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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.

Tricuspid valve agenesis/atresia (TVA) is a congenital cardiac malformation where the tricuspid valve is not formed. It is hypothesized that TVA results from a failure of the normal rightward expansion of the atrioventricular canal (AVC). We tested predictions of this hypothesis by morphometric analyses of the AVC in fetal hearts. We used high-resolution MRI and ultrasonography on a post-mortem fetal heart with TVA and with tricuspid valve stenosis (TVS) to validate the position of measurement landmarks that were to be applied to clinical echocardiograms. This revealed a much deeper right atrioventricular sulcus in TVA than in TVS. Subsequently, serial echocardiograms of in utero fetuses between 12 and 38 weeks of gestation were included (n = 23 TVA, n = 16 TVS, and n = 74 controls) to establish changes in AVC width and ventricular dimensions over time. Ventricular length and width and estimated fetal weight all increased significantly with age, irrespective of diagnosis. Heart rate did not differ between groups. However, in the second trimester, in TVA, the ratio of AVC to ventricular width was significantly lower compared to TVS and controls. This finding supports the hypothesis that TVA is due to a failed rightward expansion of the AVC. Notably, we found in the third trimester that the AVC to ventricular width normalized in TVA fetuses as their mitral valve area was greater than in controls. Hence, TVA associates with a quantifiable under-development of the AVC. This under-development is obscured in the third trimester, likely because of adaptational growth that allows for increased stroke volume of the left ventricle.

Organ laterality refers to the left-right asymmetry in disposition and conformation of internal organs and is established during embryogenesis. The heart is the first organ to display visible left-right asymmetries through its left-sided positioning and rightward looping. Here, we present a new zebrafish loss-of-function allele for tbx5a, which displays defective rightward cardiac looping morphogenesis. By mapping individual cardiomyocyte behavior during cardiac looping, we establish that ventricular and atrial cardiomyocytes rearrange in distinct directions. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Pharmacological treatment and ex vivo culture establishes that the cardiac twisting depends on intrinsic mechanisms and is independent from cardiac growth. Furthermore, genetic experiments indicate that looping requires proper tissue patterning. We conclude that cardiac looping involves twisting of the chambers around the atrioventricular canal, which requires correct tissue patterning by Tbx5a.

The atrial septum enables efficient oxygen transport by separating the systemic and pulmonary venous blood returning to the heart. Only in placental mammals will the atrial septum form by the coming-together of the septum primum and the septum secundum. In up to one of four placental mammals, this complex morphogenesis is incomplete and yields patent foramen ovale. The incidence of incomplete atrial septum is unknown for groups with the septum primum only, such as birds and reptiles. We found a low incidence of incomplete atrial septum in 11 species of bird (0% of specimens) and 13 species of reptiles (3% of specimens). In reptiles, there was a trabecular interface between the atrial septum and the atrial epicardium which was without a clear boundary between left and right atrial cavities. In developing reptiles (four squamates and one crocodylian), the septum primum initiated as a sheet that acquired perforations and the trabecular interface developed late. We conclude that atrial septation from the septum primum only results in a low incidence of incompleteness. In reptiles, the atrial septum and atrial wall develop a trabecular interface, but previous studies on atrial hemodynamics suggest this interface has a very limited capacity for shunting.

The sinus venosus is a cardiac chamber upstream of the right atrium that harbours the dominant cardiac pacemaker. During human heart development, the sinus venosus becomes incorporated into the right atrium. However, from the literature it is not possible to deduce the characteristics and importance of this process of incorporation, due to inconsistent terminology and definitions in the description of multiple lines of evidence. We reviewed the literature regarding the incorporation of the sinus venosus and included novel electrophysiological data. Most mammals that have an incorporated sinus venosus show a loss of a functional valve guard of the superior caval vein together with a loss of the electrical sinuatrial delay between the sinus venosus and the right atrium. However, these processes are not necessarily intertwined and in a few species only the sinuatrial delay may be lost. Sinus venosus incorporation can be characterised as the loss of the sinuatrial delay of which the anatomical and molecular underpinnings are not yet understood.

PURPOSE: Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. METHODS: We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. RESULTS: Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). CONCLUSION: Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF. ispartof: GENETICS IN MEDICINE vol:23 issue:10 pages:1952-1960 ispartof: location:United States status: published

Background: Genetic variants in SCN10A , encoding the neuronal voltage-gated sodium channel Na V 1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A , encoding the major essential cardiac sodium channel Na V 1.5. Methods: The expression of SCN10A was investigated in mouse and human hearts. With the use of CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by genome-wide association studies single-nucleotide polymorphism mapping and expression quantitative trait loci analysis. Results: We found that cardiomyocytes of the atria, sinoatrial node, and ventricular conduction system express a short transcript comprising the last 7 exons of the gene ( Scn10a-short ). Transcription occurs from an intronic enhancer-promoter complex, whereas full-length Scn10a transcript was undetectable in the human and mouse heart. Expression quantitative trait loci analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, whereas the expression of Scn5a , the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short –encoded Na V 1.8-short increased Na V 1.5-mediated sodium current. We propose that noncoding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts Na V 1.5-mediated sodium current and heart rhythm. Conclusions: Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that noncoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of Na V 1.8 in cardiomyocytes that impacts on Na V 1.5 function, cardiac conduction velocities, and arrhythmia susceptibility.

Congenital heart disease (CHD) is the most common birth defect. After birth, patients with CHD may suffer from cardiac stress resulting from abnormal loading conditions. However, it is not known how this cardiac burden influences postnatal development and adaptation of the ventricles. To study the transcriptional and cell-cycle response of neonatal cardiomyocytes to cardiac stress, we used a genetic mouse model that develops left ventricular volume overload within 2 weeks after birth. The increased volume load caused upregulation of the cardiac stress marker Nppa in the left ventricle and interventricular septum as early as 12 days after birth. Transcriptome analysis revealed that cardiac stress induced the expression of cell-cycle genes. This did not influence postnatal cell-cycle withdrawal of cardiomyocytes and other cell types in the ventricles as measured by Ki-67 immunostaining.

