Regulation of apoptosis in the endocardial cushions of the developing chick heart

William M. Keyes and Esmond J. Sanders

Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During the early stages of heart development, there are two main foci of cell death: outflow tract (OT) and atrioventricular (AV) endocardial cushions. These tissues contribute to the septa and valves of the mature heart and receive cell populations from neural crest (NC) cell migration and epicardial cell invasion. We examined embryonic chick hearts for expression, in the cushions, of bcl-2 family members, caspase-9, and the caspase substrate poly(ADP-ribose) polymerase. Antiapoptotic bcl-2 is expressed heavily in the OT and AV regions throughout embryonic days (ED) 4-7, with a decrease in levels at ED 4 and 5 in OT and AV cushions, respectively. Proapoptotic bax predominantly associated with the prongs of the NC-derived aorticopulmonary (AP) septum but was expressed throughout the AV cushions. Proapoptotic bak also associated with the prongs of the AP septum in the OT, while protein levels were upregulated at ED 4-5 and 4-6 in OT and AV cushions, respectively. Bid expression showed a similar time course. We found the 10-kDa cleavage fragment of active caspase-9 at ED 4-8 and 5-8 in OT and AV cushions, respectively, and the 24-kDa cleavage fragment of poly(ADP-ribose) polymerase throughout ED 3-8 and 7-8 in OT and AV cushions, respectively. Caspase-3 cleavage occurred throughout the time period examined. Using cushion cell cultures, we found that inhibitors of caspases-3 and -9 and a universal caspase inhibitor significantly reduced apoptosis, as did retroviral overexpression of bcl-2 using an RCAS expression vector. Premigratory NC cells were fluorescently labeled in vivo with 1,1-didodecyl-3,3,3',3'-tetramethylindocarbocyanine. Subsequent nuclear staining of cushion cells with 4,6-diamidino-2-phenylindole revealed the presence of apoptotic nuclei in the NC cells in the OT cushions and in the prongs of the AP septum. These results demonstrate a developmentally regulated role for the bcl-2 and the caspase families of molecules in the endocardial cushions of the developing heart and lend support to the possibility that some of the dying cells in the cushions are derived from the NC.

heart development; cell death; morphogenesis; bcl-2; caspase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC DEVELOPMENT IS A COMPLEX morphogenetic process that involves the remodeling of a simple tubular structure to form a mature four-chambered organ (4, 17, 38). Among the more complex morphogenetic events are the development of the endocardial cushions in the outflow tract (OT) and atrioventricular (AV) regions (reviewed in Ref. 27) and the invasion of the heart by neural crest (NC) cells to specific sites of remodeling (reviewed in Refs. 5 and 10).

The endocardial cushions are primarily pads of mesenchymal tissue, which are induced to form by an epithelial-to-mesenchymal cell transformation from the endocardium under the influence of myocardial stimulation (9, 26). The cushions contribute to valve development and septal alignment.

The OT is a linear myocardial tube containing paired longitudinal ridges of mesenchymal cushions that will ultimately fuse to separate the initially common lumen into the primitive pulmonary artery and aorta (46, 51). Fusion of the opposing cushions occurs via the caudal growth of the aorticopulmonary (AP) septum, which is primarily of neural crest (NC) origin, and the cranial growth of the conotruncal septum, with the outflow valves forming where these septa meet (18). The OT cushions also fuse with the anterior crest of the interventricular septum and assist in the correct alignment of the ventricular chambers with the major outflow vessels of the heart (17, 45).

The AV cushions initially form as two lateral outgrowths (superior and inferior) that fuse between the common atrium and single ventricle to divide the single lumen into the left and right AV orifices (17). Subsequent fusion and remodeling in the AV region provide the framework for the mitral and tricuspid valves (28, 49) and contribute to the correct septation of the chambers through fusion with the interatrial and interventricular septa (23, 48). Correct alignment and fusion of both sets of cushions are crucial, with many congenital heart defects resulting from improper development of these structures (7, 27).

Another complex morphogenetic event in heart development is the invasion of populations of NC cells to sites of cardiac remodeling (46). These cells originate in the cranial folds of the neural tube between the otic placode and the caudal limit of the third somite (21, 46), and they migrate through the embryo to populate the pharyngeal arches, where they will ultimately contribute to the endothelial lining of the vessels. From the arches, some crest cells continue into the cardiac outflow tract, forming the condensed mesenchyme of the AP septum. This septum grows in a caudal direction, with well-characterized lateral prongs of mesenchyme invading the truncal and conal cushions of the OT, thus facilitating their fusion (46). Below the level of the incipient semilunar valve, the prongs disperse, with only individual crest cells continuing their migration into the conal cushions and myocardium. Some crest cells, by way of subendocardial migration, reach the site of closure of the interventricular septum with the AV cushion and the, by now, partially muscularized conal cushions, which aids in correct separation of the heart chambers. Another population of NC cells reaches the AV cushions and regions of the prospective conduction system by way of the venous pole of the heart (30), where they are thought to play a role in the differentiation of these specialized tissues.

Intrinsic to normal cushion development are a number of phases of apoptotic cell death at specific sites and times of development. It has been shown that two main foci of cell death occur consecutively in the cushions of the OT and the AV regions (19, 29, 31, 43). Although the exact role of these episodes of cell death remains unclear, it has been suggested that some of the apoptotic cells may be derived from the NC (31).

