Figure 4
MF20 (red) and TUNEL (green) double staining. A: Stage 30, a typical four-chamber view of the AV cushions showing that most apoptotic cells are located in the central area distant to the myocardium. B: Stage 32, most apoptotic cells in the AV cushions are located adjacent to cardiomyocytes, especially in the upper part (arrows), often interspersed between the invading myocardial strips. C: A higher magnification of B. D: Stage 33. This right parasagittal section shows apoptotic cells in the subconal cushions (arrows) adjacent to the cardiomyocytes as well as in the subconal myocardium (arrowhead). E: Four-chamber view of stage 31 shows MF-20–positive apoptotic cells in the upper part of the interventricular septum (asterisk), corresponding to the His bundle region, as well as in the subconal myocardium (arrowhead). F: A stage 29, frontal section across the base of the tubular OT shows apoptosis in the conal regions. Note many MF-20–positive cells are localized in the myocardium of the OT (arrowheads) as well as in the cushions. ra, right atrium; lv, left ventricle; avc, atrioventricular cushions; cc, conal cushions; pa, pulmonary artery; rot, right outflow tract. Scale bars = 200 μm in A–F.
Source publication
To investigate spatial and temporal distributions of apoptosis in the embryonic chick heart and its relation to different tissue types, we examined apoptosis in the embryonic chick heart from Hamburger and Hamilton stage 17 through 3 days after hatching. MF20 antibody, alpha-smooth muscle actin (SMA) antibody and EAP-300 antibody were applied to de...
Contexts in source publication
Context 1
... 28 through 34, when apoptosis was most remarkable in the embryonic chick heart. We found that apoptosis in cardiomyocytes was much less than in cushion tissues, and was located mainly in a few specific regions. The first was the conotruncus region of OFT from stages 27 through 29, which accompanied apoptosis activity in the outflow tract cushions (Fig. 4F). The second was the subconal myocardium at stages 32-34, near the atrioventricular junction areas (Fig. 4D,E). The third was the ventric- ular trabeculae, as well as in the papillary muscles. Cell death detected in the His bundle area and the bundle branches were also MF20 positive (Fig. ...
Context 2
... was much less than in cushion tissues, and was located mainly in a few specific regions. The first was the conotruncus region of OFT from stages 27 through 29, which accompanied apoptosis activity in the outflow tract cushions (Fig. 4F). The second was the subconal myocardium at stages 32-34, near the atrioventricular junction areas (Fig. 4D,E). The third was the ventric- ular trabeculae, as well as in the papillary muscles. Cell death detected in the His bundle area and the bundle branches were also MF20 positive (Fig. ...
Context 3
... apoptosis activity in the outflow tract cushions (Fig. 4F). The second was the subconal myocardium at stages 32-34, near the atrioventricular junction areas (Fig. 4D,E). The third was the ventric- ular trabeculae, as well as in the papillary muscles. Cell death detected in the His bundle area and the bundle branches were also MF20 positive (Fig. ...
Context 4
... cells in cush- ion tissues relative to nearby cardiomyocytes. At early stages (before stage 30), the volume of the atrioventric- ular cushions continues to enlarge to occupy a signifi- cant part of the heart. At this period, apoptosis was located in the central part of the atrioventricular cush- ions, away from cardiomyocytes below and above (Fig. 4A). Later (mainly stages 32-34), the relative volume of cushions shrank as the cardiomyocytes muscularize the atrioventricular regions. At this time, apoptotic cells were often scattered between the invading myo- cardial strips and fibroblast cells adjacent to invading cardiomyocytes in cushions were the major sites of apoptosis (Fig. ...
Context 5
... and above (Fig. 4A). Later (mainly stages 32-34), the relative volume of cushions shrank as the cardiomyocytes muscularize the atrioventricular regions. At this time, apoptotic cells were often scattered between the invading myo- cardial strips and fibroblast cells adjacent to invading cardiomyocytes in cushions were the major sites of apoptosis (Fig. ...
Context 6
... the peak level of apoptosis in the OTCs was a stage earlier than in the AVCs. After that, apoptosis activity in the OTCs decreased as quickly as it built up, approaching background level by stage 33 (Fig. 7A). The decline of apoptotic cells in the AVCs after stage 30 was also very significant but less dramatic compared with that in the OTCs (Fig. 4B), partly due to enlarged volume of AV cushion-derived ...
