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Keywords:

  • reactive oxygen species;
  • hydrogen peroxide;
  • Caspase;
  • cell injury

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

This study tested the hypothesis that the remodeling of the cardiac outflow tract (OFT) may represent a developmental window of vulnerability to reactive oxygen species (ROS). Chick embryos were exposed in ovo or ex ovo to increasing concentrations of the stable oxidant hydrogen peroxide (H2O2). As assessed by trypan blue staining, H2O2 induced cell injury in the stage 25–30 OFT at concentrations as low as 1 nM. Higher concentrations were required to induce cell injury in the ventricular and atrial myocardium. Using DCFDA as an indicator of oxidant stress, H2O2 also induced a greater fluorescent signal in the OFT myocardium. H2O2 at these low concentrations also induced Caspase activity, indicative of activation of the pathway of PCD. Interestingly, the induction of Caspase-3 activity was predominately in the OFT cushion mesenchymal cells. Thus, the developing OFT is particularly sensitive to ROS-mediated injury, suggesting that ROS could play a role in the development of congenital defects of the cardiac OFT. Developmental Dynamics 236:3496–3502, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

An obligate consequence of oxygen utilization in aerobic metabolism is the generation of reactive oxygen species (ROS) that can be toxic to the cells in which they are generated (Chance et al.,1979). A number of mechanisms have evolved to defend against the potentially damaging effects of ROS, including reducing sinks such as glutathione, and enzymes such as glutathione peroxidase (Gpx), superoxide dismutase (SOD), catalase, and peroxiredoxins, which catalyze the conversion of ROS to harmless end-products (reviewed in Fridovich,1999). The roles of ROS generation and de-toxification in a number of disease states, such as ischemia-reperfusion injury, ageing, and oxygen toxicity, have been extensively studied in mature animals (Martindale and Holbrook,2002; Li and Jackson,2002; Chandra et al.,2000).

The development of the embryo represents a unique situation with regard to oxygen consumption and the disposal of its metabolic by-products (reviewed in Burggren and Reiber,2007; Fisher and Burggren,2007). In eutherian mammals, the placenta develops as the gas exchanger through which oxygen diffuses from the maternal to the embryonic circulation, while in birds it is the chorio-allantois through which oxygen diffuses from the air to the embryonic circulation. These specialized structures develop in parallel with the developing embryo, and even when fully developed, are not as efficient as the lungs of the mature animal for delivering oxygen to the tissues. In addition, the mixing of oxygenated and de-oxygenated blood in the embryo, and differences in blood hemoglobin concentrations and activities, in sum, leads to much lower rates of oxygen delivery to the tissues of the developing avian and mammalian embryo as compared to the mature animal.

The rate of generation of ROS is proportional to the concentration of oxygen in the tissues, though other factors, such as metabolic rate, may also play a role (reviewed in Turrens,2003). Cells defend themselves against potentially toxic ROS via the induction of the de-toxifying enzymes such as SOD, catalase, Gpx, and preoxiredoxins. It has been proposed that the embryo may represent a state in which oxygen concentrations, ROS generation, and anti-oxidant defenses are generally low, and susceptibility to injury induced by changes in oxygen concentration or the generation of ROS is high (reviewed in Fantel,1996). However, the role of oxygen utilization and oxidative stress in the development or mal-development of specific structures within and beyond the heart and vascular system is still poorly understood.

The embryonic cardiac outflow tract (OFT) connects the developing ventricles with the aortic sac and undergoes dramatic remodeling in the transition to the dual series circulation of birds and mammals (reviewed in Sugishita et al.,2004c). Using the nitroimidazole EF5 and the transcriptional regulatory protein HIF-1α as surrogate indicators of oxygen concentrations, we have suggested that the avian OFT myocardium is relatively hypoxic during its remodeling in the transition to a dual circulation (Sugishita et al.,2004a,b). The reason for this relative myocardial hypoxia is thought to be the large diffusion distance created by the adjacent thick mesenchymal cushions at a stage of development prior to the establishment of the coronary circulation, so that oxygen delivery is diffusion-limited. The utilization of oxygen, generation of ROS, and development of ROS defense systems are thought to have evolved and developed in a co-dependent fashion. This leads to the hypothesis that the hypoxia-dependent remodeling of the OFT from Hamburger-Hamilton stages 25–32 (Hamburger and Hamilton,1951) (ED5-8) may represent a window of particular vulnerability to ROS. To test this hypothesis, chick embryos were exposed in ovo or ex ovo to increasing concentrations of the stable oxidant hydrogen peroxide (H2O2). The OFT myocardium appeared particularly sensitive to the oxidizing effect of H2O2 as measured by DCFDA fluorescence and trypan blue exclusion as an indicator of cell injury. These observations in this animal model raise the possibility that the relatively high prevalence of congenital cardiac OFT defects is due to a particular susceptibility to oxidant stress–mediated teratogenesis during the transition to a dual circulation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

