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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.

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

Perturbations in cardiac development result in congenital heart disease, the leading cause of birth defect-related infant morbidity and mortality. Advances in cardiac developmental biology have significantly augmented our understanding of signalling pathways and transcriptional networks underlying heart formation. Cardiogenesis is initiated with the formation of mesodermal multipotent cardiac progenitor cells and is governed by cross-talk between developmental cues emanating from endodermal, mesodermal and ectodermal cells. The molecular and transcriptional machineries that direct the specification and differentiation of these cardiac precursors are part of an evolutionarily conserved programme that includes the Nkx-, Gata-, Hand-, T-box- and Mef2 family of transcription factors. Unravelling the hierarchical networks governing the fate and differentiation of cardiac precursors is crucial for our understanding of congenital heart disease and future stem cell-based and gene therapies. Recent molecular and genetic lineage analyses have revealed that subpopulations of cardiac progenitor cells follow distinctive specification and differentiation paths, which determine their final contribution to the heart. In the last decade, progenitor cells that contribute to the arterial pole and right ventricle have received much attention, as abnormal development of these cells frequently results in congenital defects of the aortic and pulmonary outlets, representing the most commonly occurring congenital cardiac defects. In this review, we provide an overview of the building plan of the vertebrate four-chambered heart, with a special focus on cardiac progenitor cell specification, differentiation and deployment during arterial pole development.

Advances made in understanding temporal changes in structure of the developing heart, along with advances in knowledge of the lineage of cardiomyocytes forming the components of cardiac chambers, permit us to draw inferences concerning substrates for arrhythmias such as atrial fibrillation and outflow tract tachycardias. We frame these insights in our description of the formation of cardiac chambers. Adult-like electrocardiograms can be generated by developing hearts before it is possible to recognize an anatomically discrete conduction system. Working components of the atrial and ventricular chambers, which are rapidly conducting, balloon from walls of the primary heart tube, themselves slowly conducting. Recognition of the locations of these populations of primary and secondary myocardial pools suggests that some potential myocardial substrates (those producing outflow tract tachycardias) initially had a primary phenotype. In contrast, cardiomyocytes forming pulmonary venous sleeves, substrates for many cases of atrial fibrillation, have never possessed a primary phenotype. This article is part of a JCTR special issue on Cardiac Anatomy.

Knowledge of complex morphogenetic processes that occur during embryonic development is essential for understanding anatomy and to get insight in the pathogenesis of congenital malformations. Understanding these processes can be facilitated by using a three-dimensional (3D) developmental series of human embryos, which we aim to create in this project. Digital images of serial sections of 34 human embryos of the Carnegie Collection between Carnegie stages 7 (15-17 days) and 23 (56-60 days) are used to create 3D reconstructions of different organ systems. The software package Amira is used to align the sections and to create the 3D reconstructions. In this midway evaluation we show the first results of the atlas, containing 34 embryos with more than 13.500 manually annotated sections. The 3D models can be interactively viewed within a 3D-pdf. This will be the first complete digital 3D human embryology atlas of this size, containing all developing organ systems. (C) 2012 Elsevier Inc. All rights reserved

On the basis of both developmental and morphological evidence, we would suggest that a ventricle is best defined as any chamber within the ventricular mass possessing an apical trabecular component. Such ventricles can be of right or left morphology, and always coexist. The ventricles are normally formed when possessing all three of the inlet, apical trabecular, and outlet components, but incomplete when lacking one or both of the inlet and outlet components. Ventricles that are incomplete because of lack of the inlet component are always hypoplastic, with incomplete right ventricles being positioned antero-superiorly within the ventricular mass, and incomplete left ventricles located postero-inferiorly. Patients having such incomplete ventricles because of the lack of the inlet component have functionally univentricular hearts, although the functionally univentricular arrangement can also be produced in the setting of normally constituted but hypertrophied ventricles. Full analysis of ventricular morphology, therefore, requires attention not only to component make-up, but also size.

Congenital heart defects (CHDs) occur mostly sporadic, but familial CHD cases have been reported. Mutations in several genes, including NKX2.5, GATA4 and NOTCH1, were identified in families and patients with CHD, but the mechanisms underlying CHD are largely unknown. We performed genome-wide linkage analysis in a large four-generation family with autosomal dominant CHD (including atrial septal defect type I and II, tetralogy of Fallot and persistent left superior vena cava) and low atrial rhythm, a unique phenotype that has not been described before. We obtained phenotypic information including electrocardiography, echocardiography and DNA of 23 family members. Genome-wide linkage analysis on 12 affected, 5 unaffected individuals and 1 obligate carrier demonstrated significant linkage only to chromosome 9q21-33 with a multipoint maximum LOD score of 4.1 at marker D9S1690, between markers D9S167 and D9S1682. This 48-cM critical interval corresponds to 39 Mb and contains 402 genes. Sequence analysis of nine candidate genes in this region (INVS, TMOD1, TGFBR1, KLF4, IPPK, BARX1, PTCH1, MEGF9 and S1PR3) revealed no mutations, nor were genomic imbalances detected using array comparative genomic hybridization. In conclusion, we describe a large family with CHD and low atrial rhythm with a significant LOD score to chromosome 9q. The phenotype is representative of a mild form of left atrial isomerism or a developmental defect of the sinus node and surrounding tissue. Because the mechanisms underlying CHD are largely unknown, this study represents an important step towards the discovery of genes implied in cardiogenesis.