Fibrosis is a hallmark of adverse cardiac remodeling, which promotes heart failure, but it is also an essential repair mechanism to prevent cardiac rupture, signifying the importance of appropriate regulation of this process. In the remodeling heart, cardiac fibroblasts (CFs) differentiate into myofibroblasts (MyoFB), which are the key mediators of the fibrotic response. Additionally, cardiomyocytes are involved by providing pro-fibrotic cues. Nuclear receptor Nur77 is known to reduce cardiac hypertrophy and associated fibrosis, however, the exact function of Nur77 in the fibrotic response is yet unknown. Here, we show that Nur77-deficient mice exhibit severe myocardial wall thinning, rupture and reduced collagen fiber density after myocardial infarction and chronic isoproterenol (ISO) infusion. Upon Nur77 knockdown in cultured rat CFs, expression of MyoFB markers and extracellular matrix proteins is reduced after stimulation with ISO or transforming growth factor–β (TGF-β). Accordingly, Nur77-depleted CFs produce less collagen and exhibit diminished proliferation and wound closure capacity. Interestingly, Nur77 knockdown in neonatal rat cardiomyocytes results in increased paracrine induction of MyoFB differentiation, which was blocked by TGF-β receptor antagonism. Taken together, Nur77-mediated regulation involves CF-intrinsic promotion of CF-to-MyoFB transition and inhibition of cardiomyocyte-driven paracrine TGF-β-mediated MyoFB differentiation. As such, Nur77 provides distinct, cell-specific regulation of cardiac fibrosis.

Summary Retinoic acid (RA) signaling plays an important role during heart development in establishing anteroposterior polarity, formation of inflow and outflow tract progenitors, and growth of the ventricular compact wall. RA is also utilized as a key ingredient in protocols designed for generating cardiac cell types from pluripotent stem cells (PSCs). This review discusses the role of RA in cardiogenesis, currently available protocols that employ RA for differentiation of various cardiovascular lineages, and plausible transcriptional mechanisms underlying this fate specification. These insights will inform further development of desired cardiac cell types from human PSCs and their application in preclinical and clinical research., In this article, Devalla and colleagues outline the role of retinoic acid (RA) signaling in the heart and provide an overview of human pluripotent stem cell-based cardiac differentiation approaches that employ RA. They offer new insights into molecular mechanisms underpinning the function of RA signaling and discuss the crosstalk with wingless-related integration site (WNT) and bone morphogenetic protein (BMP) signaling in cardiac cell fate specification.

During development, the ventricles of mammals and birds acquire a specialized pattern of electrical activation with the formation of the atrioventricular conduction system (AVCS), which coincides with the completion of ventricular septation. We investigated whether AVCS formation coincides with ventricular septation in developing Siamese crocodiles (Crocodylus siamensis). Comparisons were made with Amazon toadhead turtle (Mesoclemmys heliostemma) with a partial septum only and no AVCS (negative control) and with chicken (Gallus gallus) (septum and AVCS, positive control). Optical mapping of the electrical impulse in the crocodile and chicken showed a similar developmental specialization that coincided with full ventricular septation, whereas in the turtle the ventricular activation remained primitive. Co-localization of neural marker human natural killer-1 (HNK-1) and cardiomyocyte marker anti-myosin heavy chain (MF20) identified the AVCS on top of the ventricular septum in the crocodile and chicken only. AVCS formation is correlated with ventricular septation in both evolution and development.

Organ laterality refers to the left-right asymmetry in disposition and conformation of internal organs and is established during embryogenesis. The heart is the first organ to display visible left-right asymmetries through its left-sided positioning and rightward looping. Here, we present a new zebrafish loss-of-function allele for tbx5a, which displays defective rightward cardiac looping morphogenesis. By mapping individual cardiomyocyte behavior during cardiac looping, we establish that ventricular and atrial cardiomyocytes rearrange in distinct directions. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Pharmacological treatment and ex vivo culture establishes that the cardiac twisting depends on intrinsic mechanisms and is independent from cardiac growth. Furthermore, genetic experiments indicate that looping requires proper tissue patterning. We conclude that cardiac looping involves twisting of the chambers around the atrioventricular canal, which requires correct tissue patterning by Tbx5a.

Purpose: Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. Methods: We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. Results: Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). Conclusion: Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

Mutations and variations in and around SCN5A, encoding the major cardiac sodium channel, influence impulse conduction and are associated with a broad spectrum of arrhythmia disorders. Here, we identify an evolutionary conserved regulatory cluster with super enhancer characteristics downstream of SCN5A, which drives localized cardiac expression and contains conduction velocity-associated variants. We use genome editing to create a series of deletions in the mouse genome and show that the enhancer cluster controls the conformation of a >0.5 Mb genomic region harboring multiple interacting gene promoters and enhancers. We find that this cluster and its individual components are selectively required for cardiac Scn5a expression, normal cardiac conduction and normal embryonic development. Our studies reveal physiological roles of an enhancer cluster in the SCN5A-SCN10A locus, show that it controls the chromatin architecture of the locus and Scn5a expression, and suggest that genetic variants affecting its activity may influence cardiac function., Mutations associated with SCN5A, which encodes the major cardiac sodium channel, influence impulse conduction and are associated with arrhythmia disorders. Here, the authors identify an evolutionary conserved, super enhancer-like, regulatory cluster downstream of SCN5A and show that it controls the chromatin architecture of the locus and Scn5a expression.

