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Background: Premature restriction or closure of foramen ovale (FO) in otherwise structurally normal hearts may be associated with right ventricular dilation, tricuspid regurgitation, pericardial effusion, heart failure, even poor perinatal outcomes. Data about these rare conditions are lacking., Methods: We retrospectively reviewed the echocardiographic records of 9704 fetuses seen from 2010 to 2014 in Beijing Anzhen Hospital, a regional and national referral center, to ascertain the presence of restriction or closure of FO. We collected the fetal echocardiography and perinatal outcome data for this group of fetuses with restriction or closure of FO., Results: In this large, single-institution cohort (n = 9704), 6707 fetuses seen between 23 and 37 weeks of gestation had normal heart structures; of these, 60 (0.89%) had restrictive FO (rFO) and 5 (0.07%) had closure of FO (cFO). Fetal echocardiographic images showed right atrial dilation in 48 (73.84%), right ventricular dilation in 38 (58.46%), tricuspid regurgitation in 19 (29.23%), and pericardial effusion in 10 (15.38%). Also in this group, 50 (83.3%) with rFO and 4 (80.0%) with cFO had follow-up data. No prenatal deaths occurred in either the rFO or the cFO group, but the neonatal mortality included 1 in the rFO group and 2 in the cFO group., Conclusion: Premature rFO/cFO are rare in fetuses with otherwise structurally normal hearts. The fetal echocardiographic characteristics include right atrial and ventricular dilated, tricuspid regurgitation, and pericardial effusion. Most fetuses had a good outcome, although there was an association between rFO, especially cFO, with neonatal morality and complications (prematurity, maternal preeclampsia and placental abruption, hydrops fetalis, and necrotizing enterocolitis with perforation)., (© 2018 Wiley Periodicals, Inc.)

The four-chamber structure of the mammalian heart is established during embryonic development. While key regulators for ventricular development are well studied, regulatory mechanisms for atrial chamber morphogenesis remain poorly understood. Here, we found that angiopoietin-1 (Angpt1), a vascular maturation factor, is highly and specifically expressed in atrial myocardium during heart development. Loss of myocardial Angpt1 in mouse embryo led to severe impairment in atrial chamber morphogenesis. We revealed that Angpt1 deficiency results in excessive deposition of cardiac jelly, which disturbs regulation of myocardial growth, thereby impairing maturation of atrial chambers. Mechanistically, myocardial Angpt1 activates endocardial Tie2 and positively regulates expression of ADAMTS proteases, which is crucial for proper degradation of cardiac jelly. Accordingly, loss of Tie2 also impairs ADAMTS-mediated degradation of cardiac jelly in atrium. Collectively, myocardial Angpt1/endocardial Tie2 signaling in atrium promotes spatiotemporal degradation of cardiac jelly during early cardiac development and is therefore indispensable for atrial chamber morphogenesis., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Normal development of the heart is regulated, in part, by mechanical influences associated with blood flow during early stages of embryogenesis. Specifically, the potential for retrograde flow at the atrioventricular canal (AVC) is particularly important in valve development. However, the mechanisms causing this retrograde flow have received little attention. In this study, a numerical analysis was performed on images of the embryonic zebrafish heart between 48 and 55hpf. During these stages, normal retrograde flow is prevalent. To manipulate this flow, zebrafish were placed in a centrifuge and subjected to a hypergravity environment to alter the cardiac preload at various six-hour intervals between 24 and 48hpf. Parameters of the pumping mechanics were then analyzed through a spatiotemporal analysis of processed image sequences. We find that the loss of retrograde flow in experimentally manipulated embryos occurs in part because of a greater resistance in the form of atrial and AVC contractile closure. Additionally, during retrograde flow, these embryos exhibit significantly greater pressure difference across the AVC based on calculations of expansive and contractile rates of the atrium and ventricle. These results elucidated that the developing heart is highly sensitive to small changes in pumping mechanics as it strives to maintain normal hemodynamic conditions necessary for later cardiac development., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mutations in the T-box transcription factor TBX20 are associated with multiple forms of congenital heart defects, including cardiac septal abnormalities, but our understanding of the contributions of endocardial TBX20 to heart development remains incomplete. Here, we investigated how TBX20 interacts with endocardial gene networks to drive the mesenchymal and myocardial movements that are essential for outflow tract and atrioventricular septation. Selective ablation of Tbx20 in murine endocardial lineages reduced the expression of extracellular matrix and cell migration genes that are critical for septation. Using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we identified accessible chromatin within endocardial lineages and intersected these data with TBX20 ChIP-seq and chromatin loop maps to determine that TBX20 binds a conserved long-range enhancer to regulate versican (Vcan) expression. We also observed reduced Vcan expression in Tbx20-deficient mice, supporting a direct role for TBX20 in Vcan regulation. Further, we show that the Vcan enhancer drove reporter gene expression in endocardial lineages in a TBX20-binding site-dependent manner. This work illuminates gene networks that interact with TBX20 to orchestrate cardiac septation and provides insight into the chromatin landscape of endocardial lineages during septation.

