A frameshift mutation in LRSAM1 is responsible for a dominant hereditary polyneuropathy.

Despite the high number of genes identified in hereditary polyneuropathies/Charcot-Marie-Tooth (CMT) disease, the genetic defect in many families is still unknown. Here we report the identification of a new gene for autosomal dominant axonal neuropathy in a large three-generation family. Linkage analysis identified a 5 Mb region on 9q33-34 with a LOD score of 5.12. Sequence capture and next-generation sequencing of the region of interest identified five previously unreported non-synonymous heterozygous single nucleotide changes or indels, four of which were confirmed by Sanger sequencing. Two sequence variants co-segregated with the disease, and one, a 2 bp insertion in the last exon of LRSAM1, was also absent in 676 ethnicity-matched control chromosomes. This frameshift mutation (p.Leu708Argfx28) is located in the C-terminal RING finger motif of the encoded protein. Ubiquitin ligase activity in transfected cells with constructs carrying the patient mutation was affected as measured by a higher level of abundance of TSG101, the only reported target of LRSAM1. Injections of morpholino oligonucleotides in zebrafish embryos directed against the ATG or last splice site of zebrafish Lrsam1 disturbed neurodevelopment, showing a less organized neural structure and, in addition, affected tail formation and movement. LRSAM1 is highly expressed in adult spinal cord motoneurons as well as in fetal spinal cord and muscle tissue. Recently, a homozygous mutation in LRSAM1 was proposed as a strong candidate for the disease in a family with recessive axonal polyneuropathy. Our data strongly support the hypothesis that LRSAM1 mutations can cause both dominant and recessive forms of CMT.

Development of the human aortic arch system captured in an interactive three-dimensional reference model.

Variations and mutations in the human genome, such as 22q11.2 microdeletion, can increase the risk for congenital defects, including aortic arch malformations. Animal models are increasingly expanding our molecular and genetic insights into aortic arch development. However, in order to justify animal-to-human extrapolations, a human morphological, and molecular reference model would be of great value, but is currently lacking. Here, we present interactive three-dimensional reconstructions of the developing human aortic arch system, supplemented with the protein distribution of developmental markers for patterning and growth, including T-box transcription factor TBX1, a major candidate for the phenotypes found in patients with the 22q11.2 microdeletion. These reconstructions and expression data facilitate unbiased interpretations, and reveal previously unappreciated aspects of human aortic arch development. Based on our reconstructions and on reported congenital anomalies of the pulmonary trunk and tributaries, we postulate that the pulmonary arteries originate from the aortic sac, rather than from the sixth pharyngeal arch arteries. Similar to mouse, TBX1 is expressed in pharyngeal mesenchyme and epithelia. The endothelium of the pharyngeal arch arteries is largely negative for TBX1 and family member TBX2 but expresses neural crest marker AP2α, which gradually decreases with ongoing development of vascular smooth muscle. At early stages, the pharyngeal arch arteries, aortic sac, and the dorsal aortae in particular were largely negative for proliferation marker Ki67, potentially an important parameter during aortic arch system remodeling. Together, our data support current animal-to-human extrapolations and future genetic and molecular analyses using animal models of congenital heart disease. © 2013 Wiley Periodicals, Inc.

Impairment of the tRNA-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia.