H2O2 Exposures Ex Ovo

Stage 25–30 chick embryos were first incubated in H2O2 ex ovo so that the concentration of H2O2 to which the tissues were exposed could be precisely set. Embryos were freshly dissected and placed in individual wells of a 12–24 well plate containing either PBS or H2O2. Embryos incubated in PBS for up to 3 hr showed no induction of trypan blue staining (Fig. 1a) or Caspase activity (data not shown). Thus incubation of the embryos ex ovo in and of itself did not induce cell injury within this time frame. Embryos were incubated in a 1,000-fold range of concentrations of H2O2 (1 nM to 1 μM) for 30 min. In stage-25 embryos, at the lowest concentration of H2O2 tested, 1 nM, trypan blue staining was observed in the distal OFT and the ventricular apex (Fig. 1b, n=13). At the higher concentration of 5 nM, the entire OFT myocardium was strongly trypan blue positive (Fig. 1c, n=12). The apical region of the ventricles was again positive as was the superior aspect of the atria. At the higher concentrations of 100 nM and 1 μM H2O2, the OFT, atrial, and ventricular myocardium all were strongly positive for trypan blue staining (Fig. 1d and data not shown, n=7 at each concentration). Most of the remainder of the embryo was negative or weakly positive for trypan blue staining under these conditions of H2O2 exposure. Embryos at stage 28–30 showed similar patterns of trypan blue staining in the H2O2 dose-response experiments (n=18 at Stage 28, n= 9 at stage 30, data not shown). In separate experiments, the ventricular apex was incised and H2O2 solution infused into the ventricular chamber so that both luminal and epicardial surfaces were bathed in H2O2. A similar pattern of trypan blue staining was observed (Stage 25, n=6, data not shown).

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Figure 1. Trypan blue staining of hearts in embryos incubated with increasing concentrations of H202 ex ovo. Stage-25 embryos were incubated ex ovo over a wide range of H2O2 concentrations for 30 min prior to staining with trypan blue as described in the Experimental Procedures section. Images were captured with a Leica DMB stereomicroscope and Spot RT digital camera.A: A control embryo incubated in PBS shows no trypan blue staining.B: An embryo incubated in 1 nM H2O2 shows trypan blue staining of the distal OFT and the ventricular apex (arrows). C: An embryo incubated in 5nM H2O2 shows more intense trypan blue staining over the entire myocardial portion of the OFT and lighter staining of the ventricular apical region (arrow). The tips of the atria are also positive (arrow). D: At substantially higher concentrations of H2O2 (100 nM), the entire heart stains positive for trypan blue. OFT, outflow tract; vent, ventricles.

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H2O2 Exposures In Ovo

Since removal of the embryo from the egg could alter its sensitivity to H2O2-induced stress, we next tested the effect of injection of H2O2 into the pericardial space of the embryo in the intact egg. H2O2 was mixed with a PBS-glycerol solution and injected via a micro-pipette into the pericardial space. We have previously used this method for the delivery of recombinant adenovirus and various compounds to the developing heart (Fisher and Watanabe,1996; Watanabe et al.,2001). Control embryos received the glycerol-PBS solution alone. Injection of H2O2 to give an estimated final concentration of 10 nM H2O2 caused significant cell injury in the OFT of the stage-25 embryo (Fig. 2a, control; b, H2O2, n=7 each). Trypan blue–positive cells could also be seen extending along a blood vessel at the base of the OFT (small arrows). An antibody against myosin (MF-20) was used to identify the myocardium in sections of the heart (Fig. 2c). At higher magnification, it is evident that the trypan blue–positive cells are myocardial cells (Fig. 2d). The dose-response to H2O2 in ovo was similar to that in ex ovo, with minimal trypan blue staining at 1 nM H2O2 (n=3) and trypan blue staining throughout the atria, ventricles, and OFT at concentrations of 1 μM H2O2 (n=8, data not shown).

