Apoptosis is required for the proper formation of the ventriculo-arterial connections.

The role of apoptosis in cardiac morphogenesis has not been directly tested. Cardiomyocyte apoptosis is prevalent during the remodeling of the embryonic chicken cardiac outflow tract (OFT) in the transition from a single to a dual circulation. We tested the hypothesis that OFT cardiomyocyte apoptosis drives the shortening and rotation of the embryonic cardiac OFT and is required to achieve the mature ventriculo-arterial configuration. Chick embryos were treated with the peptide Caspase inhibitors zVAD-fmk or DEVD-cho at HH stages 15-20 (looped heart). Morphology of control and experimental embryos was assessed at HH stage 35, at which time the control hearts have developed a dual circulation. Infection of the hearts with a recombinant adenovirus expressing green fluorescent protein was used to follow the fate of the OFT cardiomyocytes. Affected embryos displayed abnormal persistence of a long infundibulum (OFT myocardial remnant) beneath the great vessels, indicating failure of OFT shortening. In some instances, the infundibulum connected both great vessels to the right ventricle in a side-by-side arrangement with transposition of the aorta, indicating a failure of rotation of the OFT, and modeling human congenital double outlet right ventricle. Defects were also observed at other sites in the heart where apoptosis is prevalent, such as in the formation of the cardiac valves and trabeculae. To more specifically target the apoptosis of the OFT cardiomyocytes, recombinant adenovirus was used to express the X-linked inhibitor of apoptosis protein in these cells. This resulted in an effect on outflow tract shortening and rotation similar to that of the peptide inhibitors, while the effects on the other cardiac structures were not observed. These results demonstrate that elimination of OFT cardiomyocytes by apoptosis is necessary for the proper formation of the ventriculo-arterial connections, and suggest apoptosis as a potential target of teratogens and genetic defects that are associated with congenital human conal heart defects.     

Coronary artery embryogenesis in cardiac defects induced by retinoic acid in mice

BACKGROUND: Although normal coronary artery embryogenesis is well described in the literature, little is known about the development of coronary vessels in abnormal hearts. METHODS: We used an animal model of retinoic acid (RA)-evoked outflow tract malformations (e.g., double outlet right ventricle [DORV], transposition of the great arteries [TGA], and common truncus arteriosus [CTA]) to study the embryogenesis of coronary arteries using endothelial cell markers (anti-PECAM-1 antibodies and Griffonia simplicifolia I (GSI) lectin). These markers were applied to serial sections of staged mouse hearts to demonstrate the location of coronary artery primordia. RESULTS: In malformations with a dextropositioned aorta, the shape of the peritruncal plexus, from which the coronary arteries develop, differed from that of control hearts. This difference in the shape of the early capillary plexus in the control and RA-treated hearts depends on the position of the aorta relative to the pulmonary trunk. In both normal and RA-treated hearts, there are several capillary penetrations to each aortic sinus facing the pulmonary trunk, but eventually only 1 coronary artery establishes patency with 1 aortic sinus. CONCLUSIONS: The abnormal location of the vessel primordia induces defective courses of coronary arteries; creates fistulas, a single coronary artery, and dilated vessel lumens; and leaves certain areas of the heart devoid of coronary artery branches. RA-evoked heart malformations may be a useful model for elucidating abnormal patterns of coronary artery development and may shed some light on the angiogenesis of coronary artery formation.     

Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger-Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.     