Pontocerebellar hypoplasia (PCH) represents a group (PCH1-6) of neurodegenerative autosomal recessive disorders characterized by hypoplasia and/or atrophy of the cerebellum, hypoplasia of the ventral pons, progressive microcephaly and variable neocortical atrophy. The majority of PCH2 and PCH4 cases are caused by mutations in the TSEN54 gene; one of the four subunits comprising the tRNA-splicing endonuclease (TSEN) complex. We hypothesized that TSEN54 mutations act through a loss of function mechanism. At 8 weeks of gestation, human TSEN54 is expressed ubiquitously in the brain, yet strong expression is seen within the telencephalon and metencephalon. Comparable expression patterns for tsen54 are observed in zebrafish embryos. Morpholino (MO) knockdown of tsen54 in zebrafish embryos results in loss of structural definition in the brain. This phenotype was partially rescued by co-injecting the MO with human TSEN54 mRNA. A developmental patterning defect was not associated with tsen54 knockdown; however, an increase in cell death within the brain was observed, thus bearing resemblance to PCH pathophysiology. Additionally, N-methyl-N-nitrosourea mutant zebrafish homozygous for a tsen54 premature stop-codon mutation die within 9 days post-fertilization. To determine whether a common disease pathway exists between TSEN54 and other PCH-related genes, we also monitored the effects of mitochondrial arginyl-tRNA synthetase (rars2; PCH1 and PCH6) knockdown in zebrafish. Comparable brain phenotypes were observed following the inhibition of both genes. These data strongly support the hypothesis that TSEN54 mutations cause PCH through a loss of function mechanism. Also we suggest that a common disease pathway may exist between TSEN54- and RARS2-related PCH, which may involve a tRNA processing-related mechanism.

Three-dimensional and molecular analysis of the arterial pole of the developing human heart.

Labeling experiments in chicken and mouse embryos have revealed important roles for different cell lineages in the development of the cardiac arterial pole. These data can only fully be exploited when integrated into the continuously changing morphological context and compared with the patterns of gene expression. As yet, studies on the formation of separate ventricular outlets and arterial trunks in the human heart are exclusively based on histologically stained sections. So as to expand these studies, we performed immunohistochemical analyses of serially sectioned human embryos, along with three-dimensional reconstructions. The development of the cardiac arterial pole involves several parallel and independent processes of formation and fusion of outflow tract cushions, remodeling of the aortic sac and closure of an initial aortopulmonary foramen through formation of a transient aortopulmonary septum. Expression patterns of the transcription factors ISL1, SOX9 and AP2α show that, in addition to fusion of the SOX9-positive endocardial cushions, intrapericardial protrusion of pharyngeal mesenchyme derived from the neural crest contributes to the separation of the developing ascending aorta from the pulmonary trunk. The non-adjacent walls of the intrapericardial arterial trunks are formed through addition of ISL1-positive cells to the distal outflow tract, while the facing parts of the walls form from the protruding mesenchyme. The morphogenetic steps, along with the gene expression patterns reported in this study, are comparable to those observed in the mouse. They confirm the involvement of mesenchymal tissues derived from endocardium, mesoderm and migrating neural crest cells in the process of initial septation of the distal part of the outflow tract, and its subsequent separation into discrete intrapericardial arterial trunks.

Multiscale modelling of Potts shunt as a potential palliative treatment for suprasystemic idiopathic pulmonary artery hypertension: a paediatric case study.

Potts shunt (PS) was suggested as palliation for patients with suprasystemic pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. PS, however, can result in poorly understood mortality. Here, a patient-specific geometrical multiscale model of PAH physiology and PS is developed for a paediatric PAH patient with stent-based PS. In the model, 7.6mm-diameter PS produces near-equalisation of the aortic and PA pressures and [Formula: see text] (oxygenated vs deoxygenated blood flow) ratio of 0.72 associated with a 16% decrease of left ventricular (LV) output and 18% increase of RV output. The flow from LV to aortic arch branches increases by 16%, while LV contribution to the lower body flow decreases by 29%. Total flow in the descending aorta (DAo) increases by 18% due to RV contribution through the PS with flow into the distal PA branches decreasing. PS induces 18% increase of RV work due to its larger stroke volume pumped against lower afterload. Nonetheless, larger RV work does not lead to increased RV end-diastolic volume. Three-dimensional flow assessment demonstrates the PS jet impinging with a high velocity and wall shear stress on the opposite DAo wall with the most of the shunt flow being diverted to the DAo. Increasing the PS diameter from 5mm up to 10mm results in a nearly linear increase in post-operative shunt flow and a nearly linear decrease in shunt pressure-drop. In conclusion, this model reasonably represents patient-specific haemodynamics pre- and post-creation of the PS, providing insights into physiology of this complex condition, and presents a predictive tool that could be useful for clinical decision-making regarding suitability for PS in PAH patients with drug-resistant suprasystemic PAH.