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Background
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Objective
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Background
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Citations
... 8,47 Morphologically, we noticed gradual relative shortening and in-folding of the AV myocardium (compare Figures 2 and 8) accompanied by epicardial invasion to the AV groove. Apoptosis was implicated in removing some of this myocardium in the chick, 48 and its inhibition resulted in presence of accessory activation pathways. 44 Since the wave of apoptosis is tightly controlled in both spatial and temporal manner, our limited series does not allow is to pronounce a firm judgment whether it is also implicated in the crocodiles (together with epicardial invasion), but we consider it likely. ...
... In reptiles 26,29 including crocodiles, it is present during the entire incubation period, while in the chick embryo, it disappears from the working myocardium well before septation. 48 In the mouse, however, SMA expression in the myocardium continues throughout the fetal development. 71,72 In our recent study, we confirmed SMA myocardial expression pattern similar to crocodiles in axolotl, 73 that is, stronger in the areas of the primary myocardium of the AV canal and the outflow tract. ...
Background
Recent reports confirmed the notion that there exists a rudimentary cardiac conduction system (CCS) in the crocodylian heart, and development of its ventricular part is linked to septation. We thus analyzed myocardial development with the emphasis on the CCS components and vascularization in two different crocodylian species.
Results
Using optical mapping and HNK‐1 immunostaining, pacemaker activity was localized to the right‐sided sinus venosus. The atrioventricular conduction was restricted to dorsal part of the atrioventricular canal. Within the ventricle, the impulse was propagated from base‐to‐apex initially by the trabeculae, later by the ventricular septum, in which strands of HNK‐1 positivity were temporarily observed. Completion of ventricular septation correlated with transition of ventricular epicardial activation pattern to mature apex‐to‐base direction from two periapical foci. Despite a gradual thickening of the ventricular wall, no morphological differentiation of the Purkinje network was observed. Thin‐walled coronary vessels with endothelium positive for QH1 obtained a smooth muscle coat after septation. Intramyocardial vessels were abundant especially in the rapidly thickening left ventricular wall.
Conclusions
Most of the CCS components present in the homeiotherm hearts can be identified in the developing crocodylian heart, with a notable exception of the Purkinje network distinct from the trabeculae carneae.
... Apart from the subepicardially located cTMs, we found another population (CD68+) to be present in endocardial cushions and overlapping with regions of intensive apoptosis. This would indicate that some of embryonic cTMs are also phagocytically active in the area of the outflow tract shortening and valvular remodeling (Cheng et al. 2002;Vicente Steijn et al. 2018). ...
The role of cardiac tissue macrophages (cTMs) during pre- and postnatal developmental stages remains in many aspects unknown. We aimed to characterize cTM populations and their potential functions based on surface markers. Our in situ studies of immunostained cardiac tissue specimens of murine fetuses (from E11to E17) revealed that a significant number of embryonic cTMs (phenotyped by CD45, CD68, CD64, F4/80, CD11b, CD206, Lyve-1) resided mostly in the subepicardial space, not in the entire myocardial wall, as observed in adult individuals. cTMs accompanied newly developed blood and lymphatic vessels adhering to vessel walls by cellular processes. A subpopulation of CD68-positive cells was found to form accumulations in areas of massive apoptosis during the outflow tract remodeling and shortening. Flow cytometry analysis at E14 and E17 stages revealed newly defined three subpopulations:CD64low, CD64highCD206-and CD64highCD206+. The levels of mRNA expression for genes related to regulation of angiogenesis (VEGFa, VEGFb, VEGFc, bFGF), lymphangiogenesis (VEGFc) and extracellular matrix (ECM) remodeling (MMP13, Arg1, Ym1/Chil3, Retlna/FIZZ1) differed among the selected populations and/or embryonic stages. Our results demonstrate a diversity of embryonic cTMs and their tissue-specific locations, suggesting their various potential roles in regulating angiogenesis, lymphangiogenesis and ECM remodeling.
... These cardiac neural crest cells form a condensed pillar in both cushions located in the middle part of the OFT and become dispersed and intermingle with the endocardially-derived mesenchymal cells in the cushions of the proximal part of the OFT [16,[22][23][24]. When the cushions in the proximal OFT are fused, the dispersed neural crest cells enter into apoptosis and the cardiomyocytes of the initially smooth-walled myocardium start to form protrusions into the flanking mesenchyme [16,20,25,26] (Figure 2). While invading the mesenchyme, they never lose their contact with the flanking OFT myocardium [16,[27][28][29]. ...