Among the key players in the apoptotic process are the bcl-2 family of regulators (1, 41) and the caspase family of effector enzymes (39). Antiapoptotic members of the family, e.g., bcl-2 and bcl-XL, which have up to four bcl-2 homology domains (BH1, BH2, BH3, and BH4), are normally present on membranes of mitochondria and the endoplasmic reticulum (33). Some proapoptotic members, such as bax and bak (possessing BH1, BH2, and BH3), reside mostly in the cytosol. On receiving stimuli, e.g., increased pH, or dimerization (14, 20), these molecules translocate to the outer mitochondrial membrane (50) and are thought to facilitate the release of cytochrome c via interaction with a voltage-dependent anion channel (35). Other proapoptotic members, e.g., bid and bim (BH3 only), also normally reside in the cytosol, where, on stimulation, they translocate and insert into the mitochondrial membrane, resulting in cytochrome c release, independent of the voltage-dependent anion channel (36). Once in the cytosol, cytochrome c activates the effector family of caspase enzymes (37). There can also be interaction between this mitochondria-dependent pathway and a cell surface death-receptor pathway via the activation of the BH3-only molecule bid, also resulting in caspase activation.

Caspases are a family of cysteine proteases that have been divided into two groups. Initiator caspases, e.g., caspase-8 and caspase-9, when activated, bring about the downstream activation of the second group of caspases, the executors, e.g., caspase-3, caspase-6, and caspase-7 (3). The latter cleave such cellular proteins as poly(ADP)-ribose polymerase (PARP) and DNA fragmentation factor, which results in the characteristic apoptotic demise of the cell.

Previous studies of apoptosis in heart development have relied on morphological assessment and vital dye staining (29) or on labeling of the fragmenting DNA in the nuclei of late-stage apoptotic cells with a nick end-labeling technique such as TdT-mediated dUTP nick end labeling (TUNEL) (19, 31). Here, we examine the mechanisms of cell death in the cushion cells and show that the expression of pro- and antiapoptotic members of the bcl-2 family are developmentally regulated in the AV and OT cushions in such a way as to suggest their involvement in cushion cell death. In addition, we demonstrate active caspase-9 and PARP cleavage in the cushions, implicating the downstream mitochondria-associated death pathway in these cells. Furthermore, in vitro experiments on cultured cushion cells indicate that bcl-2 overexpression and exogenous caspase inhibitors can block the onset of apoptosis in these cells. Finally, fluorescent labeling of migratory NC cells and assessment of their nuclear morphology in the heart lend support to the contention that some of the dying cells in the cushions may be NC in origin.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chick embryos. Fertilized White Leghorn hens' eggs were incubated at 37°C for 4-8 days, and the resulting embryos were staged according to the Hamburger and Hamilton (HH) staging system (16). The embryos were removed and washed in Tyrode saline. For immunocytochemistry, the heads were removed and the embryos were immersed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at 4°C for 4-15 h, depending on the stage. After they were fixed, the embryos were washed in PBS and stored at 4°C. For Western blotting and cell culture, the hearts were removed to Tyrode solution on ice. With the use of electrolytically sharpened tungsten needles, the AV endocardial cushions and the entire OT were dissected free from the heart without the use of digesting enzymes.

Immunocytochemistry. After they were fixed, the embryos were washed in PBS, dehydrated through a graded series of ethanol, and cleared in Hemo-De (Fisher Scientific). The embryos were then embedded in paraffin wax, sectioned at 8 µm, and mounted on glass slides. Sections were cleared in Hemo-De (twice for 10 min each), rehydrated in graded ethanol, and washed in double-distilled water (DDW). To quench endogenous peroxidase, sections were treated with 0.3% H2O2 in DDW for 30 min and then washed in DDW. Sections were blocked in 10% goat serum with 0.5% Tween 20 (Fisher Scientific) for 1 h at room temperature. Excess solution was removed, and sections were incubated with primary antibodies diluted in 1% goat serum at 4°C overnight. The primary antibodies bcl-2 (N-19, 1:200), bax (I-19, 1:200), and bak (G-23 1:100) were obtained from Santa Cruz Biotechnology. Sections were washed three times for 5 min each in PBS and then incubated with a biotinylated secondary antibody (goat anti-rabbit IgG; Vector Laboratories) at a dilution of 1:200 in 1% goat serum for 1 h at room temperature. After they were washed again three times for 5 min each in PBS, sections were incubated with the Vectastain ABC reagent (Vector Laboratories) according to the manufacturer's instructions for 1 h at room temperature. After another wash, sections were stained using 3,3'-diaminobenzidine (Sigma) with ammonium nickel sulfate. Sections were washed in PBS, dehydrated through graded ethanol, cleared in Hemo-De, and mounted with Permount (Fisher Scientific). Negative controls consisted of preincubating the primary antibody with the supplier's blocking peptide and resulted in absence of staining in all cases.

Polyacrylamide gel electrophoresis and Western blotting. The dissected AV endocardial cushions, the OT, and cultured cells were homogenized in protease inhibitor buffer containing 15 µg/ml aprotinin, 1 µg/ml leupeptin, 5 µg/ml pepstatin, and 1.74 mg/ml phenylmethylsulfonyl fluoride, and the protein concentration was determined using the Bio-Rad protein assay. Samples were loaded at 10-15 µg/lane and run on a 10% polyacrylamide gel for 45 min. The separated proteins were transferred to nitrocellulose membranes at 100 V for 2 h. The membranes were stained initially with Ponceau S to ensure even loading of lanes and a successful transfer. The membranes were then subjected to blocking in 5% skimmed milk in Tris-buffered saline + Tween 20 (TTBS) for 1 h at room temperature. The membranes were probed with the following primary antibodies in 5% skimmed milk in TTBS overnight at 4°C: Bcl-2 B46620 monoclonal (1:200; Transduction Laboratories), Bax B-9 monoclonal (1:50; Santa Cruz Biotechnologies), Bak Ab-2 monoclonal (1:50; Oncogene Research Products), Bid goat polyclonal (1:50; R & D Systems), PARP A-20 goat polyclonal (1:50; Santa Cruz Biotechnologies), caspase-3 rabbit polyclonal (1:500; Stress-Gen Biotechnologies), and activated caspase-9 AAP-109 rabbit polyclonal (1:500; Stress-Gen Biotechnologies). The membranes were washed for 3-5 min in 5% skimmed milk and probed with biotinylated secondary antibodies at dilutions of 1:1,000-1:2,000 for 1.5 h. After another washing step, the membranes were incubated with the Vectastain ABC kit for 1.5 h, washed again, and developed using enhanced chemiluminescence reagent (ECL, Amersham). The gels represent the result of a minimum of three repetitions of each experiment on different samples.