Three-dimensional and molecular analysis of the venous pole of the developing human heart.

BACKGROUND: Various congenital malformations and many abnormal rhythms originate from the venous pole of the heart. Because of rapid changes during morphogenesis, lack of molecular and lineage data, and difficulties in presenting complex morphogenetic changes in the developing heart in a clear fashion, the development of this region in human has been difficult to grasp. METHODS AND RESULTS: To gain insight into the development of the different types of myocardium forming the venous pole of the human heart, we performed an immunohistochemical and 3-dimensional analysis of serial sections of human embryos ranging from 22 through 40 days of development. Three-dimensional models were prepared in a novel interactive portable format providing crucial spatial information and facilitating interpretation. As in the mouse, the systemic venous myocardium expresses the transcription factor TBX18, whereas the pulmonary venous myocardium expresses NKX2-5. In contrast to the mouse, a systemic venous sinus is identified upstream from the atrial chambers, albeit initially with nonmyocardial walls. From the outset, as in the mouse, the pulmonary vein empties to a chamber with atrial, rather than systemic venous, characteristics. Compared with the mouse, the vestibular spine is a more prominent structure. CONCLUSIONS: The similarities in gene expression in the distinctive types of myocardium surrounding the systemic and pulmonary venous tributaries in man and mouse permit extrapolation of the conclusions drawn from transgenic and lineage studies in the mouse to the human, showing that the systemic and pulmonary venous myocardial sleeves are derived from distinct developmental lineages.

Cardiovascular anatomy in children with bidirectional Glenn anastomosis, regarding the transcatheter Fontan completion.

BACKGROUND: Transcatheter stent-secured completion of total cavopulmonary connection (TCPC) after surgical preparations during the Glenn anastomosis procedure has been reported, but complications from this approach have precluded its clinical acceptance. AIMS: To analyse cardiovascular morphology and dimensions in children with bidirectional Glenn anastomosis, regarding the optimal device design for transcatheter Fontan completion without special surgical "preconditionings". METHODS: We retrospectively analysed 60 thoracic computed tomography and magnetic resonance angiograms performed in patients with a median age of 4.1 years (range: 1.8-17.1 years). Additionally, we simulated TCPC completion using different intra-atrial stent-grafts in a three-dimensional model of the representative anatomy, and performed calculations to determine the optimal stent-graft dimensions, using measured distances. RESULTS: Two types of cardiovascular arrangement were identified: left atrium interposing between the right pulmonary artery (RPA) and inferior vena cava, with the right upper pulmonary vein (RUPV) orifice close to the intercaval axis (65%); and intercaval axis traversing only the right(-sided) atrial cavity, with the RUPV located posterior to the atrial wall (35%). In the total population, the shortest median RPA-to-atrial wall distance was 1.9mm (range: 0.6-13.8mm), while the mean intra-atrial distance along the intercaval axis was 50.1±11.2mm. Regardless of the arrangement, 83% of all patients required a deviation of at least 5.9±2.4mm (range: 1.2-12.7mm) of the stent-graft centre at the RUPV level anteriorly to the intercaval axis to avoid covering or compressing this vein. Fixing the anterior deviation of the curved stent-graft centre at 10mm significantly decreased the range of bend angle per every given RUPV-RPA distance. CONCLUSIONS: For both types of cardiovascular arrangement, after conventional bidirectional Glenn anastomosis, the intra-atrial curved stent-graft seemed most suitable for achieving uncomplicated TCPC completion percutaneously without previous surgical "preconditionings" in the majority of children. Experimental study is necessary to validate this conclusion.