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.

Interpretation of the results of anatomical and embryological studies relies heavily on proper visualization of complex morphogenetic processes and patterns of gene expression in a three-dimensional (3D) context. However, reconstruction of complete 3D datasets is time consuming and often researchers study only a few sections. To help in understanding the resulting 2D data we developed a program (TRACTS) that places such arbitrary histological sections into a high-resolution 3D model of the developing heart. The program places sections correctly, robustly and as precisely as the best of the fits achieved by five morphology experts. Dissemination of 3D data is severely hampered by the 2D medium of print publication. Many insights gained from studying the 3D object are very hard to convey using 2D images and are consequently lost or cannot be verified independently. It is possible to embed 3D objects into a pdf document, which is a format widely used for the distribution of scientific papers. Using the freeware program Adobe Reader to interact with these 3D objects is reasonably straightforward; creating such objects is not. We have developed a protocol that describes, step by step, how 3D objects can be embedded into a pdf document. Both the use of TRACTS and the inclusion of 3D objects in pdf documents can help in the interpretation of 2D and 3D data, and will thus optimize communication on morphological issues in developmental biology.

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

Aims Holt–Oram syndrome (HOS) is a heart/hand syndrome clinically characterized by upper limb and cardiac malformations. Mutations in T-box transcription factor 5 ( TBX5 ) underlie this syndrome, the majority of which lead to premature stops. In this study, we present our functional analyses of five (novel) missense TBX5 mutations identified in HOS patients, most of whom presented with severe cardiac malformations. Methods and results Functional characterization of mutant proteins shows a dramatic loss of DNA-binding capacity, as well as diminished binding to known cardiac interaction partners NKX2-5 and GATA4. The disturbance of these interactions leads to a loss of function, as measured by the reduced activation of Nppa and FGF10 in rat heart derived cells, although with variable severity. Two out of the five mutations are peculiar: one, p.H220del, is associated with additional extra-cardiac defects, perhaps by interfering with other T-box dependant pathways, and another, p.I106V, leads to limb defects only, which is supported by its normal interaction with cardiac-specific interaction partners. Conclusion Overall, our data are consistent with the hypothesis that these novel missense mutations in TBX5 lead to functional haploinsufficiency and result in a reduced transcriptional activation of target genes, which is likely central to the pathogenesis of HOS.

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.

Background Three quarters of patients with 22q11.2 Deletion Syndrome (22q11.2DS) have congenital heart disease (CHD), typically conotruncal heart defects. Although it is currently common practice to test all children with typical CHD for 22q11.2DS, many adult patients have not been tested in the past and therefore 22q11.2DS might be under-recognised in adults. Objectives To determine the prevalence of 22q11.2DS in adults with tetralogy of Fallot (TOF) and pulmonary atresia (PA)/ventricular septal defect (VSD) and to assess the level of recognition of the syndrome in adult patients. Methods Patients were identified from CONCOR, a nationwide registry for adult patients with CHD. Inclusion criteria were diagnosis of TOF or PA/VSD and the availability of DNA. Patients with syndromes other than 22q11.2DS were excluded. Multiplex ligation-dependent probe amplification was used to detect 22q11.2 microdeletions. Results 479 patients with TOF and 79 patients with PA/VSD (56% male, median age 34.7 years) were included and analysed. Twenty patients were already known to have 22q11.2DS. A 22q11.2 microdeletion was detected in a further 24 patients. Thirty-one patients with TOF (6.5%) had 22q11.2DS, whereas 13 patients with PA/VSD had 22q11.2DS (16.5%). Of all 22q11.2 microdeletions, 54% (24/44) were unknown before this study. Conclusion This study shows that although the prevalence of 22q11.2DS in adults with TOF and PA/VSD is substantial, it is unrecognised in more than half of patients. As the syndrome has important clinical and reproductive implications, a diagnostic test should be considered in all adult patients with TOF and PA/VSD.

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.

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.

Congenital cardiac malformations account for one-quarter of all human congenital abnormalities. They are caused by environmental and genetic factors. Despite increasing efforts in fundamental research, as yet, the morphogenesis of only a limited number of malformations has been elucidated. Over the last decades, new genetic modifications have made it possible to manipulate the mammalian embryo. Evidence provided using these transgenic techniques has, over the past decade, necessitated re-evaluation of several developmental processes, important in the understanding of normal as opposed to abnormal cardiac development. In this review, we discuss current understanding of the patterning of the initial heart tube, new insights into formation of the atrial and ventricular chambers, and novel information on the origin of the cells that are added to the heart after formation of the initial tube. All of these advances modify our appreciation of malformations involving the venous and arterial poles. As we demonstrate, this new information sheds light not only on normal cardiac development, but also explains the structure of several previously controversial lesions seen in malformed human hearts. Clin. Anat. 22:4–20, 2009. © 2008 Wiley-Liss, Inc.