One major obstacle to the application of stem cell-derived cardiomyocytes (CMs) for disease modeling and clinical therapies is the inability to identify the developmental stage of these cells without the need for genetic manipulation or utilization of exogenous markers. In this study, we demonstrate that Raman microspectroscopy can non-invasively identify embryonic stem cell (ESC)-derived chamber-specific CMs and monitor cell maturation. Using this marker-free approach, Raman peaks were identified for atrial and ventricular CMs, ESCs were successfully discriminated from their cardiac derivatives, a distinct phenotypic spectrum for ESC-derived CMs was confirmed, and unique spectral differences between fetal versus adult CMs were detected. The real-time identification and characterization of CMs, their progenitors, and subpopulations by Raman microspectroscopy strongly correlated to the phenotypical features of these cells. Due to its high molecular resolution, Raman microspectroscopy offers distinct analytical characterization for differentiating cardiovascular cell populations., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers., Methods and Results: In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis., Conclusion: Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction.

Rationale: Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease., Objective: To determine the role of canonical Wnt signaling in the myocardium during AVC development., Methods and Results: We used a novel allele of β-catenin that preserves β-catenin's cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that the loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with the loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiological criteria. Aberrant AVC development can lead to ventricular pre-excitation, a characteristic feature of Wolff-Parkinson-White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of β-catenin protein levels can rescue Notch-mediated ventricular pre-excitation and dysregulated ion channel gene expression., Conclusions: Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electric programming upstream of Tbx3. Our data further suggest that ventricular pre-excitation may require both morphological patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue., (© 2014 American Heart Association, Inc.)

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1(Cre)) mouse. Embryonic Wt1(Cre);Alk3(fl/fl) specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1(Cre);Alk3(fl/fl) mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1(Cre);Alk3(fl/fl) specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cardiac development arises from two sources of mesoderm progenitors, the first heart field (FHF) and the second (SHF). Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors common for both heart fields. Here, using clonal analysis of the earliest prospective cardiovascular progenitors in a temporally controlled manner during early gastrulation, we found that Mesp1 progenitors consist of two temporally distinct pools of progenitors restricted to either the FHF or the SHF. FHF progenitors were unipotent, whereas SHF progenitors were either unipotent or bipotent. Microarray and single-cell PCR with reverse transcription analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Together, these results provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors that independently express Mesp1 at different time points during their specification, revealing that the regional segregation and lineage restriction of cardiac progenitors occur very early during gastrulation.

The past decades have seen immense progress in the understanding of cardiac development. Appreciation of precise details of cardiac anatomy, however, has yet to be fully translated into the more general understanding of the changing structure of the developing heart, particularly with regard to formation of the septal structures. In this review, using images obtained with episcopic microscopy together with scanning electron microscopy, we show that the newly acquired information concerning the anatomic changes occurring during separation of the cardiac chambers in the mouse is able to provide a basis for understanding the morphogenesis of septal defects in the human heart. It is now established that as part of the changes seen when the heart tube changes from a short linear structure to the looped arrangement presaging formation of the ventricles, new material is added at both its venous and arterial poles. The details of these early changes, however, are beyond the scope of our current review. It is during E10.5 in the mouse that the first anatomic features of septation are seen, with formation of the primary atrial septum. This muscular structure grows toward the cushions formed within the atrioventricular canal, carrying on its leading edge a mesenchymal cap. Its cranial attachment breaks down to form the secondary foramen by the time the mesenchymal cap has used with the atrioventricular endocardial cushions, the latter fusion obliterating the primary foramen. Then the cap, along with a mesenchymal protrusion that grows from the mediastinal mesenchyme, muscularizes to form the base of the definitive atrial septum, the primary septum itself forming the floor of the oval foramen. The cranial margin of the foramen is a fold between the attachments of the pulmonary veins to the left atrium and the roof of the right atrium. The apical muscular ventricular septum develops concomitant with the ballooning of the apical components from the inlet and outlet of the ventricular loop. Its apical part is initially trabeculated. The membranous part of the septum is derived from the rightward margins of the atrioventricular cushions, with the muscularizing proximal outflow cushions fusing with the muscular septum and becoming the subpulmonary infundibulum as the aorta is committed to the left ventricle. Perturbations of these processes explain well the phenotypic variants of deficient atrial and ventricular septation., (Copyright © 2014 Wiley Periodicals, Inc.)