Dissociated primary cell culture. Primary cell cultures were made of dissociated dissected AV cushions. Briefly, AV cushions were dissected to ice-cold Tyrode solution from HH stage 24 hearts (2 dozen/experiment) using electrolytically sharpened tungsten needles. Cells were then dissociated in 0.2% trypsin-EDTA (Sigma) in calcium/magnesium-free Tyrode solution for 10 min at 37°C. Trypsinization was stopped with the addition of 1 ml of medium 199 with 10% fetal bovine serum (FBS; GIBCO BRL). Cells were spun in a bench-top centrifuge for 5 min, and the supernatant was discarded. The cells were washed in 1 ml of medium 199 without serum and recentrifuged for 3 min. Cells were then resuspended in 100 µl of complete medium (medium 199 with 10% FBS and 1:1,000 gentamicin), counted using a hemocytometer slide, and resuspended in complete medium at a final volume of 100 µl/coverslip. Coverslips were prepared by coating with 1 mg/ml type I rat tail collagen (GIBCO BRL; dissolved in 0.5% acetic acid and resuspended 1:20 with 60% ethanol, with NaOH added to a final concentration of 15 mM) and exposed to ultraviolet light overnight. Once seeded, cultures were incubated overnight at 37°C in a 5% CO2 incubator. Cultures were given fresh complete medium every 2nd day for 4-6 days. Serum-starved cultures were treated in the same way, with FBS absent from the medium.

Retroviral infections. Retroviral vectors were produced as described by Logan and Francis-West (25). Briefly, pRCASBP(B) and pRCASBP(B)/bcl-2 (supplied by Dr. S. H. Hughes, Frederick Cancer Research and Development Center, Frederick, MD) were transfected into primary cultures of line 0 chick embryo fibroblast (CEF) that had been produced from specific pathogen-free eggs (Hyvac, Adel, IA). Cultures were expanded for 7-10 days. For viral collection, on the second-last day of culture, when cells were reaching confluency, medium was replaced at one-half volume with medium plus 2% serum overnight. The supernatant was collected and concentrated by ultracentrifugation at 25,000 rpm at 4°C for 2.5 h. The supernatant was carefully removed, and the remaining pellet was resuspended in 100 µl of Optimem (GIBCO BRL), which was then frozen in aliquots at -70°C. Controls included staining transfected CEFs for the human bcl-2 transcript and the p19 viral coat protein, as well as immunoblotting for bcl-2. Viral titer was obtained by serial dilution and infection of CEFs for 48 h. Cells were immunostained for the viral coat protein, and the number of infectious virions was calculated as 5.0 × 107/ml. Dissociated primary cushion cultures were prepared as described above and grown under serum-starved conditions. Cells were infected with virus after the first overnight incubation. The initial 100 µl of medium were removed and replaced with 100 µl of fresh complete medium containing 5 µl of concentrated virus with 8 µg/ml polybrene. No other virus was added during the subsequent medium changes, but fresh polybrene was included in each medium change.

Immunocytochemistry on cultures. Primary cell cultures were prepared as described above. For mitochondrial labeling, 5 µl of 10 µM MitoTracker red CMXRos (Molecular Probes) were added to 1 ml of medium. Cultures were incubated in this reagent for 45 min at 37°C, washed in warm Tyrode solution, and fixed with 4% buffered paraformaldehyde for 45 min at room temperature. Cultures were washed three times for 5 min each in PBS and treated with blocking solution of 10% goat serum in PBS with 0.5% Tween 20 for 30 min at room temperature. Primary antibodies were the same as those described above for immunocytochemistry: bcl-2 at 1:200 and the monoclonal AMV-3C2 antiviral coat protein antibody supernatant (University of Iowa Developmental Studies Hybridoma Bank) overnight at 4°C in 1% goat serum with 0.5% Tween 20. Cells were then washed three times for 5 min each in PBS. Biotinylated secondary antibodies were used at 1:200 for 1 h at room temperature and then washed three times in PBS. Cells were then fluorescently labeled with streptavidin-FITC (Calbiochem) at 1:200 for 1 h at room temperature in the dark. The cultures were washed again three times for 5 min each in PBS and treated with 4,6-diamidino-2-phenylindole (DAPI) for 4 min at room temperature. Cells were washed three times for 5 min each in PBS and mounted on slides with Vectashield mounting medium. Specimens were examined using a Zeiss LSM510 confocal microscope equipped with argon, helium-neon, and ultraviolet lasers.

TUNEL. Staining was performed on fixed primary cultures. The cells were washed in PBS after they were fixed and pretreated with TdT buffer (30 mM Trizma base, 140 mM sodium cacodylate, 1 mM cobalt chloride, pH 7.2) for 5 min. Cultures were then incubated with the reaction mixture for 1.5 h at 37°C in a humid chamber using the kit from Roche Molecular Biochemicals at a volume of 20 µl per culture [100-µl volume: 81 µl of DDW, 6.5 µl of TdT buffer, 3.26 µl of cobalt chloride, 1.86 µl of biotin-16-dUTP stock (1 nmol/l), 5.5 µl of dUTP, and 2 µl of TdT (10 U/µl)]. Cultures were then washed 3 times for 5 min each in PBS and blocked with 3% skimmed milk in PBS with 0.5% Tween 20 for 30 min. Cultures were then washed three times for 5 min each in PBS, and the fluorochrome streptavidin-FITC was added at a concentration of 1:200 for 1 h at room temperature. Cultures were again washed three times for 5 min each in PBS and treated with DAPI in PBS for 4 min. After more washes, the coverslips were mounted on slides with Vectashield and imaged digitally. The total number of TUNEL-positive cells was counted in each culture and analyzed statistically using ANOVA and Tukey's multiple comparison post test on five replicates of each treatment.