Mutations in the cardiac desmosomal protein desmoglein-2 (DSG2) are associated with arrhythmogenic right ventricular cardiomyopathy (ARVC). We studied the explanted heart of a proband carrying the DSG2-N266S mutation as well as transgenic mice (Tg-NS) with cardiac overexpression of the mouse equivalent of this mutation, N271S-dsg2, with the aim of investigating the pathophysiological mechanisms involved. Transgenic mice recapitulated the clinical features of ARVC, including sudden death at young age, spontaneous ventricular arrhythmias, cardiac dysfunction, and biventricular dilatation and aneurysms. Investigation of transgenic lines with different levels of transgene expression attested to a dose-dependent dominant-negative effect of the mutation. We demonstrate for the first time that myocyte necrosis is the key initiator of myocardial injury, triggering progressive myocardial damage, including an inflammatory response and massive calcification within the myocardium, followed by injury repair with fibrous tissue replacement, and myocardial atrophy. These observations were supported by findings in the explanted heart from the patient. Insight into mechanisms initiating myocardial damage in ARVC is a prerequisite to the future development of new therapies aimed at delaying onset or progression of the disease.

Recent research, enabled by powerful molecular techniques, has revolutionized our concepts of cardiac development. It was firmly established that the early heart tube gives rise to the left ventricle only, and that the remainder of the myocardium is recruited from surrounding mesoderm during subsequent development. Also, the cardiac chambers were shown not to be derived from the entire looping heart tube, but only from the myocardium at its outer curvatures. Intriguingly, many years ago, classic experimental embryological studies reached very similar conclusions. However, with the current scientific emphasis on molecular mechanisms, old morphological insights became underexposed. Since cardiac development occurs in an architecturally complex and dynamic fashion, molecular insights can only fully be exploited when placed in a proper morphological context. In this communication we present excerpts of important embryological studies of the pioneers of experimental cardiac embryology of the previous century, to relate insights from the past to current observations.

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.

Digital reconstruction of human developmentThe detailed morphology of human development has intrigued scientists and the medical field alike. However, the scarcity of specimens hampers detailed mapping of tissue architecture. Furthermore, inaccuracies in the description of human development have crept into textbooks from observations of animal models that are extrapolated to humans. By mapping normal developmental processes and patterns, such as the growth and relative placement of organs, congenital anomalies can be better understood. de Bakkeret al.generated interactive three-dimensional digital reconstructions based on the Carnegie collection of histologically sectioned human embryos spanning the first 2 months of gestation. These interactive models will serve as educational and scientific resources for normal and abnormal human development.Science, this issue p.10.1126/science.aag0053

Background The Brugada syndrome is an inherited cardiac electrical disorder associated with a high incidence of life-threatening arrhythmias. Screening for mutations in the cardiac Na + channel–encoding gene SCN5A uncovers a mutation in approximately 20% of Brugada syndrome cases. Genetic heterogeneity and/or undetected SCN5A mutations, such as exon duplications and deletions, could be involved in the remaining 80% mutation-negative patients. Objectives Thirty-eight SCN5A mutation-negative Dutch Brugada syndrome probands were studied. The SCN5A gene was investigated for exon duplication and deletion, and a number of candidate genes (Caveolin-3, Irx-3 , Irx-4 , Irx-5 , Irx-6 , Plakoglobin, Plakophilin-2, SCN1B , SCN2B , SCN3B , and SCN4B ) were tested for the occurrence of point mutations and small insertions/deletions. Methods We used a quantitative multiplex approach to determine SCN5A exon copy numbers. Mutation analysis of the candidate genes was performed by direct sequencing of polymerase chain reaction–amplified coding regions. Results No large genomic rearrangements in SCN5A were identified. No mutations were found in the candidate genes. Twenty novel polymorphisms were identified in these genes. Conclusion Large genomic rearrangements in SCN5A are not a common cause of Brugada syndrome. Similarly, the studied candidate genes are unlikely to be major causal genes of Brugada syndrome. Further studies are required to identify other genes responsible for this syndrome.

Bone morphogenetic proteins (BMP) are involved in the regulation of a plethora of processes underlying cardiovascular development. This review summarizes the effects of BMP and the signaling pathways that regulate the differentiation of cardiomyocytes from mesoderm in the heart-forming region and at the distal borders of the heart tube from the second heart field. Subsequently, the role of BMPs in the formation of the ventricular chambers and septovalvulogenesis in the atrioventricular canal and outflow tract is described. Finally, the effects of BMPs in stem cell biology and cardiac regeneration are discussed.

