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.

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.

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.

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.

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.

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.

Septal ablation induced by transthoracic high-intensity focused ultrasound in canines.

BACKGROUND: High-intensity focused ultrasound (HIFU) can achieve accurate and focused deep tissue ablation through an extracorporeal emission. Cardiac ablation using HIFU applied transthoracically must overcome potential interference from intervening thoracic structures. The aim of this study was to explore the efficacy and safety of septal ablation that was induced using transthoracic HIFU. METHODS: Twenty-one canines were pretreated to improve acoustic transmission. Single ablations were induced by targeting transthoracic HIFU with acoustic power of 400 W for 3 sec at the middle and basal septum in eight canines. Extended ablations were performed to create larger lesions at the basal septum in eight more canines. The three-dimensional morphology of a basal septum lesion induced by a single ablation was analyzed. The temperature at the ablative targets was measured in the other five canines. RESULTS: The cardiomyocytes in the lesions underwent necrosis with a clear boundary. The three-dimensional morphology of the lesions appeared approximately as ellipsoids with a flatter endocardial side. The peak temperature at a power of 400 W for 3 sec was 93.27 ± 2.54°C, and it remained >50°C for nearly 10 sec. No procedure-related complications were observed. CONCLUSIONS: Ultrasound-guided transthoracic HIFU has the potential to safely create small dot or large mass lesions in the septum without a thoracotomy or a catheter.

[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.

Normal development of the muscular region of the interventricular septum. II. The importance of myocardial proliferation.

In a first paper, we concluded that the muscular region of the interventricular septum is developed by the trabecular branches and showed evidence that the developing interventricular septum elongates in a direction opposite to that of atria. Nevertheless, to date the literature is lacking precise information on the importance of myocardial proliferation not only in this process but also in the morphogenesis of the ventricular cavities. The aim of this study was to determine the spatial and temporal distribution of high-intensity foci of cycling myocytes in the ventricular region of the heart of chicken embryos during cardiac septation. Histological studies, detection of the proliferating cell nuclear antigen by light and confocal microscopy and flow cytometric analysis were carried out. The results corroborate that the developing interventricular septum grows in a direction opposite to that of atria. A remoulding mechanism that results in fenestrated trabecular sheets and trabecular branching is discussed. Our findings allowed us to summarize the normal morphogenesis of the muscular region of the interventricular septum in a way that is different from that suggested by other researchers.

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.

BMP4 is required in the anterior heart field and its derivatives for endocardial cushion remodeling, outflow tract septation, and semilunar valve development.

The endocardial cushions play a critical role in septation of the four-chambered mammalian heart and in the formation of the valve leaflets that control blood flow through the heart. Within the outflow tract (OFT), both cardiac neural crest and endocardial-derived mesenchymal cells contribute to the endocardial cushions. Bone morphogenetic protein 4 (BMP4) is required for endocardial cushion development and for normal septation of the OFT. In the present study, we show that anterior heart field (AHF)-derived myocardium is an essential source of BMP4 required for normal endocardial cushion expansion and remodeling. Loss of BMP4 from the AHF in mice results in an insufficient number of cells in the developing OFT endocardial cushions, defective cushion remodeling, ventricular septal defects, persistent truncus arteriosus, and abnormal semilunar valve formation.

Both Purkinje cells and left ventricular posteroseptal reentry contribute to the maintenance of ventricular fibrillation in open-chest dogs and swine: effects of catheter ablation and the ventricular cut-and-sew operation.

BACKGROUND: Radiofrequency catheter ablation (RFCA) targeting the left ventricular posteroseptum (LVPS) and posterior papillary muscle (PPM) terminates or prevents ventricular fibrillation (VF) in rabbit and dog hearts. However, whether the mechanism of VF maintenance is reentry or focal Purkinje firing is unclear. METHODS AND RESULTS: In the present study the effects of RFCA (endocardial ablation of PPM+LVPS in 7 dogs and 7 swine), left ventricular anterolateral wall ablation (LVAL in 7 dogs), and the cut-and-sew operation (CSO: along the left ventricular posterior wall (LVPW) beside PPM in 7 swine) on VF inducibility were compared. (1) VF inducibility was decreased from 100+/-0% to 21.9+/-31.2% (p<0.0001) by PPM+LVPS endocardial ablation, but not by LVAL ablation in dogs. (2) LVPW CSO reduced VF inducibility (100+/-0% to 43.6+/-9.5%, p<0.0001) in swine. (3) In contrast to the canine Purkinje network, which is mostly localized to the subendocardium, the swine Purkinje network extends to the subepicardial layer with a higher density (p<0.001). CONCLUSION: Both PPM+LVPS ablation (Purkinje destruction) in dogs and LVPW CSO (blocking reentry) in swine reduce VF inducibility, suggesting that in both species focal firing from the Purkinje network and reentry around the PPM contributes to the maintenance of VF.

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.

Expression and function of ATP-dependent potassium channels in late post-infarction remodeling.