Objective: To evaluate cardiac anatomy and function in fetuses with right-sided congenital diaphragmatic hernia (RCDH), and to compare these values with those of normal controls., Methods: Fetal echocardiography was performed in 17 consecutive cases with isolated RCDH and 17 gestational age-matched controls. Two-dimensional measurements included ventricular and outflow tract diameters. Doppler ultrasound was used to measure the flow pattern in the ductus venosus and over the pulmonary and aortic valves, right and left ventricular myocardial performance index and the E/A wave ratio over the atrioventricular valves. Stroke volume, cardiac output and shortening fraction were calculated., Results: Median gestational age at evaluation was 27.4 (interquartile range, 24.4-28.9) weeks. RCDH cases had a significantly smaller right ventricle and pulmonary valve diameter. Furthermore, stroke volume and cardiac output from the right ventricle were lower than in controls. Myocardial contractility, however, appeared normal., Conclusions: Despite significantly reduced right ventricular dimensions and cardiac output, cardiac contractility was normal in a cohort of fetuses with RCDH., (Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.)

Establishment of specific characteristics of each embryonic cardiac chamber is crucial for development of a fully functional adult heart. Despite the importance of defining and maintaining unique features in ventricular and atrial cardiomyocytes, the regulatory mechanisms guiding these processes are poorly understood. Here, we show that the homeodomain transcription factors Nkx2.5 and Nkx2.7 are necessary to sustain ventricular chamber attributes through repression of atrial chamber identity. Mutation of nkx2.5 in zebrafish yields embryos with diminutive ventricular and bulbous atrial chambers. These chamber deformities emerge gradually during development, with a severe collapse in the number of ventricular cardiomyocytes and an accumulation of excess atrial cardiomyocytes as the heart matures. Removal of nkx2.7 function from nkx2.5 mutants exacerbates the loss of ventricular cells and the gain of atrial cells. Moreover, in these Nkx-deficient embryos, expression of vmhc, a ventricular gene, fades, whereas expression of amhc, an atrial gene, expands. Cell-labeling experiments suggest that ventricular cardiomyocytes can transform into atrial cardiomyocytes in the absence of Nkx gene function. Through suggestion of transdifferentiation from ventricular to atrial fate, our data reveal a pivotal role for Nkx genes in maintaining ventricular identity and highlight remarkable plasticity in differentiated myocardium. Thus, our results are relevant to the etiologies of fetal and neonatal cardiac pathology and could direct future innovations in cardiac regenerative medicine.

Atria and ventricles exhibit distinct molecular profiles that produce structural and functional differences between the two cardiac compartments. However, the factors that determine these differences remain largely undefined. Cardiomyocyte-specific COUP-TFII ablation produces ventricularized atria that exhibit ventricle-like action potentials, increased cardiomyocyte size, and development of extensive T tubules. Changes in atrial characteristics are accompanied by alterations of 2,584 genes, of which 81% were differentially expressed between atria and ventricles, suggesting that a major function of myocardial COUP-TFII is to determine atrial identity. Chromatin immunoprecipitation assays using E13.5 atria identified classic atrial-ventricular identity genes Tbx5, Hey2, Irx4, MLC2v, MLC2a, and MLC1a, among many other cardiac genes, as potential COUP-TFII direct targets. Collectively, our results reveal that COUP-TFII confers atrial identity through direct binding and by modulating expression of a broad spectrum of genes that have an impact on atrial development and function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the pan-retinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca(2+) sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.