Valve Interventions in Utero: Understanding the Timing, Indications, and Approaches.

Efficient use of fetal echocardiography has enabled early detection of congenital heart disease and of its often irreversible complications, such as ventricular hypoplasia in case of severe stenosis of the semilunar valves. Experience of the past 25 years has proved that balloon dilatation of the severely stenotic or atretic valve in fetuses as early as the 23rd week of gestation is technically feasible with a learning curve. Reported results regarding the ultimate biventricular circulation outcome after fetal valve intervention are at best controversial, with the desired improvements in the quality of life and cost-benefits of the postnatal treatment being as yet unconfirmed. Despite acute hemodynamic success with a relatively low rate of fetal complications, the number of suitable candidates for the fetal valve intervention remains low. High valvular tissue plasticity in the fetus and difficulties of assessing the point of no return of the myocardial damage often makes the success of fetal valve intervention short-lived and unpredictable. Hopefully, future refinements of the equipment, imaging, and biodegradable tissue regeneration materials will lead to better results of the fetal valve interventions beyond their technical success.

Vascular anatomy in children with univentricular hearts regarding transcatheter bidirectional Glenn anastomosis.

BACKGROUND: Transcatheter stent-secured Glenn anastomosis, aiming to reduce the invasiveness of palliation in patients with univentricular heart defects, has been reported in large experimental animals. The advent of biodegradable stents and tissue-engineered vascular grafts will make this procedure a reality in human patients. However, the relationship between the superior vena cava (SVC) and the right pulmonary artery (RPA) is different in humans. AIM: To characterise vascular anatomy in children with univentricular hearts, regarding technical aspects and device design for this procedure. METHODS: Retrospective analysis of 35 thoracic computed tomography angiograms at a mean age of 18.1±22.4 months. RESULTS: Two types of arrangement between the SVC and the RPA were identified: anatomy convenient for immediate wire passage and stent deployment between the two vessels (60%); and pattern of early RPA branching, requiring the perforation wire to traverse the intervascular space to avoid entrance into the upper RPA branch (40%). In patients with the convenient vascular arrangement, the vessels were nearly perpendicular, having immediate contact, with the posterior SVC aspect partially "wrapping" the adjacent RPA in most patients. In patients with early RPA branching, the mean shortest SVC-to-central RPA distance was 4.3±2.7mm. For the total population, the mean length of proximal SVC that allowed stent deployment without covering the brachiocephalic vein was 15.6±5.1mm. CONCLUSIONS: A trumpet-shaped covered stent in a craniocaudal orientation reaching from the SVC into the prebranching RPA seems most suitable for achieving bidirectional Glenn anastomosis percutaneously in humans. However, the short length of the proximal SVC and the presence of early RPA branching pose challenges for optimal design of the dedicated device.

Safety and Feasibility of the Transcatheter Approach to Create a Reverse Potts Shunt in Children With Idiopathic Pulmonary Arterial Hypertension.

BACKGROUND: The reversed Potts shunt improves right ventricular (RV) function in patients with suprasystemic pulmonary arterial hypertension (PAH). The proximity of the left pulmonary artery (LPA) to the descending aorta (DAo) permits the creation of a transcatheter connection. We sought to assess the safety, feasibility, and hemodynamic efficacy of the transcatheter Potts shunt (TPS) in children. METHODS: The TPS procedure was performed using radiofrequency energy for vessel perforation and deployment of a covered stent to connect the DAo and LPA. Procedural details and clinical follow-up data were collected prospectively. RESULTS: A TPS was successfully created in 6 children (mean age, 11.0 ± 4.2 years) with drug-refractory suprasystemic PAH and deteriorating RV function. All patients exhibited nearly complete equalization of aortic and pulmonary pressures and improvement in RV contractility within days after TPS placement. Two patients with pre-existing severe biventricular dysfunction and pericardial effusion experienced acute low-output states immediately after shunt creation because of sudden reductions in left ventricular (LV) preload, resulting in cardiac arrest, irreversible brain damage, and death. Stent dislodgement and embolization into the iliac artery occurred in 1 patient. The stent was successfully secured and followed by placement of a second stent at the target location. The procedure was uncomplicated in 4 patients, who remain alive after a mean follow-up of 10 ± 2.6 months. Intravenous vasodilator therapy was weaned uneventfully after TPS in 3 patients. CONCLUSIONS: TPS creation in children is feasible and results in hemodynamic improvement. Further insights into high-risk markers, such as reduced preprocedural LV function and preload reserves, are important for guiding patient selection.