Background In human fetuses with cardiac defects and increased nuchal translucency, abnormal ductus venosus flow velocity waveforms are observed. It is unknown whether abnormal ductus venosus flow velocity waveforms in fetuses with increased nuchal translucency are a reflection of altered cardiac function or are caused by local morphological alterations in the ductus venosus. Aim The aim of this study was to investigate if the observed increased nuchal translucency, cardiac defects and abnormal lymphatic development in the examined mouse models are associated with local changes in ductus venosus morphology. Study design Mouse embryos with anomalous lymphatic development and nuchal edema ( Ccbe1 −/− embryos), mouse embryos with cardiac defects and nuchal edema ( Fkbp12 −/− , Tbx1 −/− , Chd7 fl / fl ;Mesp1Cre , Jarid2 −/− NE+ embryos) and mouse embryos with cardiac defects without nuchal edema ( Tbx2 −/− , Fgf10 −/− , Jarid2 −/− NE − embryos) were examined. Embryos were analyzed from embryonic day (E) 11.5 to 15.5 using markers for endothelium, smooth muscle actin, nerve tissue and elastic fibers. Results All mutant and wild-type mouse embryos showed similar, positive endothelial and smooth muscle cell expression in the ductus venosus at E11.5–15.5. Nerve marker and elastic fiber expression were not identified in the ductus venosus in all investigated mutant and wild-type embryos. Local morphology and expression of the used markers were similar in the ductus venosus in all examined mutant and wild-type embryos. Conclusions Cardiac defects, nuchal edema and abnormal lymphatic development are not associated with morphological changes in the ductus venosus. Ductus venosus flow velocity waveforms most probably reflect intracardiac pressure.

Objective: To perform a morphological evaluation of the ductus venosus, heart and jugular lymphatic sac (JLS) in first-trimester human fetuses with normal and abnormal ductus venosus flow velocity waveforms (DV-FVWs) and normal and increased nuchal translucency (NT). Method: Postmortem examination was performed on fetuses with increased NT or structural malformations with previous NT and DV-FVW measurements. Ductus venosus morphology was examined using markers for endothelium, smooth muscle actin (SMA), nerves and elastic fibers. Fetal hearts were studied by microscopy. The nuchal region was analyzed using markers for lymphatic vessels, endothelium, SMA and nerves. Results: Two trisomy 21 and two trisomy 18 fetuses with increased NT and abnormal DV-FVWs were analyzed. As a control, one euploid anencephalic fetus with normal NT, cardiac anatomy and DV-FVWs was examined. Similar endothelial and SMA expression was observed in the ductus venosus in all fetuses. Nerve and elastic fiber expression were not detected. Three trisomic fetuses showed cardiac defects, one trisomic fetus demonstrated normal cardiac anatomy. The JLS was abnormally enlarged or contained red blood cells in all trisomic fetuses. The control fetus showed a normal JLS. Conclusion: Abnormal DV-FVWs are not justified by alterations in ductus venosus morphology. DV-FVWs most probably reflect intracardiac pressure. What's Already Known About This Topic? Ultrasound examination of ductus venosus flow velocity waveforms is used to indirectly assess fetal cardiac function and the fetal hemodynamic condition. Abnormal first-trimester ductus venosus flow velocity waveforms are related to chromosomal abnormalities, cardiac defects and increased nuchal translucency. Abnormal formation of the jugular lymphatic sacs is involved in the development of increased nuchal translucency. What Does This Study Add? Abnormal ductus venosus flow velocity waveforms are not caused by local alterations in ductus venosus morphology but reflect a changed hemodynamic status. The morphology of the ductus venosus is not related to abnormal ductus venosus flow velocity waveforms, chromosomal abnormalities, cardiac anatomy and increased nuchal translucency. Increased nuchal translucency in first-trimester human fetuses coincides with abnormal jugular lymphatic sac development.

Mammals and birds acquired high performance hearts and endothermy during their independent evolution from amniotes with many sauropsid features. A literature review shows that the variation in atrial morphology is greater in mammals than in ectothermic sauropsids. We therefore hypothesized that the transition from ectothermy to endothermy was associated with greater variation in cardiac structure. We tested the hypothesis in 14 orders of birds by assessing the variation in 15 cardiac structures by macroscopic inspection and histology, with an emphasis on the atria as they have multiple features that lend themselves to quantification. We found bird hearts to have multiple features in common with ectothermic sauropsids (synapomorphies), such as the presence of three sinus horns. Convergent features were shared with crocodylians and mammals, such as the cranial offset of the left atrioventricular junction. Other convergent features, like the compact organization of the atrial walls, were shared with mammals only. Pacemaker myocardium, identified by Isl1 expression, was anatomically node‐like (Mallard), thickened (Chicken), or indistinct (Lesser redpoll, Jackdaw). Some features were distinctly avian, (autapomorphies) including the presence of a left atrial antechamber and the ventral merger of the left and right atrial auricles, which was found in some species of parrots and passerines. Most features, however, exhibited little variation. For instance, there were always three systemic veins and two pulmonary veins, whereas among mammals there are 2–3 and 1–7, respectively. Our findings suggest that the transition to high cardiac performance does not necessarily lead to a greater variation in cardiac structure.

Among lizards, only monitor lizards (Varanidae) have a functionally divided cardiac ventricle. The division results from the concerted function of three partial septa, which may have homology to the ventricular septum of mammals and archosaurs. We show in developing monitors that two septa, the ‘muscular ridge’ and ‘bulbuslamelle’, express the evolutionary conserved transcription factors Tbx5, Irx1 and Irx2, orthologues of which mark the mammalian ventricular septum. Compaction of embryonic trabeculae contributes to the formation of these septa. The septa are positioned, however, to the right of the atrioventricular junction and they do not partake in the separation of incoming atrial blood streams. That separation is accomplished by the ‘vertical septum’ which expresses Tbx3 and Tbx5, which orchestrate the formation of the electrical conduction axis imbedded in the ventricular septum. These expression patterns are more pronounced in monitors than in other lizards, and are associated with a deep electrical activation near the vertical septum, contrasting the primitive base-to-apex activation of other lizards. We conclude that evolutionarily conserved transcriptional programs may underlie the formation of the ventricular septa of monitors.