Tbx5 is involved in congenital heart disease, however, the mechanisms leading to organ malformation are greatly unknown. We hypothesized a model by which the Tbx5 binding protein Pdlim7 controls nuclear/cytoplasmic shuttling and function of the transcription factor. Using the zebrafish, we present in vivo significance for an essential role of Tbx5/Pdlim7 protein interaction in the regulation of cardiac formation. Knock-down of Pdlim7 results in a non-looped heart, strikingly reminiscent of the tbx5 heartstrings mutant phenotype. However, while misregulation of Pdlim7 and Tbx5 produce similar aberrant cardiac morphology, molecular and histological analysis uncovered that the Pdlim7 and Tbx5 cardiac phenotypes are due to opposite effects on valve development. Loss of Pdlim7 function causes no valve tissue to develop while lack of Tbx5 results in increased valve tissue. These opposing defects are evident before valve formation and are the result of distinct gene misregulation during specification of the atrio-ventricular (AV) boundary. We show that Pdlim7/Tbx5 interactions affect the expression of Tbx5 target genes nppa and tbx2b at the AV boundary, and their domains of misexpression directly correlate with the identified valve defects. These studies demonstrate that controlling the correct balance of Tbx5 activity is crucial for the specification of the AV boundary and valve formation., (Copyright 2009 Elsevier Inc. All rights reserved.)

Rationale: T-box transcription factors play critical roles in the coordinated formation of the working chambers and the atrioventricular canal (AVC). Tbx2 patterns embryonic myocardial cells to form the AVC and suppresses their differentiation into chamber myocardium. Tbx20-deficient embryos, which fail to form chambers, ectopically express Tbx2 throughout the entire heart tube, providing a potential mechanism for the function of Tbx20 in chamber differentiation., Objective: To identify the mechanism of Tbx2 suppression by Tbx20 and to investigate the involvement of Tbx2 in Tbx20-mediated chamber formation., Methods and Results: We generated Tbx20 and Tbx2 single and double knockout embryos and observed that loss of Tbx2 did not rescue the Tbx20-deficient heart from failure to form chambers. However, Tbx20 is required to suppress Tbx2 in the developing chambers, a prerequisite to localize its strong differentiation-inhibiting activity to the AVC. We identified a bone morphogenetic protein (Bmp)/Smad-dependent Tbx2 enhancer conferring AVC-restricted expression and Tbx20-dependent chamber suppression of Tbx2 in vivo. Unexpectedly, we found in transfection and localization studies in vitro that both Tbx20 and mutant isoforms of Tbx20 unable to bind DNA attenuate Bmp/Smad-dependent activation of Tbx2 by binding Smad1 and Smad5 and sequestering them from Smad4., Conclusions: Our data suggest that Tbx20 directly interferes with Bmp/Smad signaling to suppress Tbx2 expression in the chambers, thereby confining Tbx2 expression to the prospective AVC region.

The Pitx2 gene regulates left-right (L/R) asymmetrical cardiac morphogenesis. Constitutive Pitx2 knock out (ko) mice die before birth and display, among other defects, right atrial isomerism, atrial and ventricular septal defects, and double outlet right ventricle. The myocardial role of the gene has not been dissected. In particular, how Pitx2 regulates the differential L/R cardiac identity program is not clear. Additionally, the relation between Pitx2 ko ventricular defects and the gene expression pattern is not understood. In this article we analyze Pitx2 myocardial function during mouse heart development. By in situ hybridization analysis we show that myocardial Pitx2 expression delineates the remodeling of the left atrioventricular canal, the inner curvature, the ventral part of the interventricular ring, and the ventral portion of the right and left ventricle. By genetic analysis using an allelic series of Pitx2 mutants, among which a myocardial specific ko (ko(myo)) we show it has a crucial role in this process. Pitx2 ko(myo) mutants survive to adulthood, when they present strong cardiac morphological and functional defects. Confocal analysis of embryonic Pitx2 ko(myo) hearts reveals delayed cardiomyocyte development in the ventricular but not in the atrial Pitx2 null areas. Conversely, selective left atrial BMP10 mRNA downregulation which normally occurs at fetal stages is not found in the Pitx2 ko(myo) mice. This is the first evidence for distinct Pitx2 action in mediating L/R atrial identity and asymmetrical ventricular remodeling.

Cardiac chamber formation represents an essential evolutionary milestone that allows for the heart to receive (atrium) and pump (ventricle) blood throughout a closed circulatory system. Here, we reveal a novel transcriptional pathway between foxn4 and tbx genes that facilitates this evolutionary event. We show that the zebrafish gene slipjig, which encodes Foxn4, regulates the formation of the atrioventricular (AV) canal to divide the heart. sli/foxn4 is expressed in the AV canal, and its encoded product binds to a highly conserved tbx2 enhancer domain that contains Foxn4- and T-box-binding sites, both necessary to regulate tbx2b expression in the AV canal.