Novel materials and devices in the transcatheter creation of vascular anastomosis--the future comes slowly (part 2).

Completion of the total cavopulmonary connection and creation of the majority of vascular anastomoses are currently usually performed surgically. The major disadvantage of the surgical approach, however, is its invasiveness, as patients undergoing cardiac surgery generally need sternotomy and cardiopulmonary bypass - often with cardiac arrest - commonly resulting in a prolonged and complicated postoperative intensive care period. Transcatheter procedures, in contrast, have a lower risk of complications, shorter intensive care and total hospital stays, and do not need a cardiopulmonary bypass or sternotomy. The second part of our review focuses on new advances in transcatheter technology, which will allow safe and effective percutaneous management of patients requiring the creation of an intervascular anastomosis and completion of the total cavopulmonary connection. It will create a therapeutic alternative able to reduce the surgical burden on this group of patients.

Novel materials and devices in the transcatheter management of congenital heart diseases-the future comes slowly (part 3).

Correction of malformations affecting the right ventricular outflow tract often results in residual abnormalities that require valve implantation at a later stage to prevent right ventricular deterioration. In the paediatric population, the pathology of congenital valve stenosis or insufficiency is often complex, options for surgical repair are limited, and valve replacement remains the only-albeit unattractive-alternative. Prosthetic heart valve implantation can be performed either surgically or, nowadays, percutaneously. Current transcatheter devices allow less invasive percutaneous valve implantation in selected patients after surgical repair, but are suitable for only a small portion of paediatric patients. In addition, there is a large heterogeneous group of patients who undergo surgical constriction of the pulmonary trunk, either to reduce pulmonary blood flow or to retrain or support the left ventricle. In the third part of this review series, we focus on new biomaterials, devices and technologies that have the potential to extend transcatheter valve implantation to a broader spectrum of congenital cardiovascular lesions, with safe and durable results in children, and on transcatheter options for the creation of a partial obstruction within the pulmonary trunk (pulmonary artery banding).

Novel materials and devices in the transcatheter management of congenital heart diseases--the future comes slowly (part 1).

Management of congenital defects of the heart and great vessels constitutes the largest part of paediatric cardiology practice. Most of these defects require interventions, either corrective or palliative, to guarantee patient survival, symptom relief and/or better quality of life. Interventions can be performed either surgically or transcatheter percutaneously. The surgical repairs are invasive, with long-term results often being suboptimal for complex lesions and after the use of grafts, especially in small patients. Nowadays, various transcatheter devices allow much less invasive percutaneous management in some carefully selected patients with congenital heart disease. However, the currently available materials and devices are only suitable for a small proportion of children, while the majority of young patients with cardiac defects still need surgery, as no transcatheter alternatives exist. There are, however, numerous new biomaterials, devices and technologies that have the potential to expand the transcatheter approach to a much broader spectrum of congenital cardiovascular lesions and conditions. In this three-part review, we describe new advances in transcatheter devices and materials, which promise to extend the application of the percutaneous approach to younger and more complex patient groups with congenital heart disease. The first part focuses on new possibilities for the transcatheter treatment of vascular stenoses in growing patients and the closure of intracardiac defects.

Vascular anatomy in children with pulmonary hypertension regarding the transcatheter Potts shunt.

