts or xenografts; p = 0.002). Interventional catheterization to address conduit obstructions significantly increased conduit longevity. CONCLUSIONS: Significant improvements in PTA outcomes are evident with trends toward earlier age at assessment and complete repair.

Coronary arteries in truncus arteriosus.

The origin and distribution of the coronary arteries was described in 39 autopsy specimens of truncus arteriosus (TA). The specimens were classified according to the number and the patterns of the truncal cusps. The position of the truncal cusps was defined in relation to intracardiac structures, namely, the atrioventricular orifices. Bicuspid truncal valves were observed in 8 cases (21%), tricuspid in 22 cases (56%) and quadricuspid in 9 cases (23%). All tricuspid valves had 2 anterior and 1 posterior cusp. Great variability in the origin of the coronary arteries was observed, with a tendency for the right coronary artery to arise from the anterior right quadrant and for the left coronary artery to arise from the anterior and left quadrant. Such a tendency was observed in 50% of the bicuspid, in 59% of the tricuspid and in 66% of the quadricuspid valves. The anatomic right ventricle was always observed to be vascularized by a right coronary artery, and the anatomic left ventricle by a left coronary artery, even in cases in which there was a single coronary trunk. The anterior surface of the right ventricle was crossed by a right coronary artery in 5 cases. A single coronary artery was observed in 7 cases (18%). Embryologic considerations are offered, especially regarding the relation between the observed variability in coronary artery patterns in TA and the absence of the truncal septation.

Pathogenesis of persistent truncus arteriosus in light of observations made in a dog embryo with the anomaly.

Among 36 embryos obtained from a strain of Keeshond dogs in which there is a large incidence of spontaneously occurring conotruncal anomalies, a specimen with persistent truncus arteriosus, type 1 was found. The embryo had a crown-rump length of 20 mm. The specimen was serially sectioned and a wax plate reconstruction was made of the heart and proximal great vessels at a magnification of X100. The truncal valve was quadricuspid and dysplastic; associated anomalies were a right subclavian artery arising anomalously from the descending aorta, a single coronary artery, an absent ductus arteriosus and a small persistent left cranial (superior) vena cava. The truncus cushions were hypoplastic, had failed to fuse and each had simply produced an arterial cusp. The observations made on this embryo support the view that in persistent truncus arteriosus there is failure of septation of the truncus arteriosus. No evidence was found in favor of the concept that persistent truncus arteriosus represents a form of tetralogy of Fallot with atresia of the subpulmonary infundibulum and partial or complete absence of the aorticopulmonary septum.

Surgical significance of the coronary arterial anatomy in truncus arteriosus communis.

In truncus arteriosus communis, as in other anomalies of conotruncal development, the coronary arterial anatomy not only differs from that found in the normal heart but also is subject to unpredictable variations. A consistently distinctive pattern was found in roughly two thirds of 31 cases, a frequency approximating that with which a distinctive coronary arterial pattern occurs in complete transposition of the great vessels. Surgical injury to a major coronary artery was identified in 2 of the 30 hearts from patients with truncus arteriosus communis who died in the early postoperative period. Of particular significance are large branches of the right coronary artery crossing the upper anterior surface of the right ventricle to supply the anterobasal surface of both ventricles and the upper part of the interventricular septum. These arteries are at special risk in surgical procedures utilizing a conduit anastomosed to a right ventriculotomy.