Terbutaline is used to treat fetal bradycardia in the setting of complete heart block (CHB); however, little is known of its effects on atrial and ventricular beat rates or patterns of heart rate (HR) acceleration. Fetal atrial and ventricular beat rates were compared before and after transplacental terbutaline treatment (10 to 30 mg/day) by fetal echocardiography in 17 fetuses with CHB caused by immune-mediated damage to a normal conduction system (isoimmune, n = 8) or a congenitally malformed conduction system associated with left atrial isomerism (LAI, n = 9). While receiving terbutaline, 9 of the 17 fetuses underwent fetal magnetocardiography (fMCG) to assess maternal HR and rhythm, patterns of fetal HR acceleration, and correlation between fetal atrial and ventricular accelerations (i.e., AV correlation). Maternal HR and fetal atrial and ventricular beat rates increased with terbutaline. However, terbutaline's effects were greater on the atrial pacemaker(s) in fetuses with isoimmune CHB and greater on the ventricular pacemaker(s) in those with LAI-associated CHB. Patterns of fetal HR acceleration also differed between isoimmune and LAI CHB. Finally, despite increasing HR, terbutaline did not restore the normal coordinated response between atrial and ventricular accelerations in isoimmune or LAI CHB. In conclusion, the pathophysiologic heterogeneity of CHB is reflected in the differing effect of terbutaline on the atrial and ventricular pacemaker(s) and varying patterns of HR acceleration. However, regardless of the cause of CHB, terbutaline augments HR but not AV correlation, suggesting that its effects are determined by the conduction system defect rather than the autonomic control of the developing heart.

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.

Myosin heavy-chain (MHC) isoforms are major structural components of the contractile apparatus of the heart muscle. Their spatio-temporal patterns of expression have been used as a tool to dissect cardiac development and differentiation. Although extensively investigated, controversy still exists concerning the expression patterns of atrial (AMHC), ventricular (VMHC), and cardiac myosin heavy-chain (CMHC) during development in the heart. In this study, we describe that probe length, probe concentration, and staining time in the non-radioactive in situ hybridization procedure seriously influence the observed pattern of MHC expression and the subsequent interpretation, explaining the divergent opinions in the field. Using a variety of external and internal controls for the in situ hybridization procedure, we demonstrate that both AMHC and VMHC are expressed throughout the entire heart tube during early development. During subsequent development, VMHC becomes restricted to the ventricles, whereas AMHC remains expressed in the atria, and, at substantially lower levels, is detected in the ventricles. These results are discussed in the context of methodological constraints of demonstrating patterns of gene expression. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

The embryonic vertebrate heart is composed of two major chambers, a ventricle and an atrium, each of which has a characteristic size, shape and functional capacity that contributes to efficient circulation. Chamber-specific gene expression programs are likely to regulate key aspects of chamber formation. Here, we demonstrate that epigenetic factors also have a significant influence on chamber morphogenesis. Specifically, we show that an atrium-specific contractility defect has a profound impact on ventricular development. We find that the zebrafish locus weak atrium encodes an atrium-specific myosin heavy chain that is required for atrial myofibrillar organization and contraction. Despite their atrial defects, weak atrium mutants can maintain circulation through ventricular contraction. However, the weak atrium mutant ventricle becomes unusually compact, exhibiting a thickened myocardial wall, a narrow lumen and changes in myocardial gene expression. As weak atrium/atrial myosin heavy chain is expressed only in the atrium, the ventricular phenotypes in weak atrium mutants represent a secondary response to atrial dysfunction. Thus, not only is cardiac form essential for cardiac function, but there also exists a reciprocal relationship in which function can influence form. These findings are relevant to our understanding of congenital defects in cardiac chamber morphogenesis.