OBJECTIVE: To morphometrically characterise the region of adjacent descending aorta (DAo) and left pulmonary artery (LPA) regarding the transcatheter creation of the reverse Potts shunt. METHODS AND RESULTS: Retrospective review of the invasive haemodynamic data and measurements of the vessel diameters, distances and angles based on the thoracic CT of children with idiopathic pulmonary arterial hypertension (PAH) with pulmonary-to-systemic systolic pressure ratio ≥0.5. Forty-eight CT scans from 47 patients were analysed. Independent of the PAH severity, the diameters of DAo and LPA, and the area of tightest contact between these vessels were very similar in patients with either infrasystemic or isosystemic/suprasystemic PAH. For total population, the tightest contact area (mean±SD, 51.8±31.9 mm2, range 12.5-177.7 mm2) had an elliptic shape stretched along the DAo length and LPA width. The shortest mean DAo-LPA distance was 1.7±0.8 mm (range 1-5 mm). Only one patient, from the suprasystemic PAH group, had the DAo-LPA distance >4 mm. None had lung tissue identified between these two vessels, while in four patients (8.3%) the prominent bronchial artery was seen coursing exactly between the LPA and DAo. The difference of prevalence of the bronchial arteries between two vessels in patients with either infrasystemic PAH or isosystemic/suprasystemic PAH did not reach statistical significance. CONCLUSIONS: Children with idiopathic PAH showed no complicating anatomic or morphometric parameters of the region with adjacent DAo and LPA, which potentially determine the planning of the transcatheter creation of Potts shunt. It holds promises for standardisation of the procedure in the future.

Building foundations for transcatheter intervascular anastomoses: 3D anatomy of the great vessels in large experimental animals.

OBJECTIVES: To provide comprehensive illustrations of anatomy of the relevant vessels in large experimental animals in an interactive format as preparation for developing an effective and safe transcatheter technique of aortopulmonary and bidirectional cavopulmonary intervascular anastomoses. METHODS: Computed tomographic angiographic studies in two calves and two sheep were used to prepare 3D reconstructions of the aorta, pulmonary arteries, and caval and pulmonary veins. Based on these reconstructions, computer simulations of the creation of stent-enhanced aortopulmonary and bidirectional cavopulmonary anastomoses were made. RESULTS: We observed the following major anatomical features: (i) caudal course of the main pulmonary artery and its branches with the proximal right pulmonary artery located immediately caudal to the aortic arch, and with the central left pulmonary artery lying at a substantial distance from the descending aorta; and (ii) the distal right pulmonary artery is located dorsal to the right atrium and inferior caval vein at a substantial distance from the superior caval vein. Animations showed creation of transcatheter analogues of Waterston's and Potts' aortopulmonary shunts through placement of a covered spool-shaped stent, and the transcatheter creation of bidirectional Glenn's cavopulmonary anastomosis, by placement of a long covered trumpet-shaped stent. CONCLUSIONS: There are considerable differences in vascular anatomy between large experimental animals and humans. Given the need to elaborate new transcatheter techniques for intervascular anastomoses in suitable animal models before application to human, it is crucial to take these anatomical differences into account during testing and optimization of the proposed procedures.

Molecular analysis of patterning of conduction tissues in the developing human heart.

BACKGROUND: Recent studies in experimental animals have revealed some molecular mechanisms underlying the differentiation of the myocardium making up the conduction system. To date, lack of gene expression data for the developing human conduction system has precluded valid extrapolations from experimental studies to the human situation. METHODS AND RESULTS: We performed immunohistochemical analyses of the expression of key transcription factors, such as ISL1, TBX3, TBX18, and NKX2-5, ion channel HCN4, and connexins in the human embryonic heart. We supplemented our molecular analyses with 3-dimensional reconstructions of myocardial TBX3 expression. TBX3 is expressed in the developing conduction system and in the right venous valve, atrioventricular ring bundles, and retro-aortic nodal region. TBX3-positive myocardium, with exception of the top of the ventricular septum, is devoid of fast-conducting connexin40 and connexin43 and hence identifies slowly conducting pathways. In the early embryonic heart, we found wide expression of the pacemaker channel HCN4 at the venous pole, including the atrial chambers. HCN4 expression becomes confined during later developmental stages to the components of the conduction system. Patterns of expression of transcription factors, known from experimental studies to regulate the development of the sinus node and atrioventricular conduction system, are similar in the human and mouse developing hearts. CONCLUSIONS: Our findings point to the comparability of mechanisms governing the development of the cardiac conduction patterning in human and mouse, which provide a molecular basis for understanding the functioning of the human developing heart before formation of a discrete conduction system.