When TUNEL was combined with immunocytochemistry for bax, the TUNEL procedure was carried out first followed by immunocytochemistry, and the two secondary antibodies were applied simultaneously in mixed solution.

Caspase inhibitors. Primary dissociated cushion cultures were prepared as described above and serum starved to induce apoptosis. The cultures were then treated with specific peptide caspase inhibitors to prevent cell death. The peptide caspase inhibitors caspase-3 inhibitor II (Z-DEVD-FMK) and caspase-9 inhibitor I (Z-LEHD-FMK), both obtained from Calbiochem, were used. A universal caspase inhibitor, BOC-Asp(OME)-FMK (Enzyme Systems Products, Livermore, CA), was also used to inhibit all caspases. All inhibitors were dissolved in DMSO at a concentration of 50 mM, aliquoted, and stored at -20°C. Caspase inhibitors were added to fresh medium 199 without serum, which was added to cultures every other day at a final concentration of 50 µM. After 4-5 days, cells were washed in PBS and fixed in 4% paraformaldehyde in PBS and stained with TUNEL and DAPI.

DiI labeling of NC cells. To label premigratory NC cells, in ovo microinjection of the fluorescent lipophilic dye 1,1-didodecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI; Molecular Probes) was used (34). The dye was prepared by dissolving 3 mg of DiI in 0.1 ml of 100% ethanol. This was then diluted with 1.1 ml of 3% BSA in PBS (2). Eggs were windowed, and a Picospritzer (General Valve) was used to inject the dye into the lumen of the neural tube at HH stages 9-11. Subblastodermal injection of India ink was used as a contrast agent. After injection, the eggs were sealed with Scotch tape and reincubated for 3-4 days. After this time, the embryos were dissected and the heads were removed from the body. Specimens were then fixed in 4% paraformaldehyde in 0.1 M PBS overnight at 4°C and embedded in OCT compound. The specimens were then frozen and sectioned at 10 µm. The frozen sections were stained with DAPI for 4 min at room temperature and mounted with Vectashield mounting medium. The slides were examined using a Zeiss LSM510 confocal microscope equipped with argon, helium-neon, and ultraviolet lasers.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Immunocytochemistry of bcl-2 family molecules. Sections of the developing heart containing the endocardial cushions were stained for various members of the bcl-2 family at times when peak cell death is observed in the cushions (19). At ED 4, in the distal OT (Fig. 1, A and B), bcl-2 protein was absent from the condensed mesenchyme of the AP septum but was present in individual endocardial cushion cells in the loose mesenchyme. In the more proximal OT cushions (Fig. 1, C and D), the protein was expressed in the majority of cushion cells. A similar pattern was seen in the AV cushions at ED 6 and 4 (Fig. 1, E and F, respectively), with high bcl-2 expression in most cushion cells. In these and in all the following immunocytochemical results, negative controls in which the antibody was preabsorbed with antigen showed no staining.


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Fig. 1.   Immunoreactivity for bcl-2 at the time of peak cell death in the atrioventricular (AV) and outflow tract (OT) cushions. A and B: distal OT on embryonic day (ED) 4 showing relatively low levels of immunoreactivity in the condensed mesenchyme of the aorticopulmonary (AP) septum (aps) and higher levels in the surrounding cushion cells (ec) and myocardium (m). C and D: proximal OT cushions on ED 4 showing widespread immunoreactivity for bcl-2 protein in the cushions (ec) and myocardium (m). E and F: AV cushions at early (ED 4) and later (ED 6) stages, respectively, showing immunoreactivity in most cushion cells. Negative controls using antibodies preabsorbed with the control peptide (not shown) resulted in absence of staining in all cases. Scale bars, 100 µm (A, C, and E), 50 µm (B), and 20 µm (D and F).

Heart sections were also stained for bax, a proapoptotic family member. At ED 4, in the distal OT (Fig. 2A), bax protein was not abundant in the core of the condensed mesenchyme of the AP septum but was more highly immunoreactive in some of the adjacent cushion and myocardial cells and at the edges of the condensed mass (arrow). Bax was also seen in the fusing cushions of the distal OT, adjacent to the AP septum (Fig. 2B). In the more proximal OT cushions (Fig. 2, C and D), bax expression appeared to associate strongly with the prongs of the AP septum that protrude into the endocardial cushions but was also present in individual cells in the surrounding cushions and in the myocardium. In the AV cushions (Fig. 2E), bax immunoreactivity appeared to be scattered widely throughout the cells of the central cushion mass at ED 6, when peak cell death is occurring. TUNEL-bax double labeling (Fig. 2F) showed that bax was expressed in the cytoplasm of all apoptotic cells.


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Fig. 2.   Immunoreactivity for bax at the time of peak cell death in the cushions. A and B: positive immunoreactivity in the distal OT (ED 4), with lower levels of bax protein in the condensed core mesenchyme of the AP septum (aps) and higher expression in adjacent cushion cells (arrow) and myocardium (m). Boxed region of A is shown in B, where bax immunoreactivity can be seen in the cells at the point of cushion fusion. C and D: in the more proximal OT (ED 4), bax appears to be highly immunoreactive in the prongs of the AP septum and is scattered throughout the cells of the cushions (ec) and myocardium (m). E: expression of bax protein is widespread throughout developing AV cushions (ED 6). Negative controls (not shown) using antibodies preabsorbed with the blocking peptide resulted in absence of staining in all cases. F: confocal image of a section through the AV cushions double-labeled with TdT-mediated dUTP nick end labeling (TUNEL) for apoptotic nuclei (green) and immunocytochemistry for bax (red cytoplasm). Bax is present in the cytoplasm of most cells in the AV cushions and in the cytoplasm of all apoptotic cells. Scale bars, 100 µm (A, C, and E) and 20 µm (B and D).