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Figure 2. Trypan blue staining of hearts in embryos incubated with increasing concentrations of H202 in ovo. Serial dilutions of H2O2 in 50% glycerol were injected into the pericardial space of Stage-25 embryos as described in the Experimental Procedures section. One hour later embryos were stained with trypan blue and imaged as described above and in the Experimental Procedures section. A: A control embryo injected with vehicle shows no trypan blue staining. B: An embryo injected with H2O2 to achieve an approximate concentration of 10 nM in the pericardial space shows staining of the myocardial portion of the OFT (arrow) and also around a blood vessel at the base of the OFT (double arrows). C: The H2O2 heart was sectioned and stained with the MF20 (anti-myosin) antibody to identify the myocardium. The boxed area is shown at higher magnification in D. D: Under combined fluorescent and white light illumination, the trypan blue–positive cells co-localize with MF-20 staining, identifying the injured cells as cardiomyocytes.

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Measures of Oxidant Stress

The oxidant-sensitive fluorophore DCFDA was used to define the relationship between H2O2 exposure and oxidant stress in the target tissues. Embryos were loaded with DCFDA ex ovo prior to exposure to H2O2 as described in the Experimental Procedures section. Control stage-25 embryos incubated in PBS showed minimal fluorescence. There was faint fluorescence that was above background at the base of the OFT and in the distal OFT (Fig. 3a, n=3). This could represent production of endogenous oxidants in these tissues. At concentrations of 1–10 nM H2O2, a strong fluorescent signal was induced rather selectively from cells in the myocardial portion of the cardiac OFT (Fig. 3b, n=3 at each concentration). At higher concentrations, 100 nM–10 μM H2O2, DCFDA fluorescence was induced throughout the heart (Fig. 3c, n=3 at each concentration). The fluorescence from the OFT was greater than that from the ventricular or atrial myocardium at these higher concentrations of H2O2. Pre-incubation of the embryos with Tempol at 10 mM, an SOD-mimetic free radical scavenger, completely blocked the induction of DCFDA fluorescence by H2O2 (Fig. 3d). Pre-incubation of the embryos with Tempol at 2 mM had a partial effect. Tempol also significantly reduced the trypan blue staining in the heart (not shown). This indicates that the observed fluorescence was mediated by the oxidation of DCFDA by H2O2 diffusing into the tissues, and thus an indicator of oxidant stress in these tissues.

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Figure 3. DCFDA as an indicator of sites of oxidative stress in control and H2O2 treated stage-25 embryos. Embryos were loaded ex ovo with DCFDA for 30 min, then incubated in PBS (control) or a wide range of H2O2 concentrations. Images were captured with a DMB stereomicroscope and Spot RT camera immediately after H2O2 exposure. A: A control embryo incubated in PBS shows slight DCFDA fluorescence at the base of the OFT and the distal OFT (arrows). The bright dots on the surface of the ventricle are artifacts from residual DCFDA. B: Upon exposure to 10 nM H2O2, bright fluorescence is observed over the entire myocardial portion of the OFT. Scattered fluorescence is also observed towards the ventricular apex and at the atrial apices, in a pattern consistent with the trypan blue staining. C: Exposure to higher concentrations of H2O2 (10 μM) induced fluorescence throughout the heart. D: Pre-treatment of the embryo with the free radical scavenger Tempol blocked the fluorescence upon exposure to 10 μM H2O2. The images in C and D were acquired with the same exposure times.

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Pathway of H2O2-Induced Cell Death

Caspase activity was measured to determine if hydrogen peroxide exposure may activate the pathway of programmed cell death. There was no difference in Caspase activities in cardiac homogenates from embryos incubated in PBS ex ovo for 1 hr as compared to embryos freshly removed from the egg (data not shown). Incubation of the stage-28 embryo in 10 nM H2O2 increased the Caspase-3,7 activity in the OFT homogenates approximately 10-fold (Table 1). Incubation in 10 μM H2O2 resulted in a similar increase in Caspase activity. Caspase-3,7 activity was not induced by 10 nM H2O2 in the ventricle but was increased by exposure to 10 μM H2O2, though the activity was still significantly less than that of the OFT. Interestingly, immunostaining against activated Caspase-3 indicated that most of the increase in Caspase activity in the H2O2-treated embryo was in the cushion mesenchyme (Fig. 4). There was no increase in staining with a second indicator of apoptosis, the lysosomal marker LysoTrackerRed (data not shown), perhaps due to the short time course of the experiments.

Table 1. Caspase-3,7 Activity Induced by H2O2 Exposure
TreatmentRLU/ μg proteinFold change vs. control
  • *

    P < 0.05 vs. control.