Origin of the proximal coronary artery stems and a review of ventricular vascularization in the chick embryo

The objective of this study was to determine how the coronary artery stems develop in the chick embryo. The hearts of 51 ink-injected and cleared chick embryos, aged embryonic days 6, 6.5, 7, 7.5, 9, and 10, were dissected, examined, and selectively photographed. Two representative hearts from each group were paraffin embedded, serially sectioned at 10 microns, and examined for aortic endothelial budding. We found that the proximal coronary artery did not appear to grow outward from the aorta as commonly described in the literature. It appeared to originate from a capillary ring which encircled the aortic and pulmonary outflow tracts. On embryonic day 7.5, one to three channels arising from this ring penetrated each aortic sinus, in an area of darker textured endothelium. Histologically and grossly, multiple channels were still apparent on day 9, particularly in the left coronary artery. One of these channels always became dominant to form the stem. Each stem, which varied in length from embryo to embryo, always ended in a plexus of sinusoidal endothelial tubes. By day 10, the coronary artery stems were longer, with many major branches. Histologically, evidence of multiple channels still was visible. It is significant that channels from the bulbar vascular ring penetrated the aorta at very specific points in the aortic sinuses and did not penetrate the pulmonary trunk or other aortic sites. We believe this fact indicates that the penetration of the aortic sinuses by channels from the bulbar vascular ring represents a controlled invasion of the aorta.     

Angioarchitecture of the venous and capillary system in heart defects induced by retinoic acid in mice

BACKGROUND: Corrosion casting and immunohistochemical staining with anti‐alpha smooth muscle actin and anti‐CD34 was utilized to demonstrate the capillary plexus and venous system in control and malformed mouse hearts. METHODS: Outflow tract malformations (e.g., double outlet right ventricle, transposition of the great arteries, and common truncus arteriosus) were induced in progeny of pregnant mice by retinoic acid administration at day 8.5 of pregnancy. RESULTS: Although control hearts exhibited areas in which capillaries tended to be oriented in parallel arrays, the orientation of capillaries in the respective areas of malformed hearts was chaotic and disorganized. The major branch of a conal vein in control hearts runs usually from the left side of the conus to its right side at the root of the pulmonary trunk and opens to the right atrium below the right auricle; thus, it has a curved course. On the other hand, a conal vein in malformed hearts courses from the left side or from the anterior side of the conus and tends to traverse straight upwards along the dextroposed aorta or along the aortopulmonary groove with its proximal part located outside of the heart. Other cardiac veins in outflow tract malformations are positioned in the same locations as in control hearts. CONCLUSIONS: We postulate that the changed location of the conal vein and disorganized capillary plexus result from malformed morphogenesis of the outflow tract and/or a disturbed regulation of angiogenic growth factor release from the adjacent environment. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Anomalous course and branches of human coronary arteries

132 hearts obtained from cadavers were dissected in order to study anomalies in the course and branches of human coronary arteries. Only in 1 heart was a deviation of the circumflex branch of the left coronary artery taking a course inferior to the coronary sulcus observed. The right coronary artery travelled the entire length of the coronary sulcus on the back of the heart and gave both a posterior ventricular and a collateral branch. Circumflex branches of the right and left coronary arteries did not anastomose.     

Expression of bone morphogenetic protein 4 and its receptors in the remodeling heart

Aims

Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure.

Main methods

Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n = 15) or ischemic heart disease (CAD; n = 9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n = 7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n = 11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n = 7 at each time point).

Key findings

Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24 h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H₂O₂ injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia–reoxygenation injury.

Significance

Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes.     

Diagnosis of supravalvular stenosis of the aorta

The authors analyzed the potentialities of combined radiodiagnosis of supravalvular stenosis (SS) and concomitant diseases of the heart and major vessels (Williams-Beuren syndrome) in 7 patients aged 7 mos. to 24 yrs. Polypositional chest and heart x-ray procedure, catheterization of the cardiac cavities, pulmonary artery and aorta, left ventriculography (6), right ventriculography (4) and angiopulmonography were used. The diagnostic potentialities of each method were defined. It was proved that SS practically in all patients was accompanied by major vascular changes in the pulmonary, vertebral, coronary, carotid, subclavicular, renal and other arteries, heart failure (atrial septal defect, and mitral incompetence).     