Ventricular hypertrabeculation (noncompaction) is a poorly characterized condition associated with heart failure. The condition is widely assumed to be the retention of the trabeculated ventricular design of the embryo and ectothermic (cold-blooded) vertebrates. This assumption appears simplistic and counterfactual. Here, we measured a set of anatomical parameters in hypertrabeculation in man and in the ventricles of embryos and animals. We compared humans with left ventricular hypertrabeculation (N=21) with humans with structurally normal left ventricles (N=54). We measured ejection fraction and ventricular trabeculation using cardiovascular MRI. Ventricular trabeculation was further measured in series of embryonic human and 9 animal species, and in hearts of 15 adult animal species using MRI, CT, or histology. In human, hypertrabeculated left ventricles were significantly different from structurally normal left ventricles by all structural measures and ejection fraction. They were far less trabeculated than human embryonic hearts (15-40% trabeculated volume versus 55-80%). Early in development all vertebrate embryos acquired a ventricle with approximately 80% trabeculations, but only ectotherms retained the 80% trabeculation throughout development. Endothermic (warm-blooded) animals including human slowly matured in fetal and postnatal stages towards ventricles with little trabeculations, generally less than 30%. Further, the trabeculations of all embryos and adult ectotherms were very thin, less than 50μm wide, whereas the trabeculations in adult endotherms and in the setting of hypertrabeculation were wider by orders of magnitude. It is concluded in contrast to a prevailing assumption, the hypertrabeculated left ventricle is not like the ventricle of the embryo or of adult ectotherms. This article is part of a Special Issue entitled:Cardiomyocyte Biology: Integration of Develomental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

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.

The atrioventricular valves are formed from a complex arrangement of an annulus and leaflets, supported by a subvalvar apparatus that is composed of tendinous cords and papillary muscles. Although much has been said and written about their development, the exact nature of the process has yet to be fully elucidated. We believe that this is vital, since unraveling this complex process holds the key to the understanding of many of the congenital malformations that may afflict the valves.

In the past, our interpretations of cardiac development depended on analysis of serially sectioned embryos, supported by three-dimensional reconstructions. It was not possible, using these techniques, to trace the fate of the various embryonic building blocks. This has all changed with the advent of the new techniques in molecular biology. Combining our experience with these new techniques and our previous studies using the classic approach, we have reviewed how the recent advances clarify controversies that still exist concerning the development of the venous pole. The arguments devolve on whether the pulmonary vein is itself a new development or whether its primordium is derived from the systemic venous tributaries, the so-called sinus venosus. The new techniques show that, rather than developing in the form of a segmented tube, the heart is built up by addition of material to both its arterial and venous poles. At no stage is it possible to recognize a discrete part of the tube that can be identified as the sinus venosus. The confluence of the systemic venous tributaries does not become recognizable as a discrete anatomic entity until compartmented into the newly formed right atrium concomitant with formation of the venous valves. The new molecular techniques show that the pulmonary vein is a new structure, anatomically and developmentally, that is derived from mediastinal myocardium. It gains its connection to the morphologically left atrium between the right- and left-sided systemic venous tributaries.

The Museum Vrolik collection of anatomical specimens in Amsterdam, The Netherlands, comprises over 5,000 specimens of human and animal anatomy, embryology, pathology, and congenital anomalies. Recently, we rediagnosed a subset of the collection comprising dried human trunk skeletons and cranial base preparations presenting with homeotic transformations (vertebral phenotypic shifts) and numerical vertebral anomalies. We identified 11 trunk skeletons with either anterior or posterior homeotic transformations (AHT or PHT), 5 trunk skeletons with either less or more than the normal number of vertebrae, and well over a hundred cranial base preparations with either AHT (atlas-assimilation) or PHT (occipital vertebra). We found that, although homeotic transformations and numerical anomalies are distinct conditions, both can be described in terms of mismatch between homeotic patterning and morphological segmentation of the paraxial mesoderm. Therefore these two processes are perhaps not as tightly linked as they may seem on the basis of recent molecular studies. In homeotic transformations there is a constant mismatch between homeotic patterning and morphological segmentation throughout the affected region of the vertebral column. In numerical anomalies there is a variable mismatch between homeotic patterning and morphological segmentation, either because of stretching or squeezing of the homeotic pattern or because of oligo- or polysegmentation of the presomitic mesoderm (PSM). Homeotic transformations of the axial skeleton have an incidence of about 1%-5%, apart from their occurrence in malformation syndromes. Of the various etiological possibilities, explaining their frequent but mostly sporadic occurrence, maternal hyperthermia seems an attractive candidate.