Outflow Tract  
 Control1,279 ± 290
 10 nM H2O212,723 ± 3,384*+10.0
 10 μM H2O214,768 ± 4,055*+11.5
Ventricle  
 Control655 ± 260
 10 nM H2O2985 ± 739+1.5
 10 μM H2O21,498 ± 900*+2.3
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Figure 4. Staining for active Caspase-3 in control and H2O2 treated embryos. Stage-25 embryos were incubated ex ovo in (A) PBS or (B) 10 nM H2O2 for 30 min, processed and stained with an antibody that specifically recognizes active Caspase-3 as described in the Experimental Procedures section. There is increased staining for active Caspase-3 in the OFT cushion mesenchyme (arrows) as compared to the control embryo, and to a lesser extent in the OFT myocardium (lines). This figure is representative of three control and three H2O2-treated embryos.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

This study shows that the embryonic cardiac OFT is highly sensitive to ROS during a critical phase of its morphogenesis. The embryos were exposed to fixed concentrations of the stable cell permeant oxidant H2O2. This removed the generation of ROS as a variable so as to specifically test sensitivity to ROS. This represents an artificial system, but one that is commonly used experimentally to test the role of ROS in various biological processes (reviewed in Chandra et al.,2000). The embryonic OFT is sensitive to H2O2 in the nanomolar concentration range. The OFT myocardium was considerably more sensitive to H2O2 than was the ventricular or atrial myocardium in the same embryo, and also significantly more sensitive than post-natal and mature cardiomyocytes, where more than 1,000-fold higher concentrations of H2O2 are required for cell killing (Kwon et al.,2003). While it is possible that part of this difference stems from differences in the experimental methods, for example the ability of H2O2 to diffuse into the target cells, it is unlikely that this could account for such a large difference in sensitivity to H2O2.

That the OFT myocardium is sensitive to H2O2 in the nanomolar range is significant for several reasons. First, this is close to the range of the estimates of normal H2O2 concentrations in respiring cells (Chance et al.,1979). It is thus more likely that this concentration would be achieved under physiological or pathological conditions, as compared to the sub-millimolar concentrations used in other experimental systems. Second, the effects of oxidant stress, as assessed by H2O2 exposure, are highly dependent upon the concentration and duration of exposure (Antunes et al.,2001; Antunes and Cadenas,2001; Kurata,2000; reviewed in Forman et al.,2004). At low concentrations, H2O2 may induce reversible oxidative modification of proteins involved in various signaling pathways, for example MAP kinases and Akt, that regulate cell death and cell survival (Kwon et al.,2003; Finkel,2003). H2O2/oxidation may also induce diverse transcriptional stress responses, via AP-1, Nf-kB, p53, and other factors, depending on the concentration (Bozonet et al.,2005), though the short time course used in the current study would limit the contribution of a transcriptional response. At higher concentrations, H2O2 may cause irreversible modifications of proteins and programmed cell death, while at the highest concentrations necrotic cell death ensues from cell shock. Our finding of cell injury induced by H2O2 at nanomolar concentrations in the OFT myocardium suggests that this may be triggered by a specific signaling pathway. Interestingly, H2O2 induced cell injury in the OFT myocardium and Caspase activation in the OFT mesenchyme. This suggests that either (1) different pathways were activated in the two different cell types or (2) signaling from the myocardium to the mesenchymal cells, as is required for endothelial-to-mesenchymal differentiation (Eisenberg and Markwald,1995), also affects cell survival. The definition of the pathway(s) that lead to cell death upon oxidant exposure in the developing OFT requires further study.

