Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio-venous connections.

Recent reports suggest that mammalian embryonic coronary endothelium (CoE) originates from the sinus venosus and ventricular endocardium. However, the contribution of extracardiac cells to CoE is thought to be minor and nonsignificant for coronary formation. Using classic (Wt1(Cre)) and previously undescribed (G2-Gata4(Cre)) transgenic mouse models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardium (ST/PE)-derived endothelial cells are required for the formation of ventricular coronary arterio-venous vascular connections. Our results indicate that at least 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE compartment. Moreover, we show that conditional deletion of the ST/PE lineage-specific Wilms' tumor suppressor gene (Wt1) in the ST/PE of G2-Gata4(Cre) mice and in the endothelium of Tie2(Cre) mice disrupts embryonic coronary transmural patterning, leading to embryonic death. Taken together, our results demonstrate that ST/PE-derived endothelial cells contribute significantly to and are required for proper coronary vascular morphogenesis.

Mesodermal expression of Moz is necessary for cardiac septum development.

Ventricular septal defects (VSDs) are the most commonly occurring congenital heart defect. They are regularly associated with complex syndromes, including DiGeorge syndrome and Holt-Oram syndrome, which are characterised by haploinsufficiency for the T-box transcription factors TBX1 and TBX5, respectively. The histone acetyltransferase monocytic leukaemia zinc finger protein, MOZ (MYST3/KAT6A), is required for the expression of the Tbx1 and Tbx5 genes. Homozygous loss of MOZ results in DiGeorge syndrome-like defects including VSD. The Moz gene is expressed in the ectodermal, mesodermal and endodermal aspects of the developing pharyngeal apparatus and heart; however it is unclear in which of these tissues MOZ is required for heart development. The role of MOZ in the activation of Tbx1 would suggest a requirement for MOZ in the mesoderm, because deletion of Tbx1 in the mesoderm causes VSDs. Here, we investigated the tissue-specific requirements for MOZ in the mesoderm. We demonstrate that Mesp1-cre-mediated deletion of Moz results in high penetrance of VSDs and overriding aorta and a significant decrease in MOZ-dependant Tbx1 and Tbx5 expression. Together, our data suggest that the molecular pathogenesis of VSDs in Moz germline mutant mice is due to loss of MOZ-dependant activation of mesodermal Tbx1 and Tbx5 expression.

Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor.

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

The protein phosphatase 2A B56γ regulatory subunit is required for heart development.

BACKGROUND: Protein Phosphatase 2A (PP2A) function is controlled by regulatory subunits that modulate the activity of the catalytic subunit and direct the PP2A complex to specific intracellular locations. To study PP2A's role in signal transduction pathways that control growth and differentiation in vivo, a transgenic mouse lacking the B56γ regulatory subunit of PP2A was made. RESULTS: Lack of PP2A activity specific to the PP2A-B56γ holoenzyme, resulted in the formation of an incomplete ventricular septum and a decrease in the number of ventricular cardiomyocytes. During cardiac development, B56γ is expressed in the nucleus of α-actinin-positive cardiomyocytes that contain Z-bands. The pattern of B56γ expression correlated with the cardiomyocyte apoptosis we observed in B56γ-deficient mice during mid to late gestation. In addition to the cardiac phenotypes, mice lacking B56γ have a decrease in locomotive coordination and gripping strength, indicating that B56γ has a role in controlling PP2A activity required for efficient neuromuscular function. CONCLUSIONS: PP2A-B56γ activity is required for efficient cardiomyocyte maturation and survival. The PP2A B56γ regulatory subunit controls PP2A substrate specificity in vivo in a manner that cannot be fully compensated for by other B56 subunits.

[Trabeculae of the sinus part of the heart interventricular septum in the fetal period of human development].