Two distinct chitinases have been identified in mammals: a phagocyte-specific enzyme named chitotriosidase and an acidic mammalian chitinase (AMCase) expressed in the lungs and gastrointestinal tract. Increased expression of both chitinases has been observed in different pathological conditions: chitotriosidase in lysosomal lipid storage disorders like Gaucher disease and AMCase in asthmatic lung disease. Recently, it was reported that AMCase activity is involved in the pathogenesis of asthma in an induced mouse model. Inhibition of chitinase activity was found to alleviate the inflammation-driven pathology. We studied the tissue-specific expression of both chitinases in mice and compared it to the situation in man. In both species AMCase is expressed in alveolar macrophages and in the gastrointestinal tract. In mice, chitotriosidase is expressed only in the gastrointestinal tract, the tongue, fore-stomach, and Paneth cells in the small intestine, whereas in man the enzyme is expressed exclusively by professional phagocytes. This species difference seems to be mediated by distinct promoter usage. In conclusion, the pattern of expression of chitinases in the lung differs between mouse and man. The implications for the development of anti-asthma drugs with chitinases as targets are discussed.

Molecular imaging, which is the three-dimensional (3D) visualization of gene expression patterns, is indispensable for the study of the function of genes in cardiac development. The instrumentation, as well as the development of specific contrast agents for molecular imaging, has shown spectacular advances in the last decade. In this review, the spatial resolutions, contrast agents, and applications of these imaging methods in the field of cardiac embryology are discussed. Apart from 3D reconstructions from histological sections, not many of these methods have been applied in embryological research. This review shows that, for most methods, neither the spatial resolutions nor the specificity and applicability of the contrast agents are adequate for the reliable imaging of specific gene expression at the microscopic resolution required for embryological studies of small organs like the developing heart. Although a 3D reconstruction from sections will always suffer from imperfections, the resulting reconstructions meet the aim of most biological studies, especially since the original microscopic images are linked. With respect to imaging of gene expression, only histological sections and laser scanning microscopy provide the required resolution and specificity at the tissue and cellular level. Episcopic fluorescence image capturing and optical projection tomography are being used for microscopic phenotyping and lineage analysis, and both show potential for detailed molecular imaging. Other methods can be used very efficiently in rapid evaluation of biological experiments and high-throughput screens of large-scale gene expression profiling efforts when high spatial resolution is not required.

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.

Postnatally, heart muscle cells almost completely lose their ability to divide, which makes their loss after trauma irreversible. Potential repair by cell grafting or mobilizing endogenous cells is of particular interest for possible treatments for heart disease, where the poor capacity for cardiomyocyte proliferation probably contributes to the irreversibility of heart failure. Knowledge of the molecular mechanisms that underly formation of heart muscle cells might provide opportunities to repair the diseased heart by induction of (trans) differentiation of endogenous or exogenous cells into heart muscle cells. We briefly review the molecular mechanisms involved in early development of the linear heart tube by differentiation of mesodermal cells into heart muscle cells. Because the initial heart tube does not comprise all the cardiac compartments present in the adult heart, heart muscle cells are added to the distal borders of the tube and within the tube. At both distal borders, mesodermal cell are recruited into the cardiac lineage and, within the heart tube, muscular septa are formed. In this review, the relative late additions of heart muscle cells to the linear heart tube are described and the potential underlying molecular mechanisms are discussed.

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.

Moorman, Antoon F. M., and Vincent M. Christoffels. Cardiac Chamber Formation: Development, Genes, and Evolution. Physiol Rev 83: 1223-1267, 2003; 10.1152/physrev.00006.2003.—Concepts of cardiac development have greatly influenced the description of the formation of the four-chambered vertebrate heart. Traditionally, the embryonic tubular heart is considered to be a composite of serially arranged segments representing adult cardiac compartments. Conversion of such a serial arrangement into the parallel arrangement of the mammalian heart is difficult to understand. Logical integration of the development of the cardiac conduction system into the serial concept has remained puzzling as well. Therefore, the current description needed reconsideration, and we decided to evaluate the essentialities of cardiac design, its evolutionary and embryonic development, and the molecular pathways recruited to make the four-chambered mammalian heart. The three principal notions taken into consideration are as follows. 1) Both the ancestor chordate heart and the embryonic tubular heart of higher vertebrates consist of poorly developed and poorly coupled “pacemaker-like” cardiac muscle cells with the highest pacemaker activity at the venous pole, causing unidirectional peristaltic contraction waves. 2) From this heart tube, ventricular chambers differentiate ventrally and atrial chambers dorsally. The developing chambers display high proliferative activity and consist of structurally well-developed and well-coupled muscle cells with low pacemaker activity, which permits fast conduction of the impulse and efficacious contraction. The forming chambers remain flanked by slowly proliferating pacemaker-like myocardium that is temporally prevented from differentiating into chamber myocardium. 3) The trabecular myocardium proliferates slowly, consists of structurally poorly developed, but well-coupled, cells and contributes to the ventricular conduction system. The atrial and ventricular chambers of the formed heart are activated and interconnected by derivatives of embryonic myocardium. The topographical arrangement of the distinct cardiac muscle cells in the forming heart explains the embryonic electrocardiogram (ECG), does not require the invention of nodes, and allows a logical transition from a peristaltic tubular heart to a synchronously contracting four-chambered heart. This view on the development of cardiac design unfolds fascinating possibilities for future research.

