Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart.

RATIONALE: ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), encoded by the clustered genes Nppa and Nppb, are important prognostic, diagnostic, and therapeutic proteins in cardiac disease. The spatiotemporal expression pattern and stress-induction of the Nppa and Nppb are tightly regulated, possibly involving their coregulation by an evolutionary conserved enhancer cluster. OBJECTIVE: To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlying Nppa-Nppb coregulation in vivo. METHODS AND RESULTS: By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream of Nppb. Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof, Nppb and flanking regions or the entire genomic block spanning Nppa-Nppb, respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic, and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression of Nppa and Nppb but not of any other gene. Enhancer cluster-deficient mice showed enlarged hearts before and after birth, similar to Nppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters of Nppa and Nppb in a competitive rather than a cooperative mode, resulting in increased Nppa expression when Nppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity when its target genes are absent. In enhancer cluster-deficient animals, Nppa was induced but remained low in the postmyocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal to Nppa. CONCLUSIONS: Coordinated ventricular expression of Nppa and Nppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy.

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system.

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3+/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3+/- mutants. Notably, Tbx3+/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program.

The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3+ pacemaker cells. Here, we have isolated and characterized the Tbx3+ cells from Tbx3+/Venus knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3+ pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3+ sinoatrial nodal cells and Nppb-Katushka+ atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program, including key nodal transcription factors, BMP signaling and Smoc2, the disruption of which in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1 and HOX family members, and BMP and NOTCH signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the Tbx3+/Venus allele provides a reliable tool for visualizing the sinoatrial node, and studying its development and function.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

RATIONALE: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. OBJECTIVE: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. METHODS AND RESULTS: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. CONCLUSIONS: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction.

A small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3+ cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases.

Variation in a Left Ventricle-Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function.

RATIONALE: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5' to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1). OBJECTIVE: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. METHODS AND RESULTS: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. CONCLUSIONS: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.

Reduced sodium channel function unmasks residual embryonic slow conduction in the adult right ventricular outflow tract.

RATIONALE: In patients with Brugada syndrome, arrhythmias typically originate in the right ventricular outflow tract (RVOT). The RVOT develops from the slowly conducting embryonic outflow tract. OBJECTIVE: We hypothesize that this embryonic phenotype is maintained in the fetal and adult RVOT and leads to conduction slowing, especially after sodium current reduction. METHODS AND RESULTS: We determined expression patterns in the embryonic myocardium and performed activation mapping in fetal and adult hearts, including hearts from adult mice heterozygous for a mutation associated with Brugada syndrome (Scn5a1798insD/+). The embryonic RVOT was characterized by expression of Tbx2, a repressor of differentiation, and absence of expression of both Hey2, a ventricular transcription factor, and Gja1, encoding the principal gap-junction subunit for ventricular fast conduction. Also, conduction velocity was lower in the RVOT than in the right ventricular free wall. Later in the development, Gja1 and Scn5a expression remained lower in the subepicardial myocardium of the RVOT than in RV myocardium. Nevertheless, conduction velocity in the adult RVOT was similar to that of the right ventricular free wall. However, in hearts of Scn5a1798insD/+ mice and in normal hearts treated with ajmaline, conduction was slower in the RVOT than in the right ventricular wall. CONCLUSIONS: The slowly conducting embryonic phenotype is maintained in the fetal and adult RVOT and is unmasked when cardiac sodium channel function is reduced.

Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart.