... The epicardially derived mesenchyme seems to be an unlikely cell source for myocardial differentiation in the context of myocardialization of the OFT as epicardially derived cells arrive after the onset of muscularization in the outlet septum. Neural crest-derived mesenchyme can also be excluded because these cells undergo apoptosis prior to the onset of the muscularization [16,25,37]. Furthermore, the non-myocardial OFT, which is largely derived from cardiac neural crest cells, is not able to form myocardial networks in the in vitro 3D collagen assay [28]. ...
After the formation of the linear heart tube, it becomes divided into right and left components by the process of septation. Relatively late during this process, within the developing outflow tract, the initially mesenchymal outlet septum becomes muscularized as the result of myocardialization. Myocardialization is defined as the process in which existing cardiomyocytes migrate into flanking mesenchyme. Studies using genetically modified mice, as well as experimental approaches using in vitro models, demonstrate that Wnt and TGFβ signaling play an essential role in the regulation of myocardialization. They also show the significance of the interaction between cardiomyocytes, endocardial derived cells, neural crest cells, and the extracellular matrix. Interestingly, Wnt-mediated non-canonical planar cell polarity signaling was found to be a crucial regulator of myocardialization in the outlet septum and Wnt-mediated canonical β-catenin signaling is an essential regulator of the expansion of mesenchymal cells populating the outflow tract cushions.
... One hypothesis suggests that cushion mesenchyme is removed close to the wall, by a process of cell death. High levels of apoptotic cell death have been reported in the developing outflow cushions [84][85][86][87][88] and removal of cushion tissue by cell death in order to refine their shape continues to be an attractive model or leaflet sculpting ( Figure 5). However, apoptosis has not been described in the sculpting arterial valve leaflets, although detailed studies are lacking. ...
Although in many ways the arterial and atrioventricular valves are similar, both being derived for the most part from endocardial cushions, we now know that the arterial valves and their surrounding structures are uniquely dependent on progenitors from both the second heart field (SHF) and neural crest cells (NCC). Here, we will review aspects of arterial valve development, highlighting how our appreciation of NCC and the discovery of the SHF have altered our developmental models. We will highlight areas of research that have been particularly instructive for understanding how the leaflets form and remodel, as well as those with limited or conflicting results. With this background, we will explore how this developmental knowledge can help us to understand human valve malformations, particularly those of the bicuspid aortic valve (BAV). Controversies and the current state of valve genomics will be indicated.
... Apoptosis plays an important part of the AV myocardium remodeling (Cheng et al., 2002), occurring at the time of maturation of the ventricular conduction patterns (Reckova et al., 2003). This results in an apex to base activation of the ventricles, as opposed to an immature base to apex activation at earlier stages (Reckova et al., 2003). ...
... Sampling for optical mapping was performed at ED8, by which time most hearts should show mature ventricular activation pattern (Reckova et al., 2003). Sampling for histology was performed at ED5, ED6, and ED7 (N = 6 per stage), when apoptosis in the AV canal is the most abundant (Cheng et al., 2002). ...
Background:
During heart development, it has been hypothesized that apoptosis of atrioventricular canal myocardium and replacement by fibrous tissue derived from the epicardium are imperative to develop a mature atrioventricular conduction. To test this, apoptosis was blocked using an established caspase inhibitor and epicardial growth was delayed using the experimental epicardial inhibition model, both in chick embryonic hearts.
Results:
Chicken embryonic hearts were either treated with the peptide caspase inhibitor zVAD-fmk by intrapericardial injection in ovo (ED4) or underwent epicardial inhibition (ED2.5). Spontaneously beating embryonic hearts isolated (ED7-ED8) were then stained with voltage-sensitive dye Di-4-ANEPPS and imaged at 0.5-1 kHz. Apoptotic cells were quantified (ED5-ED7) by whole-mount LysoTracker Red and anti-active caspase 3 staining. zVAD-treated hearts showed a significantly increased proportion of immature (base to apex) activation patterns at ED8, including ventricular activation originating from the right atrioventricular junction, a pattern never observed in control hearts. zVAD-treated hearts showed decreased numbers of apoptotic cells in the atrioventricular canal myocardium at ED7. Hearts with delayed epicardial outgrowth showed also increased immature activation patterns at ED7.5 and ED8.5. However, the ventricular activation always originated from the left atrioventricular junction. Histological examination showed no changes in apoptosis rates, but a diminished presence of atrioventricular sulcus tissue compared with controls.