In the first part of our review of cardiac development,1 we discussed the initial changes involved in transformation of the heart forming regions of the embryo into the great veins, the atrial and ventricular chambers, and the arterial trunks. We showed that this first part of cardiac development could be divided into phases of formation of the primary myocardial tube, looping of the tube, during which additional parts are added that give the future compartments their definitive topography, and the assembly of these components into the cardiac chambers and arterial trunks. In this second review, we discuss the processes that complete the separation of the two sides of the definitive heart, for the most part involving septation of the parts of the primary tube not themselves directly involved in formation of the chamber-specific compartments of the atriums and ventricles. In this respect, when concluding our first review, we explained how the arterial trunks were also formed by septation within the solitary outflow tract of the primary heart tube. We also showed, however, that subsequent to formation of the two arterial trunks, there was disappearance of the cushions that initially divided them. Thus, in the definitive heart, the proximal parts of the aorta and pulmonary trunk, along with the sinuses of the arterial roots and the subpulmonary infundibulum, possess their own discrete walls, separated by extra-cardiac space. Although septation by fusion of endocardial cushions will be a topic included in this second review, septation and separation of the outflow tracts is sufficiently complicated to require special treatment. Because of this, we will devote a third review specifically to the mechanisms underscoring the remodelling of the outflow tracts. In this review, therefore, we will confine our considerations to the formation of the atrial, atrioventricular, and ventricular septal structures. As will become …

A variety of methods for high throughput analysis of differential gene expression has been developed over the past years. We have implemented the EASEL technique that adds flexibility, efficiency and wide-applicability to these methods. The EASEL procedure is unique as it integrates several well established techniques and thereby offers a combination of subtractive hybridization of 3' cDNA ends with macroarrays analysis and Serial Analysis of Gene Expression (SAGE). In addition, once a set of interesting, differentially expressed genes is identified, the material required for follow up studies to test the hypothesis that the gene is truly involved in the process of interest is readily available. In this report, we first present a step-by-step validation of the procedure, since several of the incorporated steps had to be tailored to meet specific requirements and implied drastic modifications of the original methods. Secondly, we applied EASEL to the identification of up-regulated gene products in the outflow tract region of the embryonic rat heart. Here we provide evidence that at least two among the differentially expressed genes detected, follistatin-like protein gene and membrane type 1-metallo proteinase gene, are selectively up-regulated in the outflow tract, suggesting their involvement in the development of this region during embryogenesis.

Through the 20th century, knowledge of the events occurring during cardiac development was clouded by conflicting descriptions, coupled with use of notably different terminologies. Furthermore, not all accounts were based on direct study of embryonic material, instead being constructed on the basis of interpretations of previous reports, supported by inferences made from the structure of the congenitally malformed heart. Such processes, in themselves, are understandable, since it is axiomatic that proper appreciation of the events occurring during formation of the heart will aid in the analysis of the morphogenesis of cardiac malformations, this being a desirable prerequisite in the search for optimal treatment. Over the past decade, this has all changed. There has been an explosion of work, both anatomical and molecular, devoted to cardiac development. Advances in technology, coupled with the use of suitable animal models, now enable us to provide a more accurate account of the steps involved in formation and septation of the cardiac chambers. Not all of this new information is concordant with the “classical” accounts. In these reviews, therefore, we will describe, first, the steps involved in formation of the primary heart tube, and its conversion to the four cardiac chambers and the paired arterial trunks. We will then look in greater detail at the events occurring during the separation of the initial solitary heart tube into discrete systemic and pulmonary circulations. The mesodermal tissues that give rise to the heart first become evident when the embryo is undergoing the process known as gastrulation. In the human, this occurs during the third week of development, while for the mouse, at a comparable stage of development, around seven days will have elapsed from fertilisation, and the embryo will be in the presomitic stage. The embryonic plate in humans, initially possessing two layers, is ovoid, and is …

International audience; In this review, we focus on two important steps in the formation of the embryonic heart: (i) the progressive addition of late differentiating progenitor cells from the second heart field that drives heart tube extension during looping morphogenesis, and (ii) the emergence of patterned proliferation within the embryonic myocardium that generates distinct cardiac chambers. During the transition between these steps, the major site of proliferation switches from progenitor cells outside the early heart to proliferation within the embryonic myocardium. The second heart field and ballooning morphogenesis concepts have major repercussions on our understanding of human heart development and disease. In particular, they provide a framework to dissect the origin of congenital heart defects and the regulation of myocardial proliferation and differentiation of relevance for cardiac repair.