RATIONALE: The clinically important atrioventricular conduction axis is structurally complex and heterogeneous, and its molecular composition and developmental origin are uncertain. OBJECTIVE: To assess the molecular composition and 3D architecture of the atrioventricular conduction axis in the postnatal mouse heart and to define the developmental origin of its component parts. METHODS AND RESULTS: We generated an interactive 3D model of the atrioventricular junctions in the mouse heart using the patterns of expression of Tbx3, Hcn4, Cx40, Cx43, Cx45, and Nav1.5, which are important for conduction system function. We found extensive figure-of-eight rings of nodal and transitional cells around the mitral and tricuspid junctions and in the base of the atrial septum. The rings included the compact node and nodal extensions. We then used genetic lineage labeling tools (Tbx2(+/Cre), Mef2c-AHF-Cre, Tbx18(+/Cre)), along with morphometric analyses, to assess the developmental origin of the specific components of the axis. The majority of the atrial components, including the atrioventricular rings and compact node, are derived from the embryonic atrioventricular canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium. No contributions to the conduction system myocardium were identified from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion. CONCLUSIONS: The atrioventricular conduction axis comprises multiple domains with distinctive molecular signatures. The atrial part proliferates from the embryonic atrioventricular canal, along with myocytes derived from the developing atrial septum. The atrioventricular bundle and lower nodal cells are derived from ventricular myocardium.

Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3.

The sinus node (or sinoatrial node [SAN]), the pacemaker of the heart, is a functionally and structurally heterogeneous tissue, which consists of a large "head" within the right caval vein myocardium and a "tail" along the terminal crest. Here, we investigated its cellular origin and mechanism of formation. Using genetic lineage analysis and explant assays, we identified T-box transcription factor Tbx18-expressing mesenchymal progenitors in the inflow tract region that differentiate into pacemaker myocardium to form the SAN. We found that the head and tail represent separate regulatory domains expressing distinctive gene programs. Tbx18 is required to establish the large head structure, as seen by the existence of a very small but still functional tail piece in Tbx18-deficient fetuses. In contrast, Tbx3-deficient embryos formed a morphologically normal SAN, which, however, aberrantly expressed Cx40 and other atrial genes, demonstrating that Tbx3 controls differentiation of SAN head and tail cardiomyocytes but also demonstrating that Tbx3 is not required for the formation of the SAN structure. Our data establish a functional order for Tbx18 and Tbx3 in SAN formation, in which Tbx18 controls the formation of the SAN head from mesenchymal precursors, on which Tbx3 subsequently imposes the pacemaker gene program.

The Tbx2+ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle.

The primary myocardium of the embryonic heart, including the atrioventricular canal and outflow tract, is essential for septation and valve formation. In the chamber-forming heart, the expression of the T-box transcription factor Tbx2 is restricted to the primary myocardium. To gain insight into the cellular contributions of the Tbx2+ primary myocardium to the components of the definitive heart, genetic lineage tracing was performed using a novel Tbx2Cre allele. These analyses revealed that progeny of Tbx2+ cells provide an unexpectedly large contribution to the Tbx2-negative ventricles. Contrary to common assumption, we found that the embryonic left ventricle only forms the left part of the definitive ventricular septum and the apex. The atrioventricular node, but not the atrioventricular bundle, was found to derive from Tbx2+ cells. The Tbx2+ outflow tract formed the right ventricle and right part of the ventricular septum. In Tbx2-deficient embryos, the left-sided atrioventricular canal was found to prematurely differentiate to chamber myocardium and to proliferate at increased rates similar to those of chamber myocardium. As a result, the atrioventricular junction and base of the left ventricle were malformed. Together, these observations indicate that Tbx2 temporally suppresses differentiation and proliferation of primary myocardial cells. A subset of these Tbx2Cre-marked cells switch off expression of Tbx2, which allows them to differentiate into chamber myocardium, to initiate proliferation, and to provide a large contribution to the ventricles. These findings imply that errors in the development of the early atrioventricular canal may affect a much larger region than previously anticipated, including the ventricular base.

Pitx2c and Nkx2-5 are required for the formation and identity of the pulmonary myocardium.