During heart development, chamber myocardium forms locally from the embryonic myocardium of the tubular heart. The atrial natriuretic factor (ANF) gene is specifically expressed in this developing chamber myocardium and is one of the first hallmarks of chamber formation. We investigated the regulatory mechanism underlying this selective expression. Transgenic analysis shows that a small fragment of the ANF gene is responsible for the developmental pattern of endogenous ANF gene expression. Furthermore, this fragment is able to repress cardiac troponin I (cTnI) promoter activity selectively in the embryonic myocardium of the atrioventricular canal (AVC). In vivo inactivation of a T-box factor (TBE)- or NK2-homeobox factor binding element (NKE) within the ANF fragment removed the repression in the AVC without affecting its chamber activity. The T-box family member Tbx2, encoding a transcriptional repressor, is expressed in the embryonic myocardium in a pattern mutually exclusive to ANF, thus suggesting a role in the suppression of ANF. Tbx2 formed a complex with Nkx2.5 on the ANF TBE-NKE, and was able to repress ANF promoter activity. Our data provide a potential mechanism for chamber-restricted gene activity in which the cooperative action of Tbx2 and Nkx2.5 inhibits expression in the AVC.

In mouse, atrial natriuretic factor (ANF) gene expression was shown to be a marker for chamber formation within the embryonic heart. To gain insight into the process of chamber formation in the chicken embryonic heart, we analyzed the expression pattern of cANF during development. We found cANF to be specifically expressed in the myocardium of the morphologically distinguishable atrial and ventricular chambers, similar to ANF in mouse. cANF expression was never detected in the myocardium of the atrioventricular canal (AVC), inner curvature, and outflow tract (OFT), which is lined by endocardial cushions. Expression was strictly excluded from the interventricular myocardium and most proximal part of the bundle branches, as identified by the expression of Msx-2, whereas the rest of the bundle branches, trabeculae, and surrounding working myocardium did express cANF. The myocardium that forms de novo within the cushions after looping did not express cANF. At HH9 cANF expression was first observed in a subset of cardiomyocytes, which was localized ventrally in the fused heart tube and laterally in the unfused cardiac sheets. Together, these results show that cANF expression can be used to distinguish differentiated chamber (working) myocardium, including the peripheral ventricular conduction system, from embryonic myocardium. We conclude that differentiation of chamber myocardium takes place already at HH9 at the ventral side of the linear heart tube, possibly preceded by latero-medial signals in the unfused cardiac sheets., (Copyright 2002 Wiley-Liss, Inc.)

Objective: To evaluate cardiac dimensions and function in euploid fetuses with intracardiac echogenic foci., Study Design: Forty-eight fetuses with a single cardiac echogenic focus situated in the left ventricle had echocardiography performed at 22-24 weeks of gestation. Fifty normal fetuses at 22-24 weeks' gestation served as controls. Two-dimensional and M-mode directed fetal echocardiography were used to exclude cardiac anomalies and measure right and left ventricular free walls and interventricular septal thickness and ventricular systolic and diastolic dimensions. Cardiac size was expressed as a ratio of ventricular wall thickness/biparietal diameter, and cardiac function was expressed as ventricular shortening fraction. Doppler fetal echocardiography measurements included pulmonary and aortic maximum systolic velocities and time to peak velocities as indices of ventricular systolic function, and the ratio between early ventricular filling (E-wave) and active atrial filling (A-wave) peak velocities at the level of the atrioventricular valves as an index of ventricular diastolic function., Results: Early ventricular filling/active atrial filling peak velocity ratios were significantly lower in fetuses with intracardiac echogenic foci than in control fetuses. In the mitral valve the ratio was 0.37 +/- 0.14 (0.039) (mean +/- SD (95% confidence interval for difference between the means)) vs. 0.59 +/- 0.19 (0.052) and in the tricuspid valve it was 0.42 +/- 0.16 (0.045) vs. 0.62 +/- 0.21 (0.058). No significant differences were found in cardiac dimensions, ventricular shortening fraction and Doppler systolic indices., Conclusion: Euploid fetuses with intracardiac echogenic foci show low E/A ratio values in midtrimester echocardiography. This finding might indicate cardiac diastolic dysfunction.

In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and beta-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of "primary" myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium., (Copyright 2000 Academic Press.)