The contraction of the heart is orchestrated by the components of the cardiac conduction system (CCS), which initiate and propagate the electrical impulses to coordinately activate the cardiac chambers. In the adult heart, the impulse is generated in the sinoatrial node and activates the atrial myocardium. Slow conduction of the impulse through the atrioventricular node allows for emptying of the atria and filling of the ventricles prior to ventricular contraction. Subsequent fast conduction through the atrioventricular bundle, bundle branches, and Purkinje fibre network activates the ventricular myocardium and causes the ventricles to contract. The development and function of the CCS involves complex regulatory networks of transcription factors acting in stage-, tissue-, and dose-dependent manners. Disrupted function or expression of these factors might lead to impaired development or function of the CCS components, associated with heart failure and sudden death. It is therefore crucial to understand the molecular and cellular mechanisms controlling the complex regulation of CCS development. This chapter summarizes current insight in the development and function of the different compartments of the CCS, and discusses the transcriptional networks underlying these processes.

International audience; 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.

Mammals and birds acquired high performance hearts and endothermy during their independent evolution from amniotes with many reptile characters. A literature review shows that the variation in atrial morphology is greater in mammals than in ectothermic reptiles. We therefore hypothesized that the transition from ectothermy to endothermy associated with greater variation in cardiac structure. We tested the hypothesis in birds, by assessing the variation in 15 characters in hearts from 13 orders of birds. Hearts were assessed by gross morphology and histology, and we focused on the atria as they have multiple features that lend themselves to quantification. We found bird hearts to have multiple features in common with ectothermic reptiles (synapomorphies), for instance the presence of three sinus horns. Convergent features were shared with crocodylians and mammals, such as the cranial offset of the left atrioventricular junction. Other convergent features like the compact organization of the atrial walls were shared with mammals only. Sinus myocardium expressing Isl1 was node-like (Mallard), thickened (chicken), or anatomically indistinct from surrounding myocardium (Lesser redpoll). Some features were distinctively avian (apomorphies), including the presence of a left atrial antechamber, and the ventral merger of the left and right atrium, which was found in parrots and passerine birds. Most features, however, exhibited little variation. For instance, there were always three systemic veins and two pulmonary veins, whereas among mammals there are 2-3 and 1-7, respectively. Our findings suggest that the transition to high cardiac performance does not necessarily lead to greater variation in cardiac structure.

BACKGROUND Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death.OBJECTIVES This meta-analysis sought to gain insights into the genetic determinants of myocardial mass.METHODS We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment.RESULTS We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p CONCLUSIONS Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets. (C) 2016 by the American College of Cardiology Foundation.

Objective To assess whether cardiac failure, because of cardiac defects, and abnormal jugular lymphatic development are involved in nuchal edema (NE) – the morphological equivalent of increased nuchal translucency – in various euploid mutant mouse models. Method Mouse embryos with lymphatic abnormalities and NE (Ccbe1−/−), with cardiac defects and NE (Fkbp12−/−, Tbx1−/−, Chd7fl/fl;Mesp1Cre, Jarid2−/−NE+) and with cardiac malformations without NE (Tbx2−/−, Pitx2−/−, Fgf10−/−, Jarid2−/−NE−) were examined. Embryos were analyzed from embryonic day 11.5 to 15.5. Markers for lymphatic vessels, endothelium, smooth muscle cells and nerves were used to study the nuchal region. Hematoxylin–Azophloxine staining was performed to examine cardiac morphology. Results Mouse embryos with lymphatic abnormalities and NE (Ccbe1−/−) showed no formation of the jugular lymphatic sac but normal cardiac morphology. In mouse embryos with cardiac defects and NE (Fkbp12−/−, Tbx1−/−, Chd7fl/fl;Mesp1Cre, Jarid2−/−NE+) enlarged jugular lymphatic sacs or large nuchal cavities within the NE were found. In mouse embryos with a cardiac malformation without NE (Tbx2−/−, Pitx2−/−, Fgf10−/−, Jarid2−/−NE−) normal jugular lymphatic sacs were observed. Conclusion NE consistently coincides with abnormal jugular lymphatic development in euploid mouse embryos, independent of cardiac anatomy. NE is unlikely to be caused by temporary cardiac failure solely because of a cardiac defect. © 2015 John Wiley & Sons, Ltd.

The regenerative medicine field has been revolutionized by the direct conversion of one cell type to another by ectopic expression of lineage-specific transcription factors. The direct reprogramming of fibroblasts to induced cardiac myocytes (iCMs) by core cardiac transcription factors (Gata4, Mef2c, Tbx5) both in vitro and in vivo has paved the way in cardiac regeneration and repair. Several independent research groups have successfully reported the direct reprogramming of fibroblasts in injured myocardium to cardiac myocytes employing a variety of approaches that rely on transcription factors, small molecules, and micro RNAs (miRNAs). Recently, this technology has been considered for local repair of the pacemaker and the cardiac conduction system. To address this, we will first discuss the direct reprograming advancements in the setting of working myocardium regeneration, and then elaborate on how this technology can be applied to repair the cardiac pacemaker and the conduction system.