The pulmonary vein is sleeved by myocardium, which is a major source of atrial fibrillation and is involved in congenital sinus venosus defects. Little is known about the cellular origin and mechanism of formation of the pulmonary myocardium. We observed a biphasic process of pulmonary myocardium formation in mice. Firstly, a myocardial cell population forms de novo at the connection of the pulmonary vein and the atrium. Genetic labeling revealed that atrial cells do not contribute to this population, indicating it forms by differentiation of pulmonary mesenchymal cells. Secondly, these pulmonary myocardial cells initiate a phase of rapid proliferation and form the pulmonary myocardial sleeve. Pitx2c-deficient mice do not develop a pulmonary myocardial sleeve because they fail to form the initial pulmonary myocardial cells. Genetic-labeling analyses demonstrated that whereas the systemic venous return derives from Nkx2-5-negative precursors, the pulmonary myocardium derives from Nkx2-5-expressing precursors, indicating a distinct origin of the 2 venous systems. Nkx2-5 and its target gap-junction gene Cx40 are expressed in the atria and in the pulmonary myocardium but not in the systemic venous return, which expresses the essential pacemaker channel Hcn4. When Nkx2-5 protein level was lowered in a hypomorphic model, the pulmonary myocardium switched to a Cx40-negative, Hcn4-positive phenotype resembling that of the systemic venous return. In conclusion, our data suggest a cellular mechanism for pulmonary myocardium formation and highlight the key roles played by Pitx2c and Nkx2-5 in its formation and identity.

Molecular pathway for the localized formation of the sinoatrial node.

The sinoatrial node, which resides at the junction of the right atrium and the superior caval vein, contains specialized myocardial cells that initiate the heart beat. Despite this fundamental role in heart function, the embryonic origin and mechanisms of localized formation of the sinoatrial node have not been defined. Here we show that subsequent to the formation of the Nkx2-5-positive heart tube, cells bordering the inflow tract of the heart tube give rise to the Nkx2-5-negative myocardial cells of the sinoatrial node and the sinus horns. Using genetic models, we show that as the myocardium of the heart tube matures, Nkx2-5 suppresses pacemaker channel gene Hcn4 and T-box transcription factor gene Tbx3, thereby enforcing a progressive confinement of their expression to the forming Nkx2-5-negative sinoatrial node and sinus horns. Thus, Nkx2-5 is essential for establishing a gene expression border between the atrium and sinoatrial node. Tbx3 was found to suppress chamber differentiation, providing an additional mechanism by which the Tbx3-positive sinoatrial node is shielded from differentiating into atrial myocardium. Pitx2c-deficient fetuses form sinoatrial nodes with indistinguishable molecular signatures at both the right and left sinuatrial junction, indicating that Pitx2c functions within the left/right pathway to suppress a default program for sinuatrial node formation on the left. Our molecular pathway provides a mechanism for how pacemaker activity becomes progressively relegated to the most recently added components of the venous pole of the heart and, ultimately, to the junction of the right atrium and superior caval vein.

Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18.

The venous pole of the mammalian heart is a structurally and electrically complex region, yet the lineage and molecular mechanisms underlying its formation have remained largely unexplored. In contrast to classical studies that attribute the origin of the myocardial sinus horns to the embryonic venous pole, we find that the sinus horns form only after heart looping by differentiation of mesenchymal cells of the septum transversum region into myocardium. The myocardial sinus horns and their mesenchymal precursor cells never express Nkx2-5, a transcription factor critical for heart development. In addition, lineage studies show that the sinus horns do not derive from cells previously positive for Nkx2-5. In contrast, the sinus horns express the T-box transcription factor gene Tbx18. Mice deficient for Tbx18 fail to form sinus horns from the pericardial mesenchyme and have defective caval veins, whereas the pulmonary vein and atrial structures are unaffected. Our studies define a novel heart precursor population that contributes exclusively to the myocardium surrounding the sinus horns or systemic venous tributaries of the developing heart, which are a source of congenital malformation and cardiac arrhythmias.

The human phrenic nerve serves as a morphological conduit for autonomic nerves and innervates the caval body of the diaphragm.