In the series of 91 samples of human heart obtained from fetuses at develo pmental weeks 17-28 and formed without major defects and minor anomalies, the relief of the sinus part (SP) of the interventricular septum (IVS) was studied on the side of right ventricle (RV). Myocardial trabeculae carneae (MTC) were found in SP in 96.7% of the cases. MTC, associated with the IVS myocardium along their entire length (parietal MTC). were twice as frequent as bridge-like MTC. MTC were predominantly concentrated at the posterior corner of the RV; these were e xclusively bridge-like MTC. Most frequently, MTC were absent near the IVS membranous region. An individual anatomical variability of the relief of the RV in the fetal heart was demonstrated. Depending on the number, anatomical type and mutual position of the MIC, three variants of the SP relief were distinguished: hypertrabecular, hypotrabecular and intermediate. From week 17 to week 28 of the intrauterine life, the hearts of the fetuses may differ in the form of MTC, however their number and the anatomical type within a particular variant of the SP remained constant The existence of the parietal longitudinal MTC on the right side of the IVS SP is proposed to be one of the hallmarks of the anatomically "normal" (ordinarily formed) heart in the human fetuses.

Monitoring clonal growth in the developing ventricle.

Understanding the etiology of congenital heart defects depends on a detailed knowledge of the morphogenetic events underlying cardiac development. Deciphering the developmental processes and cell behaviors resulting in the formation of a four-chambered heart requires techniques by which the destiny of individual cells can be traced during development. Ideally, such approaches provide information on progenitor cells and growth properties of clonally related myocytes. In the avian system, clonal analysis based on the use of replication-defective retroviral labeling led to a model for growth of the ventricular wall from polyclonal transmural cones of myocardial cells. In the mouse, the nlaacZ retrospective clonal analysis system has proved to be a powerful technique for studying different aspects of cardiac morphogenesis. Morphologic and histologic analyses of clonally related myocytes at early stages of development have provided genetic evidence for the formation of the heart tube from two cell lineages. Additional aspects of cardiac morphogenesis, including formation of the interventricular septum and myocardial outflow tract, and more recently, the origin of the ventricular conduction system, have been studied using this system. This brief review discusses how the nlaacZ system has provided new insights into the divergent properties of clonally related cells in these different regions of the developing heart.

Two distinct pools of mesenchyme contribute to the development of the atrial septum.

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.

The matricellular protein CCN1 is essential for cardiac development.

The matricellular protein CCN1 (formerly named CYR61) regulates cell adhesion, migration, proliferation, survival, and differentiation through binding to integrin receptors and heparan sulfate proteoglycans. Here we show that Ccn1-null mice are impaired in cardiac valvuloseptal morphogenesis, resulting in severe atrioventricular septal defects (AVSD). Remarkably, haploinsufficiency for Ccn1 also results in delayed formation of the ventricular septum in the embryo and persistent ostium primum atrial septal defects (ASD) in approximately 20% of adults. Mechanistically, Ccn1 is not required for epithelial-to-mesenchymal transformation or cell proliferation and differentiation in the endocardial cushion tissue. However, Ccn1 deficiency leads to precocious apoptosis in the atrial junction of the cushion tissue and impaired gelatinase activities in the muscular component of the interventricular septum at embryonic day 12.5, when fusion between the endocardial cushion tissue and the atrial and ventricular septa occurs, indicating that these defects may underlie the observed AVSD. Moreover, human CCN1 maps to 1p21-p31, the chromosomal location of an AVSD susceptibility gene. Together, these results provide evidence that deficiency in matrix signaling can lead to autosomal dominant AVSD, identify Ccn1(+/-) mice as a genetic model for ostium primum ASD, and implicate CCN1 as a candidate gene for AVSD in humans.

Histology and ultrastructure of pericardial cells of Scaptotrigona postica Latreille (Hymenoptera, Apidae) in workers and queens of different ages.

The paper presents a study of the pericardial cells of Scaptotrigona postica an eusocial Brazilian stingless bee. Light and electron microscopy was used in a comparative study on workers and queens of different ages, exerting different functions in the colony. The pericardial cells are found only in the pericardial sinus, mainly in groups around the dorsal vessel. Each cell is enclosed by the basal membrane and its peripheral region is characterized by folds of the plasma membrane, which form canals and loops. The points where the plasma membrane folds is frequently closed by diaphragms, that along with the basal lamina form a barrier to substances from hemolymph. Along the membrane limiting the canals and loops, an intense endocytic activity through coated vesicles takes place indicating a selective absorption of hemolymph components. In older individuals, workers or queens, the cells exhibit larger quantities of cytoplasm inclusions, heterogeneous vacuoles containing the final products of intracellular digestion, and autophagic vacuoles with concentric membranous structures. The pericardial cells general morphology is in accordance with the role in processing metabolites captured from hemolymph and storage of indigested residues.