Myocardial remodeling late after infarction is associated with increased incidence of fatal arrhythmias. Heterogeneous prolongation of the action potential in the surviving myocardium is one of the predominant causes. Sarcolemmal ATP-dependent potassium (K(ATP)) channels are important metabolic sensors regulating electrical activity of cardiomyocytes and are capable of considerably shortening the action potential. We determined whether ATP-dependent potassium channels generate or, on the contrary prevent the heterogeneity in action potential prolongation. Cardiomyocytes were obtained from the infarct border zone, the septum and the right ventricle of rat hearts 20 weeks after coronary occlusion when rats developed signs of heart failure. Expression of the conductance subunit Kir6.1, but not Kir6.2, and of all SUR regulatory subunits was increased up to 3-fold in cardiomyocytes from the infarct border zone. Concomitantly, there was a prominent response of the K(ATP) current to diazoxide that was not detectable in control cardiomyocytes. The action potential was prolonged in cardiomyocytes from the infarct border zone (74 ms) relative to sham (41 ms). However, activation of the K(ATP) channels by diazoxide reduced action potential duration to 42 ms. In myocytes of the septum and right ventricle, expression of channel subunits, duration of action potential, and sensitivity to diazoxide were only slightly increased relative to shams. In conclusion, the myocardium remodeled after infarction displays alterations of K(ATP) expression and function with spatial heterogeneity matching that of the action potential prolongation. Drugs selectively activating diazoxide-sensitive sarcolemmal K(ATP) channels should be considered in the prevention of arrhythmias in post-infarction heart failure.

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.

Identification of interventricular septum precursor cells in the mouse embryo.

Little is known about the formation of the interventricular septum (IVS), a central event during cardiogenesis. Here, we describe a novel population of myocardial progenitor cells in the primitive ventricle of the mouse embryo, which is characterized by expression of lysozyme M (lysM). Using LysM-Cre mice we show that lysozyme expressing cells give rise to the IVS and to a part of the left ventricular free wall, demonstrating that these heart regions are developmentally related. LysM+ precursors are not of hematopoietic origin and develop in the absence of transcription factors that regulate lysozyme expression in macrophages. LysM-deficient mice lack an overt cardiac phenotype, perhaps due to compensation by the related lysozyme P, which we also found to be expressed in the developing heart. Direct visualization of lysM expression, using LysM-EGFP knock-in mice, showed that ventricular septation is initiated at embryonic day 9 by the movement of myocardial trabeculae from the primitive ventricle towards the bulbo-ventricular groove and revealed the dynamics of IVS formation at later stages. Our studies predict that LysM-Cre mice will be useful to inactivate genes in the developing IVS.

Meltrin beta expressed in cardiac neural crest cells is required for ventricular septum formation of the heart.

The heart is divided into four chambers by membranous septa and valves. Although evidence suggests that formation of the membranous septa requires migration of neural crest cells into the developing heart, the functional significance of these neural crest cells in the development of the endocardial cushion, an embryonic tissue that gives rise to the membranous appendages, is largely unknown. Mice defective in the protease region of Meltrin beta/ADAM19 show ventricular septal defects and defects in valve formation. In this study, by expressing Meltrin beta in either endothelial or neural crest cell lineages, we showed that Meltrin beta expressed in neural crest cells but not in endothelial cells was required for formation of the ventricular septum and valves. Although Meltrin beta-deficient neural crest cells migrated into the heart normally, they could not properly fuse the right and left ridges of the cushion tissues in the proximal outflow tract (OT), and this led to defects in the assembly of the OT and AV cushions forming the ventricular septum. These results genetically demonstrated a critical role of cardiac neural crest cells expressing Meltrin beta in triggering fusion of the proximal OT cushions and in formation of the ventricular septum.

Postnatal lethality and cardiac anomalies in the Ts65Dn Down syndrome mouse model.

The Ts65Dn mouse is a well-studied model for Down syndrome (DS). The presence of the translocation chromosome T17 16 (referred to as T65Dn) produces a trisomic dosage imbalance for over 100 genes on the distal region of mouse Chromosome 16. This dosage imbalance, with more than half of the orthologs of human Chromosome 21 (Hsa21), causes several phenotypes in the trisomic mice that are reminiscent of DS. Careful examination of neonates in a newly established Ts65Dn colony indicated high rates of postnatal lethality. Although the transmission rate for the T65Dn chromosome has been previously reported as 20%-40%, genotyping of all progeny indicates transmission at birth is near the 50% expected with Mendelian transmission and survival. Remarkably, in litters with maternal care that allowed survival of some pups, postnatal lethality occurred primarily in pups that inherited the T65Dn marker chromosome. This selective loss within 48 h of birth reduced the transmission of the marker chromosome from 49% at birth to 34% at weaning. Gross morphologic examination revealed cardiovascular anomalies, i.e., right aortic arch accompanied by septal defects, in 8.3% of the trisomic newborn cadavers examined. This is an intriguing finding because the orthologs of the DiGeorge region of HSA22, which are posited to contribute to the aortic arch abnormalities seen in trisomy 16 mice, are not triplicated in Ts65Dn mice. These new observations suggest that the Ts65Dn mouse models DS not only in its previously described phenotypes but also with elevated postnatal lethality and congenital heart malformations that may contribute to mortality.

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.