Immunoreactivity for another proapoptotic family member, bak, was also examined in sections at times when peak cell death is occurring. In the OT, bak appeared to localize intensely to the prongs of the AP septum (Fig. 3, A and B) and was also present in some parts of the myocardium and the loose mesenchyme of the cushions. Bak also appeared to have a distribution in the AV cushions (Fig. 3C), corresponding to regions that normally show extensive apoptosis.


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Fig. 3.   Immunoreactivity for bak at the time of peak cell death in the AV and OT cushions. A and B: bak protein expression in some regions of the OT cushions (ec) and myocardium (m) at ED 4. Strong expression can be seen in the prongs of the AP septum (aps). C: bak expression in the AV cushions (ED 6) showing little immunoreactivity in the myocardium compared with the cushions (ec). Negative controls (not shown) using the antibody preabsorbed with the blocking peptide resulted in an absence of staining in all cases. Scale bars, 100 µm (A), 20 µm (B), and 50 µm (C).

Western blotting for Bcl-2 family, caspase-9, caspase-3, and PARP. The antibody to bcl-2 revealed the 26-kDa band of bcl-2 throughout the time course examined in the AV and OT cushions at ED 4-7 (Fig. 4A). A decrease in protein expression was consistently seen at ED 5 in the AV region, and when repeated blots were densitometrically measured and analyzed, this decrease was found to be significant (P < 0.05; data not shown). This decrease corresponds with, or slightly precedes, the time of peak cell death in these regions that we showed previously. In the OT, expression at 26 kDa consistently decreased at ED 4. The antibody also consistently recognized a ~23-kDa band, which corresponds to another bcl-2 isoform of uncertain significance (40). The positive controls (Jurkat cells) showed a 26-kDa band, as expected, and often the 23-kDa form, but at much lower levels than in the embryonic tissue, suggesting the possible involvement of this isoform in developing tissue.


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Fig. 4.   Western blot on dissected AV cushions and whole OT probed for members of the bcl-2 family through ED 4-8. A: levels of bcl-2 protein at 26 kDa show a decrease at ED 5 in the AV cushions and at ED 4 in the OT. Significance of the 23-kDa isoform is unknown. B: bax protein is consistently expressed throughout the time course examined in AV cushions and the OT at ED 4-8. C: bak protein expression appears to be upregulated at ED 4-6 in AV cushions and at ED 4-5 in the OT. D: bid protein at 18 kDa becomes upregulated between ED 4 and 7 in AV cushions and is expressed throughout this period in OT cushions. +, Jurkat cell positive control; WE, positive control using lysate from the whole embryo.

In similar samples, a cross-reactive monoclonal antibody for the proapoptotic molecule, bax, revealed constitutive expression of the 23-kDa protein in the AV and OT regions (Fig. 4B) at levels similar to the whole embryo positive controls throughout the time course examined. Bax is known to be constitutively present in many tissues and becomes proapoptotic on activation.

The proapoptotic protein bak was also present. The 28-kDa protein was consistently upregulated in the AV cushions at ED 4-6 and in the OT region at ED 4-5 (Fig. 4C). These times correspond to peak cell death in both regions, implicating bak as a proapoptotic signal in this tissue.

The 18-kDa protein, bid, indicative of the death-receptor pathway, was also expressed in these tissues during this embryonic period (Fig. 4D). In the AV cushions, bid was most highly expressed at ED 6 and 7, while in the OT cushions it appeared throughout ED 4-7.

The characteristic demise of the apoptotic cell is brought about by cleavage of specific substrates by members of the caspase family of enzymes. Immunoblotting of the AV and OT regions revealed the presence of the 10-kDa cleavage fragment of active caspase-9 throughout the time course examined (Fig. 5A), providing evidence for involvement of this initiator caspase in these tissues. Evidence for the downstream activation of caspase-3 was also obtained in the AV and OT cushions between ED 4 and 7 by detection of the 12-kDa cleavage fragment of this molecule throughout this period (Fig. 5B).


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Fig. 5.   Western blot on dissected AV cushions and whole OT probed for activation cleavage fragments of caspase-9 and caspase-3. A: immunoblotting reveals the presence of the 10-kDa cleavage fragment at ED 5-8 in AV cushions, with a slightly extended expression in the OT at ED 4-8. Expression of the cleavage fragment is more intense in the cushions than in the lysate of the whole embryo (WE). B: 12-kDa cleavage fragment of activated caspase-3 is detectable in AV and OT cushions throughout ED 4-7.

One known substrate of the caspase family, which is characteristically cleaved during apoptosis, is the DNA repair enzyme PARP. Immunoblotting for this protein revealed the 24-kDa cleavage fragment to be dramatically upregulated at ED 7-8 in the AV cushions and to be present throughout ED 3-8 in the OT (Fig. 6), suggesting that the apoptotic characteristics of these cells are caspase dependent.


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Fig. 6.   Western blot on dissected AV cushions and whole OT probed for poly(ADP-ribose) polymerase (PARP). Immunoblotting reveals that expression of the 24-kDa PARP cleavage fragment is dramatically upregulated in the AV cushions at ED 7-8 only, while it is present throughout the time course examined in the complete OT (ED 3-8). In the negative control (PAb), preabsorption of the antibody with control peptide resulted in complete abolition of the 24-kDa band.

Inhibition of apoptosis in AV cushion cell cultures by caspase inhibitors. Primary cultures of dissociated AV cushions were made and were induced to undergo apoptosis by serum withdrawal. Peptide inhibitors of caspase-3 and caspase-9 and a universal caspase inhibitor were added to the cultures. After this treatment, nuclei were stained with DAPI (Fig. 7A), and apoptotic nuclei were labeled with the TUNEL technique (Fig. 7B). The total number of nuclei and the number of TUNEL-positive nuclei were then counted. Figure 7C shows the effects of the inhibitors on the number of TUNEL-labeled nuclei in each treatment. Each treatment significantly inhibited cell death in these cultures, with the universal and caspase-9 inhibitors being more effective than the inhibitor of the farther-downstream caspase-3.