Cardiocyte nucleus shape as an indicator of heart graft aging.

UNLABELLED: Morphometric publications based on the measurement of cardiocyte nuclei indicated their progressive hypertrophy ignoring, however, their shape, which is a deciding factor for the microscopic-based diagnosis of hypertrophy. We sought was to demonstrate how the shapes of cardiocyte nuclei change over time and correlate them with the thickness of the interventricular septum, (IVS) the biopsy site. MATERIAL: We evaluated myocardial biopsies taken in the first week, first month, and then annually until posttransplant year 10. Only biopsies with no rejection were considered: grade "0" ISHLT (122 biopsies). The control group encompassed fragments from seven donor hearts. METHODS: Cardiomyocyte nuclei were evaluated morphometrically. We calculated the length, breadth, perimeter, roundness, elongation, and fullness factors for correlation with the IVS thickness, and selected indices. The relationships between karyometry and IVS thickness (measured by ultrasound) as well as time were calculated by Spearman's correlation test. RESULTS: Among the examined indices, only nuclear length did not correlate significantly with follow-up time. Among the remaining indices, the strongest correlations with time were observed with regard to breadth (r = 0.214), perimeter (r = 0.150), roundness (r = -0.06) and fullness (r = 0.06), and finally elongation (r = 0.02). The decreasing thickness of the interventricular septum (r = -0.31) showed a weak correlation only with the cardiocyte nuclear length (r = -0.05). CONCLUSION: Graft aging imitates hypertrophy inasmuch as cardiocyte nuclei become wider despite the decreased thickness of the interventricular septum. Therefore, karyometric measurements do not reflect myocardial morphology.

[Morphometry of human myocardium in senile individuals]. / Morfometria do miocárdio humano em indivíduos senis.

OBJECTIVE: To carry out a quantitative assessment in human myocardium cells of senile individuals, in right, left and septal ventricular regions. METHODS: Five hearts from corpses of individuals without heart diseases, of both sexes, with age between 67 and 87 years old were used. The following parameters were assessed: myocyte unit cross section area (myoc. a(o)); myocyte unit perimeter length (myoc. l(o)); myocyte unit volume (myoc. v(o)); myocyte volumetric density (myoc. V(v)); number of myocytes per volume unit (Nmm 3myoc.). The t-test of Student was used in statistic analysis. RESULTS: The analysis of differences (p < 0.05) among right (RV), left (LV) and septal (S) ventricular regions of human heart showed that myoc. a(o) values were lower in RV (1.51 +/- 0.10 microm2) and in S (1.55 +/- 0.07 microm2) in relation to LV (1.84 +/- 0.24 microm2). Values of myoc. l(o) were also shown lower in S (5.11 +/- 0.46 microm) comparing to LV (6.2 +/- 0.97 microm). Likewise, myoc. v(o) and myoc. V(v) showed lower values in RV (88.75 +/- 25.37 microm3; 0.39 +/- 0.03%) in relation to LV (122.41 +/- 16.31 microm3; 0.41 +/- 0.01%). CONCLUSION: Results obtained show that there may be changes in dimensions of left ventricular wall myocyte cell during senescent stage. However, those differences are subtle and seem to mean the adjustment of tissue to functional changes that install along life.

A novel application of myocardial contrast echocardiography to evaluate angiogenesis by autologous bone marrow cell transplantation in chronic ischemic pig model.

OBJECTIVES: We investigated the feasibility of myocardial contrast echocardiography (MCE) to evaluate regional perfusion after bone marrow cell transplantation. BACKGROUND: The myocardial microvessels improved by cell transplantation are too small to visualize with conventional angiography. METHODS: Fourteen mini-pigs from the Nippon Institute for Biological Science were used. The proximal left anterior descending coronary artery was ligated. One month later, nine pigs survived. Six pigs received autologous cell transplantation into the left ventricular anterior wall: bone marrow mononuclear cells (BMMNCs) (n = 3) and bone marrow stromal cells (BMSCs) (n = 3). The other three pigs received saline (control group, n = 3). The pigs were sacrificed one month later. Myocardial contrast intensity (MCI) with a contrast agent was measured using the SONOS 5500 system (Philips). Capillary density (CD) and MCI were measured at four areas: anteroseptum (nontransplanted infarct area), anterior wall (transplanted infarct area), septum (border zone), and lateral wall (normal). We compared the anteroseptum with the anterior wall by MCI and CD. RESULTS: In the BMMNC and BMSC subsets, the CD of the anterior wall was higher than that of the anteroseptum (p < 0.001). There was a linear relation between MCI and CD (acoustic unit [AU2] = 0.234 CD + 0.010, r = 0.92, p < 0.001). At one month after cell transplantation, MCI of the anterior wall increased in the BMMNC and BMSC subsets (p < 0.05), although it did not change in the control group. The ratio of wall thickness (systole/diastole) in the transplanted infarct area was larger than that in the nontransplanted infarct area (p < 0.01). CONCLUSIONS: Myocardial contrast echocardiography is useful to evaluate regional perfusion, which was enhanced by bone marrow cell transplantation.