Mammals and birds have a specialized cardiac atrioventricular conduction system enabling rapid activation of both ventricles. This system may have evolved together with high heart rates to support their endothermic state (warm-bloodedness) and is seemingly lacking in ectothermic vertebrates from which first mammals then birds independently evolved. Here, we studied the conduction system in crocodiles (Alligator mississippiensis), the only ectothermic vertebrates with a full ventricular septum. We identified homologues of mammalian conduction system markers (Tbx3-Tbx5, Scn5a, Gja5, Nppa-Nppb) and show the presence of a functional atrioventricular bundle. The ventricular Purkinje network, however, was absent and slow ventricular conduction relied on trabecular myocardium, as it does in other ectothermic vertebrates. We propose the evolution of the atrioventricular bundle followed full ventricular septum formation prior to the development of high heart rates and endothermy. In contrast, the evolution of the ventricular Purkinje network is strongly associated with high heart rates and endothermy., eLife digest Mammals and birds are referred to as ‘warm-blooded’ animals, because they maintain a constant high body temperature. This requires a lot of energy, so their bodies need to be well supplied with blood at all times. The hearts of mammals and birds contain two important structures that help them do this. The first is a full wall of muscle – called the ventricular septum – that divides the heart into left and right sides. The second is an electrical circuit made of specialized muscle cells that ensures that the heart beats fast enough by sending rapid electrical signals to the rest of the heart muscle. The circuit contains one group of cells in the ventricular septum, called the bundle of His, and another group termed the Purkinje network. Reptiles, however, do not maintain high body temperatures and are instead often thought of as ‘cold-blooded’ animals. The hearts of reptiles do not need to pump blood around the body as quickly and have different structures from warm-blooded animals. For example, most reptile hearts do not have a fully developed ventricular septum. The only exceptions are crocodiles, alligators and their relatives (the ‘crocodilians’), which do. Jensen, Boukens et al. therefore wanted to determine if a crocodilian heart also contained a specialized electrical circuit like those of birds and mammals. Previous studies that attempted to answer this question using only anatomical and electrical methods had yielded ambiguous results. As such, Jensen, Boukens et al. combined these methods with genetic techniques for a more detailed study. First, the ventricular septum of American alligators, a species of crocodilian, was examined, and found to contain a narrow tissue structure that strongly resembled the bundle of His. Indeed, if this presumptive bundle of His was cut, the electrical circuit was broken. Additional genetic analysis of this structure confirmed that genes similar to those active in the mammalian bundle of His were also switched on in alligators. However, recordings of heart activity showed that heart rates and the spread of electrical signals were both slower in alligators than in warm-blooded animals. This suggests that, although alligators have evolved some specialized muscle cells (in the form of a bundle of His), their electrical circuit is still ‘incomplete’. The lack of a Purkinje network, for example, would explain why their heart rates remain slow like other reptiles’. Together these findings add to the current understanding of how the heart works in different animals with varying requirements for energy and blood flow. Also, since crocodiles and warm-blooded birds both evolved from ancient reptiles, detailed descriptions of their heart structures could shed more light on how warm-bloodedness first developed.

Mammals evolved from reptile-like ancestors, and while the mammalian heart is driven by a distinct sinus node, a sinus node is not apparent in reptiles. We characterized the myocardial systemic venous pole, the sinus venosus, in reptiles to identify the dominant pacemaker and to assess whether the sinus venosus remodels and adopts an atrium-like phenotype as observed in mammals. Anolis lizards had an extensive sinus venosus of myocardium expressing Tbx18. A small sub-population of cells encircling the sinuatrial junction expressed Isl1, Bmp2, Tbx3, and Hcn4, homologues of genes marking the mammalian sinus node. Electrical mapping showed that hearts of Anolis lizards and Python snakes were driven from the sinuatrial junction. The electrical impulse was delayed between the sinus venosus and the right atrium, allowing the sinus venosus to contract and aid right atrial filling. In proximity of the systemic veins, the Anolis sinus venosus expressed markers of the atrial phenotype Nkx2-5 and Gja5. In conclusion, the reptile heart is driven by a pacemaker region with an expression signature similar to that of the immature sinus node of mammals. Unlike mammals, reptiles maintain a sinuatrial delay of the impulse, allowing the partly atrialized sinus venosus to function as a chamber.

Atrial septal defect (ASD) is the most common congenital heart defect clinically characterized by an opening in the atrial septum. Mutations in GATA4, TBX5, and NKX2-5 underlie this phenotype. Here, we report on the identification of a novel -6 G>C mutation in the highly conserved Kozak sequence in the 5'UTR of GATA4 in a small family presenting with two different forms of ASD. This is the first time a mutation in the Kozak sequence has been linked to heart disease. Functional assays demonstrate reduced GATA4 translation, though the GATA4 transcript levels remain normal. This leads to a reduction of GATA4 protein level, consequently diminishing the ability of GATA4 to transactivate target genes, as demonstrated by using the GATA4-driven Nppa (ANF) promoter. In conclusion, we identified a mutation in the GATA4 Kozak sequence that likely contributes to the pathogenesis of ASD. In general, it points to the importance of accurate protein level regulation during heart development and emphasizes the need to analyze the entire transcribed region when screening for mutations.

Rationale: Cardiac progenitor cells from the second heart field (SHF) contribute to rapid growth of the embryonic heart, giving rise to right ventricular and outflow tract (OFT) myocardium at the arterial pole of the heart, and atrial myocardium at the venous pole. Recent clonal analysis and cell-tracing experiments indicate that a common progenitor pool in the posterior region of the SHF gives rise to both OFT and atrial myocytes. The mechanisms regulating deployment of this progenitor pool remain unknown. Objective: To evaluate the role of TBX1 , the major gene implicated in congenital heart defects in 22q11.2 deletion syndrome patients, in posterior SHF development. Methods and Results: Using transcriptome analysis, genetic tracing, and fluorescent dye-labeling experiments, we show that Tbx1 -dependent OFT myocardium originates in Hox -expressing cells in the posterior SHF. In Tbx1 null embryos, OFT progenitor cells fail to segregate from this progenitor cell pool, leading to failure to expand the dorsal pericardial wall and altered positioning of the cardiac poles. Unexpectedly, addition of SHF cells to the venous pole of the heart is also impaired, resulting in abnormal development of the dorsal mesenchymal protrusion, and partially penetrant atrioventricular septal defects, including ostium primum defects. Conclusions: Tbx1 is required for inflow as well as OFT morphogenesis by regulating the segregation and deployment of progenitor cells in the posterior SHF. Our results provide new insights into the pathogenesis of congenital heart defects and 22q11.2 deletion syndrome phenotypes.