Defective Tbx2-dependent patterning of the atrioventricular canal myocardium causes accessory pathway formation in mice.

Ventricular preexcitation, a feature of Wolff-Parkinson-White syndrome, is caused by accessory myocardial pathways that bypass the annulus fibrosus. This condition increases the risk of atrioventricular tachycardia and, in the presence of atrial fibrillation, sudden death. The developmental mechanisms underlying accessory pathway formation are poorly understood but are thought to primarily involve malformation of the annulus fibrosus. Before birth, slowly conducting atrioventricular myocardium causes a functional atrioventricular activation delay in the absence of the annulus fibrosus. This myocardium remains present after birth, suggesting that the disturbed development of the atrioventricular canal myocardium may mediate the formation of rapidly conducting accessory pathways. Here we show that myocardium-specific inactivation of T-box 2 (Tbx2), a transcription factor essential for atrioventricular canal patterning, leads to the formation of fast-conducting accessory pathways, malformation of the annulus fibrosus, and ventricular preexcitation in mice. The accessory pathways ectopically express proteins required for fast conduction (connexin-40 [Cx40], Cx43, and sodium channel, voltage-gated, type V, α [Scn5a]). Additional inactivation of Cx30.2, a subunit for gap junctions with low conductance expressed in the atrioventricular canal and unaffected by the loss of Tbx2, did not affect the functionality of the accessory pathways. Our results suggest that malformation of the annulus fibrosus and preexcitation arise from the disturbed development of the atrioventricular myocardium.

Lack of Gata3 results in conotruncal heart anomalies in mouse.

The transcription factor Gata3 is an important regulator of the development of thymus, the nervous system, ear, kidney, and adrenal glands. This study analyzes the role of Gata3 in the developing heart using a mouse strain containing an nlsLacZ reporter gene fused in frame to the Gata3 gene by homologous recombination. Using in situ hybridization, RT-PCR and Gata3-LacZ histochemistry, Gata3 expression was shown in various cardiac structures up to newborn stage. During looping stages (E9.5-E11.5) Gata3-LacZ activity recapitulated endogenous Gata3 and was abundantly expressed in the endocardial ridges and endothelium of distal outflow tract. Strong reporter gene expression was also noted in the mesenchyme of ventral branchial arches, and in the epithelium. In the atrioventricular canal expression was relatively lower. In the four-chambered heart stages (E13.5-E17.5) the LacZ-staining did not recapitulate the endogenous Gata3 transcript and showed rather lineage tracing of formerly Gata3-expressing cells in the hearts. beta-Galactosidase activity was detected in the cusps of semilunar valves, aorta, pulmonary trunk, innominate and common carotid arteries, and faintly in the atrioventricular valves. Gata3-null embryos die normally between E11 and E12. Pharmacological treatment with sympathomimetic beta-adrenergic receptor agonist lengthens the survival up to E18 when malformations of the heart such as ventricular septal defect (VSD), double-outlet of right ventricle (DORV), anomalies of the aortic arch (AAA) and persistent truncus arteriosus (PTA) were detected. The specified malformations correlate with the normal developmental pattern of Gata3-LacZ expression. The short outflow tract and insufficient rotation of truncus arteriosus during looping stages might be the main reasons underlying these malformations.