Percutaneous transvenous cellular cardiomyoplasty. A novel nonsurgical approach for myocardial cell transplantation.

OBJECTIVES: The study evaluated a nonsurgical means of intramyocardial cell introduction using the coronary venous system for direct myocardial access and cell delivery. BACKGROUND: Direct myocardial cell repopulation has been proposed as a potential method to treat heart failure. METHODS: We harvested bone marrow from Yorkshire swine (n = 6; 50 to 60 kg), selected culture-flask adherent cells, labeled them with the gene for green fluorescence protein, expanded them in culture, and resuspended them in a collagen hydrogel. Working through the coronary sinus, a specialized catheter system was easily delivered to the anterior interventricular coronary vein. The composite catheter system (TransAccess) incorporates a phased-array ultrasound tip for guidance and a sheathed, extendable nitinol needle for transvascular myocardial access. A microinfusion (IntraLume) catheter was advanced through the needle, deep into remote myocardium, and the autologous cell-hydrogel suspension was injected into normal heart. Animals were sacrificed at days 0 (n = 2), 14 (n = 1, + 1 control/collagen biogel only), and 28 (n = 2), and the hearts were excised and examined. RESULTS: We gained widespread intramyocardial access to the anterior, lateral, septal, apical, and inferior walls from the anterior interventicular coronary vein. No death, cardiac tamponade, ventricular arrhythmia, or other procedural complications occurred. Gross inspection demonstrated no evidence of myocardial perforation, and biogel/black tissue dye was well localized to sites corresponding to fluoroscopic landmarks for delivery. Histologic analysis demonstrated needle and microcatheter tracts and accurate cell-biogel delivery. CONCLUSIONS: Percutaneous intramyocardial access is safe and feasible by a transvenous approach through the coronary venous system. The swine offers an opportunity to refine approaches used for cellular cardiomyoplasty.

Regional cardiac ganglia projections in the guinea pig heart studied by postmortem DiI tracing.

Our purpose was to identify and localize intrinsic cardiac ganglia innervating distinct regions of the heart using postmortem tracing of nerve projections with DiI, a method not previously used to study the intrinsic cardiac nervous system. We also investigated the possibility of collateral innervation of myocardium and intrinsic ganglia. In isolated paraformaldehyde-fixed guinea pig hearts, crystals of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were inserted into the posterior ventricular myocardium below the atrioventricular groove, the right atrium, or the left ventricular septum. Hearts were placed in the dark at 37 degrees C for 2-14 weeks to allow DiI diffusion within neuronal membranes. Labeled neurons were observed in intracardiac ganglia after at least 4 weeks of dye exposure. Labeling was restricted to the inferior-most ganglia (those near the atrioventricular groove) when DiI was inserted into the posterior ventricular myocardium and to ganglia near the sinus node after right atrial DiI placement. Application of DiI to the left ventricular septum resulted in neuron labeling in ganglia primarily in the interatrial septum near the atrioventricular node. After 8 weeks, DiI-labeled nerve fibers and varicosities were seen surrounding unlabeled neurons in some ganglia, suggesting that axons terminating in or passing through the DiI application site in posterior ventricular tissue had collateral branches innervating these ganglia. These results indicate that intrinsic innervation of major cardiac subdivisions is accomplished by regionally segregated cardiac ganglia. Also, tracing with DiI has provided evidence for collateral nerve projections that could be the substrate for novel intracardiac regulatory circuits.

Normal distribution of melanocytes in the mouse heart.