Conclusions:
Apoptosis in the atrioventricular canal myocardium and controlled replacement of this myocardium by epicardially derived HCN4-/Trop1- sulcus tissue are essential determinants of mature ventricular activation pattern. Disruption can lead to persistence of accessory atrioventricular connections, forming a morphological substrate for ventricular pre-excitation. Developmental Dynamics 247:1033-1042, 2018. © 2018 Wiley Periodicals, Inc.
... Apoptosis and proliferation are two necessary morphogenic processes for valve development or disease [41][42][43]. Although, there was only a decrease trend in the protein level of CASP3 and CASP10 in aortic valves of female patients, TUNEL assay indicated reduced apoptotic cells in aortic valves of female RHD patients. In addition, decreased mRNA level of pro-apoptotic genes (BAX and CASP10) was found in aortic valves of female RHD patients, whereas the mRNA level of anti-apoptotic gene, BCL2, was increased. ...
Background
Rheumatic heart disease is an autoimmune disease caused by group A streptococci infection and frequently affects the aortic valve. Sex differences are common in the disease progression, treatment, and outcome. However, little is known about the sex differences in the pathology of aortic valves in rheumatic heart disease.
Design
We studied the end-stage calcific aortic valves from male versus female patients to reveal the sex-dependent pathology differences and molecular changes associated with requiring valve replacement.
Methods
Aortic valves from 39 patients with rheumatic heart disease (19 males and 20 females) were collected at the time of aortic valve replacement for comparative pathology, immunohistochemistry, and gene expression analyses. Clinical characteristics were also analyzed and compared between the two groups.
Results
Aortic valves from female patients exhibited increased expression of collagens, infiltration of monocytes/macrophages and neovascularization. Aortic valves from female patients also had increased expression of inflammatory genes involved in the NFKB pathway (phosphorylated NFKB p65 subunit, IL8, and NOS3) and Th1 cytokine genes (IFNA and IL12B). The severe valve pathology in female patients was correlated with a higher serum level of anti-streptolysin O antibodies.
Conclusion
Inflammation is more prominent in aortic valves of female patients with rheumatic heart disease. This sex difference may contribute to the severe valve pathology and worse outcome of female patients.
... In-situ detection of apoptosis TUNEL assay was done on paraformaldehyde-fixed, paraffin embedded liver sections as described previously [29,30]. Endogenous peroxidase activity was blocked with 2% H 2 O 2 , 5 minutes at room temperature. ...
Hepatic ischemia/reperfusion (l/R) injury continues to be a critical problem. The role of nitric oxide in liver I/R injury is still controversial. This study examines the effect of endothelial nitric oxide synthase (eNOS) over-expression on hepatic function following I/R. Adenovirus expressing human eNOS (Ad-eNOS) was administered by tail vein injection into C57BL/6 mice. Control mice received either adenovirus expressing LacZ or vehicle only. Sixty minutes of total hepatic ischemia was performed 3 days after adenovirus treatment, and mice were sacrificed after 6 or 24 hrs of reperfusion to assess hepatic injury. eNOS over expression caused increased liver injury as evidenced by elevated AST and ALT levels and decreased hepatic ATP content. While necrosis was not pervasive in any group, TUNEL demonstrated significantly increased apoptosis in Ad-eNOS infected livers. Western blotting demonstrated increased levels of protein nitration and upregulation of the pro-apoptotic proteins bax and p53. Our data suggest that over-expression of eNOS is detrimental in the setting of hepatic I/R.
... The myocardium of the atrioventricular canal initially ful fi lls, together with the atrioventricular cushions, valve function in the preseptation heart. In higher vertebrates, the atrioventricular canal myocardium gradually regresses, at least in part through apoptosis (Cheng et al. 2002 ) , and is replaced by fi brous insulating tissue derived from the epicardium (Kolditz et al. 2008 ) . The function of delay generator between the atrium and ventricle is then localized to the atrioventricular node Challice 1977a, 1977b ) . ...