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Fig. 7.   Primary cultures of dissociated AV cushion cells were serum-starved and treated with caspase peptide inhibitors to assess the role of caspases in the apoptosis of these cells. A and B: 4,6-diamidino-2-phenylindole (DAPI, blue) labeling of nuclei and TUNEL (green), respectively, of apoptotic nuclei in the same field of view. Condensed nuclei labeled by DAPI also label with TUNEL, confirming that they are apoptotic. C: significant reduction in the number of TUNEL-positive nuclei in cultures treated with a universal caspase inhibitor (Univ CI), caspase-3 inhibitor (C3I), and caspase-9 inhibitor (C9I) compared with untreated serum-starved cells (Ctrl). n = 5 for each treatment.

Inhibition of apoptosis in AV cushion cell cultures by bcl-2 overexpression. As described above, primary cultures of dissociated AV cushions were made and induced to undergo apoptosis by serum withdrawal. The cultures were infected with the replication-competent retrovirus RCASBP(B), carrying the human bcl-2 transcript insert or no insert as a negative control. Cultures were then incubated for a further 5 days. The cultures were stained with DAPI to label all the nuclei and TUNEL to show the apoptotic nuclei. Cultures were also stained (green) with the monoclonal antibody AMV-3C2 to immunolabel the viral coat protein, to confirm the infection efficiency, or for the overexpressed bcl-2 protein. Uninfected control cells were negative for viral coat protein (not shown) and contained endogenous bcl-2, as reflected by faint green fluorescence (Fig. 8A). All cells in the infected cultures were positive for bcl-2 overexpression, as seen by increased green fluorescence (Fig. 8B), and for viral coat protein (Fig. 8C). Because bcl-2 protein is associated with mitochondria, cultures were labeled with MitoTracker red. Unlike the endogenous bcl-2, the overexpressed bcl-2 was seen to partially colocalize with mitochondria (Fig. 8B, yellow). Figure 8D shows the number of TUNEL-positive cells in each treatment. Infection with the bcl-2-carrying virus significantly inhibited apoptosis compared with the untreated control cultures and cultures infected with the negative insert control. Bcl-2 overexpression by RCAS/bcl-2 was also shown to abolish caspase-9 and caspase-3 cleavage in cultured cells (Fig. 8E) compared with cultures treated with the negative insert RCAS/(-). Untreated cultures showed a background level of caspase-9 cleavage but no detectable caspase-3 cleavage, in contrast to uncultured OT cushion tissue, in which cleavage of both caspases was detectable (Fig. 8E).


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Fig. 8.   Primary cultures of dissociated AV cushion cells were serum starved to induce apoptosis, and bcl-2 protein was retrovirally overexpressed. A: combined confocal image of cells stained with DAPI (blue) and MitoTracker (red) and bcl-2 immunoreactivity (green) showing low levels of endogenous bcl-2 in uninfected control cells. B: increased green fluoresence in cultures infected with RCASBP(B)/bcl-2 virus, immunolabeled for bcl-2 protein. Colocalization of overexpressed bcl-2 with mitochondria is indicated by yellow. C: same as B, but stained for viral coat protein (green). D: effect of infection of cushion cells with RCASBP(B)/bcl-2 virus and negative control RCASBP(B)/(-) virus on the number of TUNEL-positive cells in culture. Bcl-2 infection significantly inhibits apoptosis compared with uninfected control (Ctrl) and negative-insert control (n = 5 for each treatment). E: Western blots for cleavage fragments of caspase-9 (at 10 kDa) and caspase-3 (at 12 kDa) in cultured cushion cells. Lane 1, cells treated with RCAS/bcl-2; lane 2, cells treated with RCAS/(-); lane 3, untreated cultures in medium + fetal bovine serum; lane 4, uncultured cushion tissue from OT.

DiI labeling of NC cells. Premigratory NC cells were labeled by injection of the lipophilic dye DiI into the lumen of the neural tube. This procedure permanently labels all cells that were in contact with the lumen at the time of injection, including the premigratory NC cells (Fig. 9A). The cardiac NC cells enter the distal OT by way of the pharnygeal arches and form the AP septum, which facilitates division of the OT. Our results confirm the presence of NC cells in the septum of the distal OT (Fig. 9B, arrows). Sections through the cushions were stained with DAPI to identify whether the nuclei of the DiI-labeled cells were apoptotic. In the OT cushions, many apoptotic cells were seen, as evidenced by their characteristically condensed and fragmenting nuclei. Some of these dying cells were seen to be carrying the DiI label (Fig. 9C), indicating that NC cells were indeed dying in the environment of the cushions. Dying NC cells were also seen in the prongs of the AP septum (Fig. 9D), but we saw no overlap of DiI labeling and apoptotic cells in the condensed mesenchyme of the AP septum (not shown). These findings further support the contention that some NC cells undergo apoptosis in the cushions and distal tips of the AP septum.