Loss of connexin45 causes a cushion defect in early cardiogenesis

At around embryonic day 9, the primitive heart of a mouse embryo undergoes spectacular alterations within 24 hours. We created mice harboring an nls-lacZ gene in place of connexin45, which encodes the only known gap junction protein in the primitive heart before embryonic day 9, using the Cre-loxP system. Connexin45-deficient mice died of heart failure at around embryonic day 10. They initiated heart contractions, but conduction block appeared within 24 hours after the first contractions. Their cardiac walls displayed an endocardial cushion defect, while the cardiac jelly was present. These abnormalities were caused by impairment of the epithelial-mesenchymal transformation of the cardiac endothelium. Activation of the cardiac endothelium depended on the presence of the connexin45 gap junctions since signaling through Ca(2+)/calcineurin and NF-ATc1 (originally named NF-ATc) was disrupted in the mutant hearts. These results indicate a requirement for gap junction channels during early cardiogenesis and hence implicate connexin45 in congenital heart diseases. http://www. biologists.com/Development/movies/dev4369.html     

Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression

It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.     

Maternal nicotine exposure induces congenital heart defects in the offspring of mice

Maternal cigarette smoking is a risk factor for congenital heart defects (CHDs). Nicotine replacement therapies are often offered to pregnant women following failed attempts of smoking cessation. However, the impact of nicotine on embryonic heart development is not well understood. In the present study, the effects of maternal nicotine exposure (MNE) during pregnancy on foetal heart morphogenesis were studied. Adult female mice were treated with nicotine using subcutaneous osmotic pumps at 0.75 or 1.5 mg/kg/day and subsequently bred with male mice. Our results show that MNE dose‐dependently increased CHDs in foetal mice. CHDs included atrial and ventricular septal defects, double outlet right ventricle, unguarded tricuspid orifice, hypoplastic left ventricle, thickened aortic and pulmonary valves, and ventricular hypertrophy. MNE also significantly reduced coronary artery size and vessel abundance in foetal hearts. Moreover, MNE resulted in higher levels of oxidative stress and altered the expression of key cardiogenic regulators in the developing heart. Nicotine exposure reduced epicardial‐to‐mesenchymal transition in foetal hearts. In conclusion, MNE induces CHDs and coronary artery malformation in mice. These findings provide insight into the adverse outcomes of foetuses by MNE during pregnancy.     

Morphology of the embryonic and hatchling american alligator ductus arteriosi and implications for embryonic cardiovascular shunting

The ductus arteriosi (DA) are embryonic blood vessels found in amniotic vertebrates that shunt blood away from the pulmonary artery and lungs and toward the aorta. Here, we examine changes in morphology of the right and left DA (LDA), and right and left aorta (LAo) from embryonic and hatchling alligators. The developing alligator has two‐patent DA that join the right and LAo. Both DA exhibit a muscular phenotype composed of an internal smooth muscle layer (2–4 cells thick). At hatching, the lumen diameter of both DA decreases as the vessels begin to close within the first 12 h of posthatch life. Between day 1 and day 12 posthatching, the vessel becomes fully occluded with endothelial and smooth muscle cells filling the lumen. A number of DA from hatchlings contained blood clots along their length. The lumen of the full term alligator DA is reduced in comparison with the full term chicken DA. The developing alligator embryo has an additional right‐to‐left shunt pathway in the LAo arising from the right ventricle. The embryonic LAo diameter is twice the diameter of either the right DA or LDA, providing a lower resistance pathway for blood leaving the right ventricle. On the basis of these findings, we propose that the paired DA of the embryonic alligator have a reduced role in the embryonic right‐to‐left shunt of blood from the right ventricle when compared with the avian DA. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

Myocardial fiber architecture in the human heart. Anatomical demonstration of modifications in the normal pattern of ventricular fiber architecture in a malformed adult specimen

Ventricular myocardial fiber architecture has been considered an important factor in heart dynamics. Most anatomical studies however have focussed on the analysis of normal hearts. The present study compares ventricular myocardial fiber architecture patterns in dissections of 5 normal hearts and a malformed human heart with membranous ventricular septal defect, overriding right aorta, pulmonic stenosis, with absent pulmonary valve and hypertrophied right ventricle. Qualitative and quantitative changes in ventricular myocardial fiber architecture were noted in the malformed heart.     