—T-cell activation in atherosclerotic plaques is thought to be initiated by plaque-derived antigens, such as oxidized LDL (oxLDL). An alternative pathway of T-cell activation independent of antigen stimulation, mediated by the cytokine interleukin (IL)-15, was recently described. We investigated IL-15 expression in atherosclerotic plaques in relation to plaque morphology, inflammatory cells, T-cell activation, and oxidation-specific epitopes by use of immunohistochemistry. In situ hybridization was used to evaluate IL-15 mRNA expression. We also studied the proliferative response of plaque-derived T-cell lines to IL-15 in vitro using [3H]thymidine incorporation. Fresh-frozen specimens were classified as fibrous (n=9), fibrolipid (n=8), and lipid-rich (n=14) plaques; normal vessels (n=4) served as reference. Expression of IL-15 mRNA and protein was found almost solely in fibrolipid and lipid-rich plaques, associated with oxLDL-positive macrophages. Sequential immunostains revealed colocalization between IL-15– and CD40L-positive T cells. Moreover, plaque-derived T-cell lines were highly responsive to IL-15. Hence, IL-15 could provide a pathway for antigen-independent T-cell activation.

To follow a candidate gene approach for the involvement of the KCND2 and KCND3 genes (Kv4.2 and Kv4.3) in the pathogenesis of the long QT syndrome (LQTS) and Brugada syndrome, it is necessary to determine the genomic organisation of KCND2 and KCND3. We therefore resolved the intron-exon boundaries and flanking intronic sequences and found that KCND2 consisted of six exons and KCND3 of seven exons. Subsequently, we designed the oligonucleotide primers needed for amplifying the coding exons of both KCND2 and KCND3 and established conditions for polymerase chain reaction amplification of each exon from genomic DNA. Furthermore, the chromosomal localisation of KCND2 and KCND3 was determined as 7q31 and 1p13.2, respectively. This information should facilitate the systematic screening of KCND2 and KCND3 exons for mutations in (inherited) arrhythmia syndromes, such as LQTS and Brugada.

Abstract —The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase–polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.

Closure of the primary atrial foramen is achieved by fusion of the atrioventricular cushions with the mesenchymal cap on the leading edge of the muscular primary atrial septum. A fourth component involved is the vestibular spine, originally described by His in 1880 as an intra-cardiac continuation of the extra-cardiac mesenchyme of the dorsal mesocardium. The morphogenesis of this area is of great clinical interest, because of the high incidence of atrial and atrioventricular septal defects. Nonetheless, the origin of the participating components is largely unknown. Here we report that the primary atrial foramen is surrounded in its entirety by mesenchyme derived from endocardium. A second population of mesenchyme not derived from endocardium was observed at the caudal margin of the mesenchymal atrial cap, entirely embedded within the mesenchyme derived from endocardium and contiguous with the mesenchyme of the dorsal mesocardium. Our reconstructions show this second population does indeed take the form of a short spine, albeit that it is the right pulmonary ridge, rather than this spine, that protrudes into the atrial lumen. From the stance of morphological description, therefore, there is little thus far to substantiate the existence of an atrial spine.

Background The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates.Results We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development.Conclusions We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.

Combined methodologies of histochemistry, immunohistochemistry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), reverse transcriptase polymerase chain reaction (RT-PCR) and a histochemical method specific for myofibrillar ATPase (mATPase) of the type IIX myosin heavy chain (MyHC) isoform were used to study human and rat single fibres to examine the homology between type II MyHC isoform-based fibres of both species. We demonstrate that human type II fibres exhibit antigenic mATPase and 3’-untranslated region (3’-UTR) sequence determinants homologous to the IIA and IIX but not the IIB MyHC isoforms of the rat. Both immunolabelling with anti-MyHC monoclonal antibodies and the mATPase method used with frozen sections confirmed that all human type II fibres express type IIA and/or type IIX MyHC. Quantitative immunohistochemistry failed to recognize human fibres with antigenic characteristics corresponding to hybrid IIXB MyHC-based fibres. Ca2+-stimulated maximum myosin ATPase activity, determined by quantitative histochemistry, revealed that human IIX fibres (with an optical density or OD = 0.707) display enzyme activity which is comparable to that of the rat type IIX (OD = 0.687) but lower than that of the rat type IIB fibres (OD = 0.836). The results do not support the notion that MyHC IIB is expressed in human limb muscles, even in hybrid fibres. We conclude that human type II fibres have been misclassified in numerous previous publications and that this has important implications in attempts to compare the physiological characteristics of fibre types, particularly when animal models are used.

The embryonic heart functions as a pump without one-way valves. To accomplish this, a long, slowly conducting myocardial structure, the outflow tract, functions as a sphincter at the arterial pole of the heart. During subsequent development tissue remodeling in the outflow tract and immigrating cells of the neural crest are responsible for connecting the right ventricle with the pulmonary trunk and the left ventricle with the aorta, that is, for the developmental formation of the ventriculoarterial junction. Most congenital malformations of the ventriculoarterial junction stem from disturbances that result in developmental arrest or in abnormal pattern formation (”real” teratology). Abnormal pattern formation can in turn originate from problems with laterality or from aberrant or incomplete formation of structural elements. Genetically modified animals with well-defined gene deficiencies are beginning to provide insight in the signal-transduction pathways and structural elements that are responsible for normal development.