We report the consistent distribution of a population of pigmented trp-1-positive cells in several important septal and valvular structures of the normal mouse (C57BL/6) heart. The pigmented cell population was first apparent by E16.5 p.c. in the right atrial wall and extended into the atrium along the interatrial septum. By E17.5, these cells were found along the apical membranous interventricular septum near or below the surface of the endocardium. The most striking distribution of dark pigmented cells was found in the tricuspid and mitral valvular leaflets and chordae tendineae. The normal distribution of pigmented cells in the valvuloseptal apparatus of C57BL/6 adult heart suggests that a premelanocytic lineage may participate in the earlier morphogenesis of the valve leaflets and chordae tendineae. The origin of the premelanocyte lineage is currently unknown. The most likely candidate populations include the neural crest and the epicardially derived cells. The only cell type in the heart previously shown to form melanocytes is the neural crest. The presence of neural crest cells, but not melanocytes, in some of the regions we describe has been reported by others. However, previous reports have not shown a contribution of melanocytes or neural crest derivatives to the atrioventricular valve leaflets or chordae tendineae in mouse hearts. If these cells are of neural crest origin, it would suggest a possibly greater contribution and persistence of neural crest cells to the valvuloseptal apparatus than has been previously understood.

Neural crest-derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation.

In mammals, the outflow tract (OFT) of the developing heart septates into the base of the pulmonary artery and aorta to guide deoxygenated right ventricular blood into the lungs and oxygenated left ventricular blood into the systemic circulation. Accordingly, defective OFT septation is a life-threatening condition that can occur in both syndromic and nonsyndromic congenital heart disease. Even though studies of genetic mouse models have previously revealed a requirement for VEGF-A, the class 3 semaphorin SEMA3C, and their shared receptor neuropilin 1 (NRP1) in OFT development, the precise mechanism by which these proteins orchestrate OFT septation is not yet understood. Here, we have analyzed a complementary set of ligand-specific and tissue-specific mouse mutants to show that neural crest-derived SEMA3C activates NRP1 in the OFT endothelium. Explant assays combined with gene-expression studies and lineage tracing further demonstrated that this signaling pathway promotes an endothelial-to-mesenchymal transition that supplies cells to the endocardial cushions and repositions cardiac neural crest cells (NCCs) within the OFT, 2 processes that are essential for septal bridge formation. These findings elucidate a mechanism by which NCCs cooperate with endothelial cells in the developing OFT to enable the postnatal separation of the pulmonary and systemic circulation.

The vagal origin of preganglionic fibers containing nitric oxide synthase in the guinea-pig heart.

The origin of nerve fibers projecting to the guinea pig heart that contain nitric oxide synthase (NOS) were studied by unilateral cervical vagotomy. Three kinds of NOS-immunoreactive (NOS-ir) nerve fibers are distributed in the control guinea pig heart: the sparse network covering the right atrium, the basket-like endings around intracardiac neuronal cell bodies in the small ganglia located in the left atrium and the interatrial septum, and the axons situated in the septal region. The sparse network in the right atrium did not change after vagotomy of right or left side. In the whole mount preparations of right atrium, we often traced labeled axons from the somata to join the network covering the right atrium. Therefore, most of this network of labeled fibers must be of intrinsic origin. Because the basket-like endings around neuronal cell bodies in the ganglia in the left atrium and the septum disappeared completely after vagotomy of left side, we conclude that they are parasympathetic preganglionic fibers originating from the left vagus nerve. NOS-ir cell bodies and the positive fibers in the atrioventricular nodal region survived after vagotomy. All of such nerve fibers were unmyelinated axons. Therefore, they seem to be the postganglionic fibers arising from the ganglia located in the left atrium or the septum.

[Quantitative analysis of tissue and cellular components of the specialized internodal conductive pathways of the heart]. / Kolichestvennyi analiz tkanevykh i kletochnykh komponentov spetsializirovannykh provodiashchikh mezhuzlovykh putei serdtsa.

A light-optic and electron-microscopic study of internodal specialized pathways in the rat interatrial cardiac septum is reported. The topography of conductive pathways is described. Morphometric investigation yielded comparative quantitative characteristics of tissue and cellular composition of the working and conductive septal myocardium. A single type of myocytes was demonstrated in specialized conductive pathways. The findings were compared with those obtained in other species. Principles of identification of specialized conductive pathways are discussed.

The development of septation in the four-chambered heart.

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.