Urokinase-type plasminogen activator (uPA) activation of plasminogen is an important mediator of cell migration in many cell types. In the developing avian heart, uPA has been implicated as a mediator of atrioventricular (AV) cushion cell migration; however, the role of the plasminogen/plasmin system has not been examined. The purpose of this study was to test the hypothesis that uPA conversion of plasminogen to plasmin mediates AV cushion cell migration in vitro. Stage 17/18 chicken atrioventricular tissue lysates converted plasminogen into plasmin through uPA activity but no tissue-type plasminogen activator activity was detected. Zymograms on living cultured AV explants also activated plasminogen producing plasmin that degraded extracellular protein. The migratory capacity of cushion cells was assessed in the presence or absence of various test reagents known to alter the plasminogen/plasmin system. Addition of either human or chicken plasminogen or aprotinin (an inhibitor of plasmin) had no effect on cell migration. However, an anti-catalytic uPA antibody that blocked AV uPA activity, significantly decreased cell migration at all concentrations tested. These results showed that uPA mediated a portion of cushion cell migration in vitro. Although AV segments activated plasminogen and degraded extracellular proteins, uPA's functional role in cushion cell migration did not involve the plasminogen/plasmin system., (Copyright 1999 Wiley-Liss, Inc.)

The data on the chamber-specific expression of cardiac genes in the animal and human heart are reviewed. The positional specification of atrial and ventricular gene expression observed in adults is initiated early during embryogenesis. The existence of a cascade of genes is assumed, beginning with genes whose products form an anterior-posterior gradient along the heart-forming regions or along the linear heart tube derived from these regions, and ending with genes that encode the contractile proteins and display chamber-specific expression.

The vertebrate heart consists of two types of chambers, the atria and the ventricles, which differ in their contractile and electrophysiological properties. Little is known of the molecular mechanisms by which these chambers are specified during embryogenesis. Here a chicken iroquois-related homeobox gene, Irx4, was identified that has a ventricle-restricted expression pattern at all stages of heart development. Irx4 protein was shown to regulate the chamber-specific expression of myosin isoforms by activating the expression of the ventricle myosin heavy chain-1 (VMHC1) and suppressing the expression of the atrial myosin heavy chain-1 (AMHC1) in the ventricles. Thus, Irx4 may play a critical role in establishing chamber-specific gene expression in the developing heart.

A classical genetic approach, in which mutagenized organisms are screened for phenotypes of interest, is appealing for the analysis of developmental processes. Here, we describe the advantages of zebrafish genetics for the study of heart development. As an example of the utility of this strategy, we discuss its potential to illuminate the molecular mechanisms of cardiac chamber formation. The signals that specify ventricular and atrial lineages and the differentiation pathways that produce distinct chambers are poorly understood. Recently identified zebrafish mutations that disrupt ventricular or atrial development promise to reveal genes essential for these processes.

We sought to determine the sensitivity of detecting congenital heart defects with routine antenatal ultrasound including the four-chamber view in a population at low risk for cardiac anomalies. Neonatal outcome and anomaly databases were reviewed to identify cases of morphologic cardiac defects from 1988-1992. Of 176 cases identified, 62 (35%) had routine antenatal ultrasound including the four-chamber view. Thirteen of these 62 (21%) were diagnosed by ultrasound and 18 of the 116 (15%) total defects present were seen. Of these, only 16 of 25 (64%) defects reasonably expected to be seen by the four-chamber cardiac view alone were detected. The routine ultrasound four-chamber cardiac evaluation is limited and detection depends on the type of cardiac defect present.

We have earlier reported partial cloning of a cDNA of a chick atrial myosin heavy chain (MHC) gene, CCSV2 and its expression pattern in embryonic chick hearts (Oana et al (1995) Eur J Cell Biol 67, 42-49). In this study, five overlapping cDNA clones (including CCSV2) which together encode the entire open reading frame of the chick atrial MHC gene were characterized, and both the entire nucleotide sequence consisting of 5825 bases and the deduced amino acid sequence consisting of 1931 amino acids determined. Reinvestigation of the nucleotide sequence of the previously reported and presumably different chick atrial specific MHC cDNA clone, AMHC1 (Yutzey et al (1994) Development 120, 871-883), revealed that our clone and AMHC1 encoded the same MHC. The chick atrial MHC gene was strongly expressed in developing chick atria from a very early stage (Hamburger and Hamilton stage 9, 29-33 h) to the adult stage. This gene was also expressed, although weakly, in the ventricle, somite (the precursor to skeletal muscle) and skeletal muscle during embryonic stages but not in adults.