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Fig. 9.   1,1-didodecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) and DAPI labeling of cardiac neural crest (NC) cells. A: neural tube and NC-derived structures are DiI-positive after taking up injected dye. NC cells have migrated to the dorsal root ganglia (arrow) and the subectodermal melanocytes (arrowhead) as expected. B: DiI-positive cardiac NC cells (red) in the septum dividing the distal OT (arrowhead) and the endothelial layers of the great vessels (arrows). C: DAPI-labeled nuclei in the OT cushions (blue). Shrunken or fragmenting apoptotic nuclei are seen in some of the NC cells carrying the red fluorescent label (arrows). D: same as C; apoptotic NC cells in the prongs of the aorticopulmonary septum (arrows) are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interest in the mechanisms and significance of episodes of apoptosis in heart development has dramatically increased in recent years (11, 19, 32, 43). Much of this recent work has concentrated on the distribution and occurrence of apoptosis in the developing heart, with a reassessment of the detailed work of Pexieder (29) using more modern techniques. These techniques indicate a more localized distribution of dying cells than previously thought, with a smaller number of main foci of cell death than Pexieder described. There seems to be little cell death in atrial and ventricular tissue in very early development, while the AV and the OT cushions seem to be the principal foci, with the largest numbers of dying cells during ED 4-8 (19, 31). OT myocytes have also been shown to be eliminated in large numbers during this time (47), and some NC cells undergo apoptosis in the AV region after entering the heart through its venous pole (30). A large number of dying cells were also reported in the superior aspect of the interventricular septum, at the site of its fusion with the atrial septum and the OT septum (11, 43). This area includes the sites of formation of the AV node, the bundle of His, and the left and right bundle branches. This site is the final destination of some of the cardiac NC cells. However, understanding of the regulation of the apoptotic process or the exact developmental role of cell death in these tissues is rudimentary.

In this study, we demonstrate the involvement in the endocardial cushions of the main, and best-characterized, regulators and effectors of cell death pathways. We also lend support to the idea that some of the apoptotic cells in the endocardial cushions may be of NC origin. Abnormalities in the development of the endocardial cushions and OT are implicated in many life-threatening congenital heart defects, which arise from structural defects or impaired alignment (e.g., tetralogy of Fallot, persistent truncus arteriosus, double-outlet right ventricle, and ventricular septal defects). An understanding of the molecular aspects of programmed cell death in these areas will be essential to understanding the etiology of some of these defects.

Members of the bcl-2 and caspase families are involved in regulation of apoptosis in endocardial cushions of the developing heart. The suggested involvement of the bcl-2 family of cell death regulators in heart development arises from inconclusive results using knockout mice or mRNA localization in heart extracts (reviewed in Ref. 43). Some evidence for caspase involvement in heart development has also been demonstrated by deletion of the caspase-8 gene in mice, which results in, among other features, impaired ventricular musculature (44). Disruption of the upstream receptor linked to caspase-8, FADD/MORT1 (53, 54), results in a similar phenotype, as does inactivation of casper (c-FLIP), the upstream inhibitor of this pathway (52). These gene knockouts do not, however, appear to affect cushion development, and it has therefore been suggested that these molecules may act in a cell death-independent manner (52). Also, some evidence for caspase-3 activity has been found by Watanabe et al. (47), who demonstrated the activity of this enzyme in homogenates of whole chick OT.

Here we show that antiapoptotic bcl-2 and proapoptotic bax, bak, and bid proteins are present in the endocardial cushions of the heart. Bcl-2 is present throughout the main phase of apoptosis (ED 4-8), with a transient downregulation of the protein at ED 4 in the OT cushions and at ED 5 in the AV cushions, corresponding with times of elevated cell death in these tissues. Bcl-2 acts in a cytoprotective manner in cells by interacting with, and hindering, the function of proapoptotic stimuli (41). The proapoptotic molecule bak appears to be upregulated at the time of the highest incidence of cell death in the cushions, further implicating members of this family in regulating cell death in these tissues. By contrast, levels of the proapoptotic bax protein appear to remain constant throughout the time course examined. Such constant levels of bax during episodes of apoptosis have been reported previously in other tissues (50). Bid, a proapoptotic molecule linking the death-receptor pathway with the mitochondrial pathway, is also upregulated in the cushions at times of high levels of cell death, implying that the receptor-mediated cell death pathway may also be involved in apoptosis in these tissues.

The proapoptotic bax and bak appear to associate predominantly with the prongs of the differentiating AP septum. However, the previously shown distribution patterns of apoptotic cells appear not to overlap exactly with the pattern of these proapoptotic molecules (19). One possible explanation for this is that the proapoptotic members may not be immediately active at this site. Inactive bax and bak are often normally found to reside in the cytosol of healthy cells. On receiving an appropriate signal, these molecules are activated, and they translocate to the mitochondria, where they contribute to the downstream apoptotic signaling pathways (15). The antibodies used in the present study do not distinguish between the inactive and active forms of these molecules. Our results show that bax is present throughout the cushions and that it is expressed in the cytoplasm of all apoptotic cells. It is possible that migrating and morphogenetically active cells express inactive proapoptotic molecules during the early development of the heart, notably in the endocardial cushions, and at the time of differentiation they receive signals that bring about translocation and activation of the proapoptotic molecules.

We also show that bcl-2 overexpression can protect primary cultures of cushion cells from serum starvation-induced apoptosis and concomitantly abolishes caspase-9 and caspase-3 activation. Bcl-2 has been shown to protect numerous cell types from a variety of insults. One interesting observation in our culture model was the fact that when the serum-starved cells were stained using the TUNEL technique, only 3-5% of the cells in the cultures were ever apoptotic at any one time (data not shown), even though bcl-2 seems to be present in the majority of the cells. This suggests that it is a specific population of cells in these cultures that is dying. Whether these are the NC-derived cells remains to be determined.

Western blotting of the dissected AV cushions and the OT provides evidence of caspase activation in these tissues. The caspase enzymes are the downstream effectors of cell death that bring about the demise of the cell by cleaving and inactivating cellular proteins necessary to maintain cellular homeostasis or by activating other cell death proteins (39). Different pathways of caspase activation may be present, with processes being receptor mediated or mitochondria initiated. Here we show evidence of activation of one of the upstream mitochondria-associated caspases and also a downstream effector caspase. Caspase-9 normally resides in an inactive state and is activated via the release of cytochrome c from the mitochondria. Activation results in cleavage of caspase-9 by a cytochrome c-Apaf complex (24), which then activates the downstream caspases, such as caspase-3 (37). The antibody used in this study recognized the 10-kDa cleavage fragment characteristic of activated caspase-9. We also provide evidence for the downstream activation of caspase-3 uniformly throughout the period examined. The activation of these two caspases does not always directly reflect the levels of expression of molecules of the bcl-2 family. However, proapoptotic bax and the caspase cleavage fragments are found in the cushions throughout ED 4-7. Further evidence of caspase activity in the endocardial cushion cells is demonstrated by the in vitro studies on primary cultures of dissociated cushions. An inhibitor of the upstream mitochondrial pathway-associated caspase-9 was as effective as a universal inhibitor, while an inhibitor of the downstream caspase-3 was significant but less effective, further suggesting the probable involvement of other caspases downstream from caspase-9.