Variants in SCN10A, which encodes a voltage-gated sodium channel, are associated with alterations of cardiac conduction parameters and the cardiac rhythm disorder Brugada syndrome; however, it is unclear how SCN10A variants promote dysfunctional cardiac conduction. Here we showed by high-resolution 4C-seq analysis of the Scn10a-Scn5a locus in murine heart tissue that a cardiac enhancer located in Scn10a, encompassing SCN10A functional variant rs6801957, interacts with the promoter of Scn5a, a sodium channel-encoding gene that is critical for cardiac conduction. We observed that SCN5A transcript levels were several orders of magnitude higher than SCN10A transcript levels in both adult human and mouse heart tissue. Analysis of BAC transgenic mouse strains harboring an engineered deletion of the enhancer within Scn10a revealed that the enhancer was essential for Scn5a expression in cardiac tissue. Furthermore, the common SCN10A variant rs6801957 modulated Scn5a expression in the heart. In humans, the SCN10A variant rs6801957, which correlated with slowed conduction, was associated with reduced SCN5A expression. These observations establish a genomic mechanism for how a common genetic variation at SCN10A influences cardiac physiology and predisposes to arrhythmia.

The sinus venosus, the cardiac chamber upstream of the (right) atrium, is a severely underinvestigated structure. Yet, its myocardium harbors the cardiac pacemaker in all vertebrates. In human, ectopic pacemaking and subsequent pathologies may originate from sinus venosus-derived myocardium surrounding the coronary sinus and the superior caval vein. In ectothermic vertebrates, i.e., fishes, amphibians and reptiles, the sinus venosus aids atrial filling by contracting prior to the atrium (atria). This is facilitated by the sinuatrial delay of approximately the same duration as the atrioventricular delay, which facilitates atrial filling of the ventricles. In mammals, the sinuatrial delay is lost, and the sinus venosus-derived myocardium persists as an extensive myocardial sheet surrounding the caval veins, which is activated in synchrony with the myocardium of the atria. The caval vein myocardium is hardly of significance in the healthy formed heart, but we suggest that the sinus venosus functions as a chamber during development when cardiac output, heart rate, blood pressure and architecture is much more like that of ectothermic vertebrates. The remodeling of the sinus venosus in mammals may be an adaptation associated with the high heart rates necessary for postnatal endothermy. If so, the endothermic birds should exhibit a similar remodeling as mammals, which remains to be investigated.

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10(-68); rs9388451, P = 5.1 × 10(-17)) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10(-14)). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10(-81)). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Item does not contain fulltext AIMS: Transgenic mice are frequently used to investigate the role of genes involved in cardiac conduction. The QRS duration calculated from the electrocardiogram (ECG) is a commonly used measure for ventricular conduction time. However, the relation between ventricular activation and QRS duration calculated from a mouse surface ECG is not well understood. We aim to relate ventricular activation and repolarization patterns with the mouse ECG. METHODS AND RESULTS: Ventricular activation and repolarization patterns generated by high-density optical mapping and a six-lead pseudo-ECG were compared in isolated mouse hearts. In addition, mouse ECGs were simulated in silico. Right-ventricular activation ends later than left-ventricular activation. Final activation coincided with the end of the QRS complex in leads III and aVF, but not in leads I, II, aVR, and aVL. The pattern of early repolarization (at 20% of repolarization, RT20) but not of RT50 or RT80 followed the activation pattern. After sodium channel blockade by ajmaline, total ventricular activation time increased by 10.0 ms, whereas QRS duration increased by only 2.1 ms. In mice carrying a mutation in Scn5a (1798insD), ventricular activation ended after the end of the QRS complex (12.9 +/- 0.1 vs. 10.8 +/- 0.3). CONCLUSION: In the mouse, ventricular myocardium activation and early repolarization waves are simultaneously present. This hampers unequivocal interpretation of the duration of the QRS complex as a measure of ventricular activation duration, especially when conduction is slowed. Under these conditions mapping of local activation and repolarization patterns is required for correct interpretation of the ECG.

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 ( Scn5a 1798insD/+ ). 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 Scn5a 1798insD/+ 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.

Item does not contain fulltext BACKGROUND: Doppler flow velocities of the ductus venosus are increasingly used to assess fetal increased nuchal translucency, growth-restriction and monochorionic twins, and might contribute to screening for cardiac defects. It is disputed whether a sphincter at the ductus venosus inlet actively regulates blood flow. AIMS: This study aims to define the morphogenesis of the developing mouse and human ductus venosus and to address the existence of a sphincter. STUDY DESIGN: The presence of endothelium, smooth muscle, elastic fibers and nerves in the ductus venosus of E10.5-15.5 mouse embryos and in three corresponding human embryos (CS16, CS19 and CS23) was examined using immunohistochemistry. Three-dimensional reconstructions of the ductus venosus of E11.5-15.5 mouse and CS14-23 human embryos were generated and examined. RESULTS: The ductus venosus lumen was narrowed from ventral-caudal to dorsal-cranial in E13.5-15.5 mouse and CS16-23 human embryos. Mouse embryos showed positive endothelial Pecam1 expression from E11.5-15.5 and smooth muscle actin staining in the ventral-caudal part of the ductus venosus from E12.5-15.5. At all developmental stages, elastic fiber and nerve marker expression was not detected in the ductus venosus (Fig. 2). In human embryos endothelial Pecam1 and smooth muscle actin expression was found in the ductus venosus from CS16 and CS19 onwards. Elastic fiber and nerve marker expression was not detected in all stages (Fig. 4). Morphogenesis and staining results of the ductus venosus were similar in both species. CONCLUSIONS: The ductus venosus lacks a sphincter at its inlet as no accumulation of smooth muscle cells, elastic fibers or nerve innervation was found in mouse embryos from E11.5-15.5 and in human embryos from CS14-23.