The inaugural Kjell Johansen Lecture in the Zoophysiology Department of Aarhus University (Aarhus, Denmark) afforded the opportunity for a focused workshop comprising comparative cardiovascular physiologists to ponder some of the key unanswered questions in the field. Discussions were centred around three themes. The first considered function of the vertebrate heart in its various forms in extant vertebrates, with particular focus on the role of intracardiac shunts, the trabecular ('spongy') nature of the ventricle in many vertebrates, coronary blood supply and the building plan of the heart as revealed by molecular approaches. The second theme involved the key unanswered questions in the control of the cardiovascular system, emphasizing autonomic control, hypoxic vasoconstriction and developmental plasticity in cardiovascular control. The final theme involved poorly understood aspects of the interaction of the cardiovascular system with the lymphatic, renal and digestive systems. Having posed key questions around these three themes, it is increasingly clear that an abundance of new analytical tools and approaches will allow us to learn much about vertebrate cardiovascular systems in the coming years.

We conducted a genome-wide association study to search for risk alleles associated with Tetralogy of Fallot (TOF), using a northern European discovery set of 835 cases and 5159 controls. A region on chromosome 12q24 was associated (P = 1.4 × 10-7) and replicated convincingly (P = 3.9 × 10-5) in 798 cases and 2931 controls [per allele odds ratio (OR) = 1.27 in replication cohort, P = 7.7 × 10-11 in combined populations]. Single nucleotide polymorphisms in the glypican 5 gene on chromosome 13q32 were also associated (P = 1.7 × 10-7) and replicated convincingly (P = 1.2 × 10-5) in 789 cases and 2927 controls (per allele OR = 1.31 in replication cohort, P = 3.03 × 10-11 in combined populations). Four additional regions on chromosomes 10, 15 and 16 showed suggestive association accompanied by nominal replication. This study, the first genome-wide association study of a congenital heart malformation phenotype, provides evidence that common genetic variation influences the risk of TOF.

Background— Association between the C677T polymorphism of the methylene tetrahydrofolate reductase ( MTHFR ) gene and congenital heart disease (CHD) is contentious. Methods and Results— We compared genotypes between CHD cases and controls and between mothers of CHD cases and controls. We placed our results in context by conducting meta-analyses of previously published studies. Among 5814 cases with primary genotype data and 10 056 controls, there was no evidence of association between MTHFR C677T genotype and CHD risk (odds ratio [OR], 0.96 [95% confidence interval, 0.87–1.07]). A random-effects meta-analysis of all studies (involving 7697 cases and 13 125 controls) suggested the presence of association (OR, 1.25 [95% confidence interval, 1.03–1.51]; P =0.022) but with substantial heterogeneity among contributing studies (I 2 =64.4%) and evidence of publication bias. Meta-analysis of large studies only (defined by a variance of the log OR 2 =0). Moreover, meta-analysis of 1781 mothers of CHD cases (829 of whom were genotyped in this study) and 19 861 controls revealed no evidence of association between maternal C677T genotype and risk of CHD in offspring (OR, 1.13 [95% confidence interval, 0.87–1.47]). There was no significant association between MTHFR genotype and CHD risk in large studies from regions with different levels of dietary folate. Conclusions— The MTHFR C677T polymorphism, which directly influences plasma folate levels, is not associated with CHD risk. Publication biases appear to substantially contaminate the literature with regard to this genetic association.

Using immunohistochemical staining, the distribution of connexin40 (Cx40) and connexin43 (Cx43) was studied in rat, guinea pig, porcine, bovine and human hearts. These species display differences in the degree of morphological differentiation of the conduction system. This study was performed in the anticipation that comparison of the distributions of Cx40 and Cx43 in young and adult specimens may provide clues as to the physiological role of connexins in the heart. To a large extent, the distribution patterns of Cx40 and Cx43 are comparable between species. In neonates and adults, Cx43 was immunolocalized throughout the working myocardium, but in the conduction system Cx43 was detected only after birth. Cx40 was found to appear slightly earlier in development than Cx43 and to disappear when levels of Cx43 became more abundant. This time course was seen in working myocardium and in the ventricular conduction system. Together these data suggest that expression of Cx40 induces or facilitates expression of Cx43, while abundant expression of Cx43 in turn leads to suppression of Cx40 expression. The exceptions to this may represent blocks in this potential regulatory sequence. A second conclusion is that Cx40 and Cx43 containing gap junctions appear in the ventricular conduction system from distal to proximal and only after birth. This indicates that terminal differentiation of the conduction system occurs unexpectedly late in development. © 1995 Wiley-Liss, Inc.