The induction of DCFDA fluorescence and cell injury at low concentrations of H2O2 suggest that the OFT myocardium is less able to de-toxify ROS than mature cardiomyocytes, and thus is more susceptible to oxidant-induced injury. The de-toxification of H2O2 is dependent upon the reducing enzyme glutathione peroxidase (Gpx), which catalyzes the oxidation of the abundant intra-cellular peptide glutathione (present in millimolar concentration), and the enzyme Catalase, which catalyzes the reaction of two H2O2 to form water and oxygen. If not de-toxified, H2O2 reacts with transition metals such as iron or copper to form highly reactive hydroxyl radicals. In general, expression of anti-oxidants such as Catalase and Superoxide Dismutase is generally lower in the embryo or fetus as compared to adult tissues; however, each organ may display unique profiles of anti-oxidant expression during development (Harris et al.,2003; de Haan et al.,1994; Fantel et al.,1995; Baek et al.,2005; Kobayashi et al.,2001; Venturini and Sparber,2001; Surai,1999). Some of the anti-oxidant genes appeared to be induced at ED10 of mouse development (approximately co-incident with ED5-6 of chick development), the period when oxygen delivery to the tissues and oxygen and substrate utilization by the tissues, particularly the working heart, are increasing (reviewed in Fisher and Burggren,2007). These tissues may be in a critical balance, where oxygen supplies are barely sufficient to meet demands, or in the case of the chicken Stage 25–32 OFT myocardium, insufficient, resulting in tissue hypoxia. At the same time, because there has been little endogenous generation of ROS, there is low expression of the anti-oxidant genes, which are induced by ROS through the Nrf transcription factors (Scarpulla,2002; Dhakshinamoorthy et al.,2000). This scenario would leave these tissues vulnerable to ROS, which may be generated in this critical developmental window as oxygen utilization increases. Some support for this model comes from gene inactivation studies in the mouse. Compound inactivation of Nrf1 and Nrf2 genes results in embryos that are non-viable at embryonic days 9–10 and exhibit elevated ROS, extensive apoptosis, and induction of Noxa, a death effector p53 target gene (Leung et al.,2003). Inactivation of the anti-oxidant genes Thioredoxin-2 (Nonn et al.,2003) and Glutathione peroxidase-4 (Yant et al.,2003) also results in demise of the embryo around this stage of development. Whether ROS produced during development may be toxic and teratogenic, or alternatively may play a critical role in morphogenesis, as suggested for cell death-dependent remodeling of limb mesenchyme (Schnabel et al.,2006), requires further study.

Could the generation of ROS play a role in the development of congenital heart defects? We tested a number of in ovo H2O2 exposure regimens, but were not able to find any that were compatible with the survival of the embryo and caused structural defects in the OFT after prolonged incubation. This may reflect a limitation of the experimental method of injection of H2O2 into the pericardial space, and more experimentation is indicated. Other studies in rodent and avian models have suggested that the teratogenicity of heavy metals (e.g., Arsenite) and organic chemicals such as ethanol and methanol, metabolic conditions such as diabetes, and drug exposures such as phenytoin and hydroxyurea, may in part be due to the primary or secondary generation of ROS (Boyer et al.,2000; Fantel and Person,2002; Fantel et al.,1992; Venturini and Sparber,2001), though detailed cell and molecular mechanisms are generally lacking (reviewed in Jenkins et al.,2007). Both neural crest cells, required for the septation of the OFT, and cushion mesenchymal cells are sensitive to ROS-mediated injury induced by retinoids and ethanol (Davis et al.,1990a,b) and trichloroethylene (Boyer et al.,2000), respectively. Studies in mice have suggested a two-hit model: mice with targeted genetic defects may be phenotypically normal but more sensitive to teratogens. Mice lacking one copy of p53, a DNA repair and tumor suppressor gene, are 2–4-fold more sensitive to the teratogenic effects of the oxidizing chemical benzo(a)pyrene (Nicol et al.,1995). Similarly the teratogenic effects of drugs/chemicals such as diphenylhydantoin, arsenite, and 2-Nitrosofluorene may be experimentally modulated by altering oxygen concentrations or addition/depletion of anti-oxidants (glutathione, Superoxide Dismutase, Catalase), in experiments generally done in rat whole embryo culture (Millicovsky and Johnston,1981; Winn and Wells,1995). Further study of genetic susceptibility and environmental exposures during critical morphogenetic developmental windows should provide new insights into the cell and molecular bases of congenital conotruncal heart defects. This information may allow for preventive strategies to reduce the incidence of these life-threatening defects in the human population.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Exposure to Hydrogen Peroxide Ex Ovo

Chicken embryos were exposed to a wide range of concentrations of hydrogen peroxide both in ovo and ex ovo. Embryos from HH stages 25–30 were used, encompassing the developmental window of hypoxia- and apoptosis-dependent remodeling of the cardiac outflow tract (OFT). Dose-response studies were performed at concentrations from 1 nM to 1 μM H2O2. The stock solution of H2O2 (30% H2O2, Fisher Scientific) was serially diluted in PBS and placed into a well containing a freshly dissected intact chick embryo. Embryos were incubated in H2O2 at 38°C for 10–30 min. Control embryos were incubated under identical conditions in PBS alone. Embryos were then processed for indicators of cell injury and oxidant stress as described below.