The mammalian heart expresses two closely related natriuretic peptide (NP) hormones, atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). The excretion of the NPs and the expression of their genes strongly respond to a variety of cardiovascular disorders. NPs act to increase natriuresis and decrease vascular resistance, thereby decreasing blood volume, systemic blood pressure and afterload. Plasma levels of BNP are used as diagnostic and prognostic markers for hypertrophy and heart failure (HF), and both ANF and BNP are widely used in biomedical research to assess the hypertrophic response in cell culture or the development of HF related diseases in animal models. Moreover, ANF and BNP are used as specific markers for the differentiating working myocardium in the developing heart, and the ANF promoter serves as platform to investigate gene regulatory networks during heart development and disease. However, despite decades of research, the mechanisms regulating the NP genes during development and disease are not well understood. Here we review current knowledge on the regulation of expression of the genes for ANF and BNP and their role as biomarkers, and give future directions to identify the in vivo regulatory mechanisms. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.

Defects originating from the atrioventricular canal region are part of a wide spectrum of congenital cardiovascular malformations that frequently affect newborns. These defects include partial or complete atrioventricular septal defects, atrioventricular valve defects, and arrhythmias, such as atrioventricular re-entry tachycardia, atrioventricular nodal block, and ventricular preexcitation. Insight into the cellular origin of the atrioventricular canal myocardium and the molecular mechanisms that control its development will aid in the understanding of the etiology of the atrioventricular defects. This review discusses current knowledge concerning the origin and fate of the atrioventricular canal myocardium, the molecular mechanisms that determine its specification and differentiation, and its role in the development of certain malformations such as those that underlie ventricular preexcitation. Birth Defects Research (Part A), 2011. © 2011Wiley-Liss, Inc.

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, alpha [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

Disorders of the cardiac conduction system occur frequently and may cause life-threatening arrhythmias requiring medication or electronic pacemaker implantation. Repair or regeneration of conduction system components is currently not possible due to limited knowledge of the molecular regulation of pacemaker myocardium. Origin and development of the cardiac conduction system have been subject to debate for many decades. This review will discuss recent advances in our understanding of the molecular regulation of the development of the conduction system. We conclude that the components of the cardiac conduction system originate from embryonic myocardium that has maintained essential features of its primitive phenotype, whereas the adjacent myocardium differentiates into working myocardium.

The sinus node generates the electrical impulse, which spreads rapidly over both atria, causing them to contract simultaneously. In the normal heart, a layer of connective tissue electrically insulates the atria and ventricles. The only pathway that crosses this plane is the atrioventricular conduction axis, through which the impulse reaches the ventricles. Within the axis, the atrioventricular node delays the impulse, allowing the ventricles to be filled before their contraction is initiated. Moreover, the atrioventricular node protects the ventricles from rapid atrial arrhythmias and may take over pacemaker function when the sinus node fails. In pathological conditions, these complex physiological properties contribute to several types of arrhythmias that originate from the atrioventricular conduction system. One example is atrioventricular block, which requires electronic pacemaker implantation because there is currently no cure for this arrhythmia. Because conduction system defects may arise during embryonic development, the mechanisms of conduction system development have been intensively studied. Nevertheless, its developmental origin, molecular composition, and phenotype have remained fertile subjects of research and debate. Lineage and expressional analyses have indicated that the atrioventricular node develops from a subpopulation of precursor cells in the dorsal part of the embryonic atrioventricular canal. These cells become distinct early in development, are less well differentiated compared to the developing working myocardium, and, in addition to their cardiogenic gene program, activate and maintain a neurogenic gene program.

Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Nav1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Nav1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Nav1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Nav1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean ± SEM 309 ± 32 V/s; n = 14) compared to subendocardial myocytes (394 ± 32 V/s; n = 11; P

Reentry is the main mechanism of life-threatening ventricular arrhythmias, including ventricular fibrillation and tachycardia. Its occurrence depends on the simultaneous presence of an arrhythmogenic substrate (a preexisting condition) and a “trigger,” and is favored by electrophysiological heterogeneities. In the adult heart, electrophysiological heterogeneities of the ventricle exist along the apicobasal, left–right, and transmural axes. Also, conduction is preferentially slowed in the right ventricular outflow tract, especially during pharmacological sodium channel blockade. We propose that the origin of electrophysiological heterogeneities of the adult heart lies in early heart development. The heart is formed from several progenitor regions: the first heart field predominantly forms the left ventricle, whereas the second heart field forms the right ventricle and outflow tract. Furthermore, the embryonic outflow tract consists of slowly conducting tissue until it is incorporated into the ventricles and develops rapidly conducting properties. The subepicardial myocytes and subendocardial myocytes run distinctive gene programs from their formation onwards. This review discusses the hypothesis that electrophysiological heterogeneities in the adult heart result from persisting patterns in gene expression and function along the craniocaudal and epicardial–endocardial axes of the developing heart. Understanding the developmental origins of electrophysiological heterogeneity contributing to ventricular arrhythmias may give rise to new therapies.

Aims T-box factors Tbx2 and Tbx3 play key roles in the development of the cardiac conduction system, atrioventricular canal, and outflow tract of the heart. They regulate the gap-junction-encoding gene Connexin43 ( Cx43 ) and other genes critical for heart development and function. Discovering protein partners of Tbx2 and Tbx3 will shed light on the mechanisms by which these factors regulate these gene programs. Methods and results Employing an yeast 2-hybrid screen and subsequent in vitro pull-down experiments we demonstrate that muscle segment homeobox genes Msx1 and Msx2 are able to bind the cardiac T-box proteins Tbx2, Tbx3, and Tbx5. This interaction, as that of the related Nkx2.5 protein, is supported by the T-box and homeodomain alone. Overlapping spatiotemporal expression patterns of Msx1 and Msx2 together with the T-box genes during cardiac development in mouse and chicken underscore the biological significance of this interaction. We demonstrate that Msx proteins together with Tbx2 and Tbx3 suppress Cx43 promoter activity and down regulate Cx43 gene activity in a rat heart-derived cell line. Using chromatin immunoprecipitation analysis we demonstrate that Msx1 can bind the Cx43 promoter at a conserved binding site located in close proximity to a previously defined T-box binding site, and that the activity of Msx proteins on this promoter appears dependent in the presence of Tbx3. Conclusion Msx1 and Msx2 can function in concert with the T-box proteins to suppress Cx43 and other working myocardial genes.