Function of developing heart is underlined by its morphology. Here we focus on development of cardiac pumping function, conduction, and metabolism in higher vertebrates. For simplicity, we distinguish three prenatal stages with different morphology, contraction mechanics, and conduction parameters: tubular heart, trabeculated one, and fully septated heart with coronary perfusion. Postnatal maturation involves namely quantitative changes and appearance of gender differences in parameters like tolerance to ischemia. The straight or looped tubular hearts operate as suction pumps and possess a caudally localized pacemaker and slow, conduction and contraction with a complete occlusion of lumen during systole. With the appearance of atrial and ventricular chambers, the preseptation trabeculated heart shares many similarities with the adult heart, but the same function is achieved by different means. The early embryonic heart is significantly more tolerant to oxygen deprivation than the fetal one. Even after septation, considerable maturation of cardiac morphology and function occurs during fetal and early postnatal period. The principal changes include spiraling of ventricular myoarchitecture, increase in capillary number, more regular arrangement of myofibrils and mitochondria and metabolic switch related to dramatically increased oxygen tension after birth. The newborn heart shows a higher resistance to hypoxia than the adult one, and it cannot be increased by ischemic preconditioning or adaptation to chronic hypoxia. © 2012 Springer Science+Business Media, LLC. All rights are reserved.
... Such macromolecular leaks, much larger than stretch-sensitive ion channels, can occur under normal and extreme physiological conditions and do not always lead to cell death (reviewed Miyake and McNeil, 2003). In light of the location of such leaks near sites of subsequent apoptosis in inner trabeculae in the developing chick (Cheng et al., 2002) and mouse (Barbosky et al., 2006), and strain-induced apoptosis demonstrated with cardiac myocytes in vitro (Cheng et al., 1995), we speculate here that strain-induced cell death may play a mechanistic role in the normal pruning of trabecular and conduction arborizations as the early heart matures. ...
The hypothesis that inner layers of contracting muscular tubes undergo greater strain than concentric outer layers was tested by numerical modeling and by confocal microscopy of strain within the wall of the early chick heart. We modeled the looped heart as a thin muscular shell surrounding an inner layer of sponge-like trabeculae by two methods: calculation within a two-dimensional three-variable lumped model and simulated expansion of a three-dimensional, four-layer mesh of finite elements. Analysis of both models, and correlative microscopy of chamber dimensions, sarcomere spacing, and membrane leaks, indicate a gradient of strain decreasing across the wall from highest strain along inner layers. Prediction of wall thickening during expansion was confirmed by ultrasonography of beating hearts. Degree of stretch determined by radial position may thus contribute to observed patterns of regional myocardial conditioning and slowed proliferation, as well as to the morphogenesis of ventricular trabeculae and conduction fascicles. Developmental Dynamics 238:1535-1546, 2009. (c) 2009 Wiley-Liss, Inc.
... Scale bar 5 100 mm. that apoptosis was similar under hypoxic and control conditions. In comparison between the right and left ventricle we observed a trend toward increased apoptosis in right ventricle in both normoxic (P 5 0.06) and hypoxic condition (P 5 0.002), similar to observations by others (Cheng et al., 2002)5 . Rich vascular plexus in hypoxic hearts fails to make a connection to the aorta. ...
... Expressing the proliferative index in cells per volume of tissue results in less time-consuming cell counting and validation by counting in one group (ED6) also the total number of cell nuclei showed that the values were within the range of 15%–50% of positive myocytes, comparable with our previous studies in the embryonic chick (Sedmera et al., 2002a; deAlmeida et al., 2007). Apoptosis is a rare event in the developing ventricular myocardium and is considered a morphogenetic mechanism used for elimination of no longer needed subpopulations of myocytes in the AV region, outflow tract, and conduction system (Cheng et al., 2002; Sugishita et al., 2004). Unlike in the adult heart, embryonic ventricular cardiomyocytes show remarkable resistance to acute anoxia/reoxygenation (Sedmera et al., 2002b). ...
Freshly isolated quail embryonic heart at Hamburger-Hamilton stage 28, stained with voltage sensitive dye and optically mapped to reveal ventricular activation pattern (indicated by color isochrones in 1 ms intervals). The activation wave sweeps from left to right in the direction of the arrow. Maturation of ventricular activation patterns is accelerated by hypoxic incubation, as described in detail together with other changes in ventricular angio- and myoarchitecture. From "Abnormal Myocardial and coronary Vasculature Development in Experimental Hypoxia," by Ondrej Nanka, et al., on page 1187, in this issue.