Vertebrate cardiogenesis is a complex process involving multiple, distinct tissue types which interact to form a four-chambered heart. Molecules have been identified whose expression patterns co-segregate with the maturation of the atrial and ventricular muscle cell lineages. It is not currently known what role intrinsic events versus external influences play in cardiac chamber morphogenesis. We developed novel, fluorescent-based, myocardial, cellular transplantation systems in order to study these questions in murine embryos and report the irreversible nature of chamber specification with respect to the downregulation of atrial myosin light chain 2 (MLC-2a) and alpha myosin heavy chain (alpha-MHC). Grafting ventricular cells into the atrial chamber does not result in upregulation of MLC-2a expression in ventricular cells. Additionally, wild-type ventricular muscle cells grafted into the wild-type background appropriately downregulate MLC-2a and alpha-MHC. Finally, grafting of RXRalpha gene-deficient ventricular muscle cells into the ventricular chambers of wild-type embryos does not rescue the persistent expression of MLC-2a, providing further evidence that ventricular chamber maturation is an early event. These studies provide a new approach for the mechanistic dissection of critical signaling events during cardiac chamber growth, maturation and morphogenesis in the mouse, and should find utility with other approaches of cellular transplantation in murine embryos. These experiments document the irreversible nature of the downregulation of atrial markers after the onset of cardiogenesis during ventricular chamber morphogenesis and temporally define the response of cardiac muscle cells to signals regulating chamber specification.

We have isolated murine embryonic atrial and ventricular cells derived from timed-pregnant females at different periods and used patch-clamp procedures to investigate age- and chamber-specific expression of ionic channels in the developing fetal mouse. Our data indicate that L-type Ca2+ channels play a dominant role in excitation during early murine cardiac embryogenesis and that Na+ channel expression increases dramatically just before birth. K+ channel expression is particularly sensitive to changes during development. Neither atrial nor ventricular cells express a slowly activating component of delayed rectification (IKs) until just before birth, and inwardly rectifying channel activity, associated with determination of cellular resting potential, is not markedly apparent until late stages of embryogenesis. Instead, we find robust expression of the ATP-regulated K+ channel at early and late states of embryonic development, which may indicate a novel functional role for this channel during morphogenesis of the heart. These results have important implications for the physiology and development of the murine cardiac conduction system and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in the myocardium of both wild-type and genetically modified mice.

In an effort to isolate novel genes that may be involved in the development of the cardiac cushions and then the formation of cardiac valves and septa, we utilized the differential mRNA display method in conjunction with the whole-mount in situ hybridization. The total RNAs used to differentially display were prepared from atrioventricular (AV) canal regions of stage 15 and stage 21 chicken hearts because critical events known to be important for the AV valve and septum formation occur during this period of the development. We have successfully obtained 14 potential candidate genes. Three examples, 15H16 (phospholamban), E13 (skeletal alpha-tropomyosin) and 21C (a novel gene), are discussed here. Levels of mRNA expression in developing hearts were determined by Northern blot analysis and their expression patterns were revealed and compared using whole-mount in situ hybridization. Both phospholamban and skeletal alpha-tropomyosin messages in the myocardium of the AV canal region showed significant decrease during this period of the development. The 21C differential display product detects a novel 9.5 Kb message whose expression is cardiac-specific at early stages of development. The expression level of the 21C gene appeared to be increased from stage 15 to stages 21 and 25 as determined by both Northern blot analysis and in situ hybridization. From these data, we demonstrate that the differential display method together with the whole-mount in situ hybridization could be an effective means for the isolation of novel and differentially expressed genes.

Atrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses.

The hearts of human embryos, ranging from 3.6 to 25 mM crown-rump length, have been studied in view of the problem of the possible contribution of the atrioventricular endocardial cushions to septation. Serial sections and graphic reconstructions were used. It is concluded that the cushions do not materially contribute to the mature muscular septum.

Morphology of the cardiac chambers and great vessels of fetal and neonatal rats was studied using the whole body freezing technique and by sectioning through the short axis of the heart with a freezing microtome. Compared to the fetal heart, the neonatal heart showed rapid change 2 to 8 days after birth. The ventricular sinus septum was straight in the fetus and became concave to the left ventricle after birth. The right ventricular wall was as thick as the left in the fetus and became thinner rapidly after birth. At the same time, the right ventricular cavity dilated. The right and left pulmonary arteries and pulmonary veins were small in the fetus and enlarged soon after birth. At the same time, the foramen ovale was closed and the diameters of the inferior vena cava and descending aorta were diminished. One-half-mm thick sections were cut serially and then photographed. Ventricular volumes and masses were calculated from summation of the areas of each chambers. Left ventricular mass per body weight increased rapidly after birth, whereas right ventricular mass per body weight remained constant from 0 to 8 days after birth.