The sinoatrial node initiates the heartbeat and controls the rate and rhythm of contraction, thus serving as the pacemaker of the heart. Despite the crucial role of the sinoatrial node in heart function, the mechanisms that underlie its specification and formation are not known. Tbx3, a transcriptional repressor required for development of vertebrates, is expressed in the developing conduction system. Here we show that Tbx3 expression delineates the sinoatrial node region, which runs a gene expression program that is distinct from that of the bordering atrial cells. We found lineage segregation of Tbx3-negative atrial and Tbx3-positive sinoatrial node precursor cells as soon as cardiac cells turn on the atrial gene expression program. Tbx3 deficiency resulted in expansion of expression of the atrial gene program into the sinoatrial node domain, and partial loss of sinoatrial node-specific gene expression. Ectopic expression of Tbx3 in mice revealed that Tbx3 represses the atrial phenotype and imposes the pacemaker phenotype on the atria. The mice displayed arrhythmias and developed functional ectopic pacemakers. These data identify a Tbx3-dependent pathway for the specification and formation of the sinoatrial node, and show that Tbx3 regulates the pacemaker gene expression program and phenotype.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The genetics of AF are complex, but recent GWAS have revealed a number of AF risk loci, including TBX5 . TBX5 plays a critical role in heart development, and continues to be expressed in the adult atria. We assessed the role of Tbx5 in the adult heart by removing Tbx5 from the mouse at 6 weeks-of-age. Adult Tbx5 mutant mice developed spontaneous sustained atrial fibrillation, characterized by surface electrocardiogram (ECG), intracardiac ECGs, and ex vivo whole heart optical mapping. Tbx5 mutant isolated cardiomycytes showed prolonged action potentials, disrupted calcium handling, and abnormal automaticity. Tbx5 mutant atrial gene expression analysis revealed down-regulation of numerous conduction system effectors genes including Scn5a, Ryr2, Gja1, Atp2a2 , Dsp , and numerous potassium channels. We also observed Tbx5 dependence of the transcription factor gene Pitx2 , the number one locus associated with AF by GWAS. We identified an enhancer upstream of PITX2 that interacts with the PITX2 promoter by 4C and whose activity is TBX5 dependent and modulated by common SNP rs1906595, which alters a canonical TBX5 binding site. We also identified cis-regulatory elements at Scn5a, Ryr2, Gja1, Atp2a2 , and Dsp , locus co-regulated by Tbx5 and Pitx2. We describe a TBX5-driven atrial molecular network including Pitx2 that determines atrial conduction properties.

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 endocrine function of the heart was shown by the identification of a potent inhibitor of renal tubular NaCl reabsorbtion, atrial natriuretic peptide/factor (ANP), in atrial extracts. Since then, Nppa, the gene encoding ANP, has become an important tool for molecular and developmental biologists. Expression of Nppa is an early and specific marker for the differentiating working myocardium of the atria and ventricles of the developing heart. Around the time of birth, ventricular Nppa expression is down-regulated, to be reactivated in response to a variety of cardiovascular disorders. Many aspects of the regulatory pathways that control Nppa gene expression during cardiac development or disease have been revealed. However, several fundamental issues remain to be resolved. Particularly, the regulatory mechanisms underlying the ventricular Nppa expression in the embryonic heart and the reactivation in the failing ventricle remain unclear. Here we review current knowledge on the transcriptional regulation of the Nppa gene.

In this communication we discuss the formation of the synchronously contracting chambered heart from a peristaltically contracting linear heart tube. It is proposed that members of the T-box family of transcription factors play a crucial role in the formation of the building plan of the formed heart. Tbx5 may confer venoarterial polarity to the heart tube, whereas Tbx2 initially and Tbx3 in later developmental stages prevent the cardiac inflow tract, atrioventricular region, outflow tract, as well as the cardiac inner curvatures from chamber differentiation. With the exception of the outflow tract that becomes incorporated into the ventricles, these regions contribute to the cardiac conduction system.

Conduction across the atrioventricular junctions had long been controversial, with arguments raging as to whether such conduction was myogenic or neurogenic. The landmark study of Tawara in 1906 established the existence of an axis of histologically specialized myocardial cells contained within fibrous sheaths as being responsible for rapid conduction from the atrial to the ventricular myocardium. Keith and Flack then demonstrated that the atrial impulse was generated in the sinus node, with subsequent slowing within the atrioventricular node of Tawara. The detailed reviews of Aschoff and Monckeberg then emphasized the morphological features that permit unequivocal recognition of the nodes, along with rapidly conducting tracts. It is now pertinent that we remember these original criteria for anatomical recognition as we discuss the steps involved in the development of these conducting structures from their embryologic primordiums. In this review, we restate the criteria of Aschoff and Monckeberg, then discuss their relevance to the developmental issues, emphasizing that all myocardial cells have the potential to conduct. The important issues, therefore, are the changes that occur as the heart itself changes from a solitary muscular tube, already possessing the capacity t generate an electrocardiogram, to the four-chambered structure we recognize in postnatal life.

Specific regions of the embryonic heart tube differentiate into atrial and ventricular chamber myocardium, whereas the inflow tract, atrioventricular canal, inner curvatures, and outflow tract do not. These regions express Tbx2, a transcriptional repressor. Here, we tested its role in chamber formation. The temporal and spatial pattern of Tbx2 mRNA and protein expression in mouse hearts was found to be complementary to that of chamber myocardium-specific genes Nppa, Cx40, Cx43, and Chisel, and was conserved in human. In vitro, Tbx2 repressed the activity of regulatory fragments of Cx40, Cx43, and Nppa. Hearts of transgenic embryos that expressed Tbx2 in the prechamber myocardium completely failed to form chambers and to express the chamber myocardium-specific genes Nppa, Cx40, and Chisel, whereas other cardiac genes were normally expressed. These findings provide the first evidence that Tbx2 is a determinant in the local repression of chamber-specific gene expression and chamber differentiation.

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 (αMHC-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.