Significance of the site of premature excitation for the left and right ventricular performance in open chest dogs

The haemodynamic response to premature excitation was studied in open-chest dog hearts. Intraventricular pressure, dP/dt and outflow rate of the left and right heart (5 experiments each) were compared at variable preextrasystolic intervals and with the stimulation at three different sites (left ventricle--LV apex and base, right ventricle--RV free wall). In both ventricles and at any driving interval the reduction of all extrasystolic parameters is significantly more pronounced on ipsilateral stimulation. This reduction is apparent even at the fusion interval, demonstrating the importance of normal spread of excitation. The differences between apex and base stimulation are, however, only insignificant. The outflow valves opening interval greatly differs in aorta and pulmonary artery, namely if LV is stimulated, which results in a considerable disproportion between LV and RV extrasystolic stroke volumes. The extrasystolic augmentation is revealed by all parameters in the right heart but is surprisingly absent in the peak pressure and relaxation rate (dP/dt) in the LV.     

A novel method for obtaining semi-thin cross sections of the Drosophila heart and their labeling with multiple antibodies

The Drosophila heart has become an exciting model for elucidating the molecular basis for cardiac function in higher organisms. To complement the genetic approaches that have recently identified an array of genes essential for cardiac function, we developed a method to obtain optimal semi-thin cross sections of embryonic, larval, and adult fly hearts in a desired orientation. A procedure for fluorescent labeling of these sections with multiple markers has also been developed, allowing the detection of proteins at high subcellular resolution. Sections obtained by our method reveal changes in cell shape between embryonic heart and aorta cardioblasts and elucidate the morphology of the adult heart. Analysis of the adult heart reveals the precise cardiac tube morphology, differential distribution of the extracellular matrix protein Laminin within the cardiac tube, as well as individual hand-positive, and Held Out Wings (HOW)-positive luminal cells that might represent blood cells. In summary, our method enables visualization of cross sections of the embryonic and adult hearts at high resolution while maintaining the ability to co-label the sections with multiple markers, thereby facilitating the analysis of cardiac tube formation and maintenance at different developmental stages.     

Analysis of proteins synthesized by 9.5 day mouse embryos: determination of cardiac and noncardiac proteins.

To catalog polypeptides that were specific to developing hearts, we separated 35S-methionine-labeled 9.5 day mouse embryos into cardiac and noncardiac (carcass) components. Two-dimensional gels were then used to analyze the polypeptides synthesized in these two fractions. As a result, we were able to distinguish polypeptides that were specific to or increased in the heart as well as those polypeptides that were specific to or increased in the embryo minus the dissected heart. Using this analysis, there were two polypeptides that were cardiac-specific and 17 that were expressed at increased levels by at least twofold in the heart. The cardiac-specific polypeptides may be used in further studies to identify early cardiac tissue. Conversely, there were 26 polypeptides unique to noncardiac structures and an additional 15 that were increased in the carcass more than twofold relative to the heart. The noncardiac-specific polypeptides may be used to define contamination of putative cardiac tissue with noncardiac material. Two of the polypeptides expressed more abundantly in the carcass appeared to correspond to known proteins in the mouse fibroblast database, cyclin and tropomyosin 4. Thus the heart at 9.5 days of murine development can be distinguished readily from the remainder of the embryonic mouse both macroscopically and on two-dimensional gels.     

Coronary sinus of the heart in various anomalies of the conotruncus

Thirty-one hearts with anomalous conotruncus (common arterial trunk, hypoplasia of the aorta with transposition, atresia of the pulmonary trunk) have been studied. There are some peculiarities in anatomy and topography of the coronary sinus, concerning its sources, that is veins forming the venous sinus, position and interrelations with the venous sulcus and with the interatrial septum, size and form of the ostium and valve of the coronary sinus. The most amount of the anatomical peculiarities of the sinus are observed in the preparations, where the anomalous conotruncus is combined with absence of one or both cardiac septa.