Communicating fibres between the phrenic nerve and sympathetic nervous system may exist, but have not been characterized histologically and immunohistochemically, even though increased sympathetic activity due to phrenic nerve stimulation for central sleep apnoea may entail morbidity and mortality. We, therefore, conducted a histological study of the phrenic nerve to establish the presence of catecholaminergic fibres throughout their course. The entire phrenic nerves of 35 formalin-fixed human cadavers were analysed morphometrically and immunohistochemically. Furthermore, the right abdominal phrenic nerve was serially sectioned and reconstructed. The phrenic nerve contained 3 ± 2 fascicles in the neck that merged to form a single fascicle in the thorax and split again into 3 ± 3 fascicles above the diaphragm. All phrenic nerves contained catecholaminergic fibres, which were distributed homogenously or present as distinct areas within a fascicle or as separate fascicles. The phrenicoabdominal branch of the right phrenic nerve is a branch of the celiac plexus and, therefore, better termed the "phrenic branch of the celiac plexus". The wall of the inferior caval vein in the diaphragm contained longitudinal strands of myocardium and atrial natriuretic peptide-positive paraganglia ("caval bodies") that where innervated by the right phrenic nerve.

Lineage and morphogenetic analysis of the cardiac valves.

We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.

Overexpression of arginase I in enterocytes of transgenic mice elicits a selective arginine deficiency and affects skin, muscle, and lymphoid development.

BACKGROUND: Arginine is required for the detoxification of ammonia and the synthesis of proteins, nitric oxide, agmatine, creatine, and polyamines, and it may promote lymphocyte function. In suckling mammals, arginine is synthesized in the enterocytes of the small intestine, but this capacity is lost after weaning. OBJECTIVE: We investigated the significance of intestinal arginine production for neonatal development in a murine model of chronic arginine deficiency. DESIGN: Two lines of transgenic mice that express different levels of arginase I in their enterocytes were analyzed. RESULTS: Both lines suffer from a selective but quantitatively different reduction in circulating arginine concentration. The degree of arginine deficiency correlated with the degree of retardation of hair and muscle growth and with the development of the lymphoid tissue, in particular Peyer's patches. Expression of arginase in all enterocytes was necessary to elicit this phenotype. Phenotypic abnormalities were reversed by daily injections of arginine but not of creatine. The expression level of the very arginine-rich skin protein trichohyalin was not affected in transgenic mice. Finally, nitric oxide synthase-deficient mice did not show any of the features of arginine deficiency. CONCLUSIONS: Enterocytes are important for maintaining arginine homeostasis in neonatal mice. Graded arginine deficiency causes graded impairment of skin, muscle, and lymphoid development. The effects of arginine deficiency are not mediated by impaired synthesis of creatine or by incomplete charging of arginyl-transfer RNA.

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.

The sinus venosus progenitors separate and diversify from the first and second heart fields early in development.

AIMS: During development, the heart tube grows by differentiation of Isl1(+)/Nkx2-5(+) progenitors to the arterial and venous pole and dorsal mesocardium. However, after the establishment of the heart tube, Tbx18(+) progenitors were proposed to form the Tbx18(+)/Nkx2-5(-) sinus venosus and proepicardium. To elucidate the relationship between these contributions, we investigated the origin of the Tbx18(+) sinus venosus progenitor population in the cardiogenic mesoderm and its spatial and temporal relation to the second heart field during murine heart development. METHODS AND RESULTS: Explant culture revealed that the Tbx18(+) cell population has the potential to form Nkx2-5(-) sinus venosus myocardium. Three-dimensional reconstruction of expression patterns showed that during heart tube elongation, the Tbx18(+) progenitors remained spatially and temporally separate from the Isl1(+) second heart field, only overlapping with the Isl1(+) domain at the right lateral side of the inflow tract, where the sinus node developed. Consistently, genetic lineage analysis revealed that the Tbx18(+) descendants formed the sinus venosus myocardium, but did not contribute to the pulmonary vein myocardium that developed in the Isl1(+) second heart field. By means of DiI labelling and expression analysis, the origin of the sinus venosus progenitor population was traced to the lateral rim of splanchnic mesoderm that down-regulated Nkx2-5 expression approximately 2 days before its differentiation into sinus venosus myocardium. CONCLUSION: Our data indicate that the cardiogenic mesoderm contains an additional progenitor subpopulation that contributes to the sinus venosus myocardium. After patterning of the cardiogenic mesoderm, this progenitor population remains spatially separated and genetically distinctive from the second heart field subpopulation.