A further hallmark of caspase activity is the cleavage of PARP, a DNA repair enzyme. In dying cells, this protein is cleaved and inactivated from a 113-kDa molecule to 89- and 24-kDa cleavage products (8). PARP cleavage is seen throughout ED 3-8 in the OT, which may reflect the prolonged time course of apoptosis in the OT myocardium as well as the slightly earlier phase in the cushions. In the dissected AV cushions, PARP was consistently cleaved during ED 7-8, which closely follows the phase of peak cell death. The cleavage of PARP did not directly correlate with the apparent activation of caspase-9 and caspase-3. This points to the likelihood that PARP cleavage is associated with the activity of factors other than caspase-3, such as inhibitors of apoptosis (13) or caspase-7 (12), or with caspase-independent mechanisms, such as apoptosis-inducing factor (6).

Another question that needs to be addressed is whether similar mechanisms of cell death are occurring in both sets of cushions. The derivation of the endocardial cushions in the AV and OT regions appears to be similar (27), and a number of our findings would suggest that similar apoptotic pathways are in effect in both sets of cushions. Bcl-2 appears to be downregulated at the onset times of peak cell death in both areas, whereas the proapoptotic bak appears to be upregulated in each area. Immunoblotting also shows the cleavage fragment of active caspase-9 in both sets of cushions. Evidence of PARP cleavage in the AV cushions and the OT further supports the idea that it is indeed apoptosis that is occurring in the cushions of the heart and that similar processes are in effect in both regions.

Are the dying cells of NC origin? The pathways of NC cell migration have been well studied, and the cardiac contribution of NC cells is the focus of much ongoing attention (5, 18, 45). There is debate as to whether the apoptotic cells observed during cushion morphogenesis are of NC origin. Retroviral and TUNEL labeling of NC cells (31) suggests that some of the apoptotic cells in the conal cushions of the OT may be crest derived. However, quail chick chimeras imply a fate of differentiation as opposed to apoptosis for most NC cells (42, 46). Furthermore, the mismatch of timing of invasion and arrival at their final destination of migrating NC cells, with the distribution and numbers of apoptotic cells, further adds to the complexity. Probably, some NC cells are eliminated by apoptosis (31), but whether it is the final fate of the majority of them remains to be shown conclusively.

Here we provide further evidence that NC cells in the OT cushions and in the prongs of the AP septum undergo apoptosis. The colocalization of DiI labeling with the fragmenting nuclei, characteristic of apoptotic cells, suggests that, for at least some NC cells, cell death is the final fate. This is further supported by the conspicuous association of the proapoptotic bax and bak proteins with the prongs of the AP septum, which is the NC-derived mesenchyme that invades the endocardial cushions. Immunoreactivity for these proteins was much lower in the condensed mesenchyme of the AP septum. The significance of this is uncertain, but it is known that the prongs of the AP septum are located in an area that will become muscularized, as opposed to the mesenchymal fate of the condensed septum itself (32). Poelmann et al. (31) contend that only a subpopulation of NC cells in the distal tips of the prongs undergoes apoptosis, and it is this view that best fits our results. However, the patterns of apoptosis shown by us previously (19) do not exactly overlap with NC distribution. This would suggest a more complex process, in which the dying cells are not exclusively NC cells. In NC-ablated embryos, the cushions of the OT fail to fuse, resulting in a heart defect known as persistent truncus arteriosus (22, 46), among other phenotypic changes, depending on the completeness of ablation. However, it remains to be seen whether levels of apoptosis in the developing heart are affected in NC-ablated embryos.

What is the role of apoptosis in the endocardial cushions? One prevailing view is that the apoptotic cells in the cushions are important for the process of muscularization of this tissue, which occurs during the differentiation of the cushions into the valve leaflets (30). It has also been suggested that these myocardializing apoptotic cells are derived from the NC (31). During cushion differentiation, myocardial cells invade the mesenchymal tissue by migration from the adjacent muscle layer (42, 51). The timing of this muscle cell migration follows the period of peak cell death in the cushions and also corresponds with the timing of invasion of NC cells. Poelmann and Gittenberger-de Groot (30) postulate that apoptotic NC cells may release "molecules" that stimulate myocardialization of the valves and septa. However, there is little evidence of apoptotic cells acting in such a signaling manner. Similar roles have also been postulated for some of the other apoptotic populations of cells in the heart. For example, apoptosis of the NC cells that enter the heart via the venous pole is thought to induce the final differentiation of cardiomyocytes into the specialized conduction system (30). Our results suggest that the apoptotic cells in the developing heart are not exclusively of NC origin and that it is premature to ascribe a central role for crest-derived cell death in cushion morphogenesis.


    ACKNOWLEDGEMENTS

The authors thank Dr. C. Logan for valuable assistance with the retroviral work and E. Parker and D. Lee for assistance.


    FOOTNOTES

This work was funded by an operating grant to E. J. Sanders and a studentship to W. M. Keyes from the Canadian Institutes of Health Research and a studentship from the Alberta Heritage Foundation for Medical Research.

Address for reprint requests and other correspondence: E. J. Sanders, Dept. of Physiology, University of Alberta, Edmonton, AB, Canada T6G 2H7 (E-mail: esmond.sanders{at}ualberta.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 23, 2002;10.1152/ajpcell.00509.2001

Received 25 October 2001; accepted in final form 18 January 2002.


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