Exposure to Hydrogen Peroxide In Ovo

H2O2 was serially diluted in 50% glycerol in PBS to increase the viscosity of the solution. A portion of the shell was removed from the Stage-25 egg and under stereomicroscopic guidance a hole was cut in the membrane surrounding the heart. Then 0.5 μl of H2O2 containing solution was injected into the pericardial space. The volume of the pericardial space was estimated to be approximately 5 μl. H2O2 solutions were made to give estimated final concentrations after injection into the epicardial space that were similar to those in the ex ovo experiments (1 nM to 1 μM). Eggs were returned to the incubator for 30 min to 1 hr and then processed for indicators of cell injury and oxidant stress as described below.

Indicators of Cell Injury

Permeability to trypan blue was used as an indicator of cell injury. After exposure to H2O2, embryos were incubated in trypan blue by standard methods. In brief, each embryo was incubated in a well containing 4 ml of 0.3% trypan blue in PBS (GibcoBRL) for 15 min. The embryos were then washed for 10 min in PBS in each of 3 successive wells before fixation in 10% formalin and further washing. These steps were all performed at room temperature with rocking. Embryos were observed in whole mount using a Leica DMB stereomicroscope and images captured with a Spot RT digital camera. Selected embryos were then embedded in paraffin, sectioned, and co-stained with an antibody directed against cardiac myosin (MF20) to delineate the myocardium, as previously described (Sugishita et al.,2004a). For the detection of active Caspase-3 by immunohistochemistry, paraformaldehyde-fixed embryos were dehydrated in serial concentrations of sucrose, embedded in OCT, and 7–10-μ-thick sections generated in a sagittal plane. The slides were blocked in PBS with 5% normal goat serum, and then incubated with anti-active Caspase 3 antibody (R&D Systems Inc, Minneapolis, MN) for 1 hr at room temperature followed by PBS washing. The slides were incubated with a fluorescein conjugated secondary antibody and viewed with a Leica FLIII microscope. Images were captured with a Spot RT digital camera. Caspase-3/7 activity was measured using its peptide recognition sequence DEVD conjugated to a bioluminescent probe (Lumiglo, Promega) according to the manufacturer's instructions and our previous protocol with minor modifications. In brief, after incubation of embryos in H2O2 or PBS ex ovo for 30 min, OFT and ventricular tissues were dissected directly into 200 μl of lysis solution (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100) on ice and immediately triturated. After the collection was complete, the samples were sonicated, aliquoted, and frozen at −80°C. Depending on the stage, 6–12 hearts were used for each sample, and three separate collections were performed. Twenty-five microliters of each sample were mixed with 25 μl of the reaction mix containing substrate in a 96-well plate. After a 1-hr room incubation at room temperature, luminescence was measured for 1 sec per well (Wallac Trilux Microbeta 1450 luminometer). All samples were measured in duplicate and the intra-sample variation was less than 10%. As negative controls, selected samples were boiled or spiked with the competitive inhibitor DEVD-CHO. Activity in these samples was similar to background luminescence (data not shown). Protein concentrations were measured using the BCA method (Pierce) and Caspase-3/7 activity expressed as RLU (relative luminescent units)/μg protein.

Indicators of Oxidant Stress

The fluorophore 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein di-acetate, acetyl ester (CM-H2DCFDA, Molecular Probes) was used as an indicator of oxidant stress. These indicators are cell permeant and become “trapped” within the cell after cleavage of the lipophilic blocking groups by intra-cellular esterases. These compounds fluoresce when oxidized to di-chlorofluorescein and thus are commonly used indicators of oxidative stress. The DCFDA was re-constituted in DMSO as a stock concentration of 10 mM. The stock concentration of DCFDA was diluted into PBS immediately before use so that the final concentration of DMSO did not exceed 0.1%. Pilot experiments were performed to determine the optimum conditions for loading of the embryo with DCFDA ex ovo. In all subsequent experiments, embryos were incubated in 2 ml of 10 μM DCFDA for 30 min at 37°C and washed three times with PBS prior to incubation in H2O2 or PBS. Embryos were then exposed to varying concentrations of H2O2 as described above. DCFDA fluorescence was examined in whole mount immediately after exposure to H2O2 with the Leica DMB stereomicroscope and images captured with the Spot RT digital camera. In order to show that DCFDA fluorescence after H2O2 exposure was indeed an indicator of oxidant stress, embryos were pre-incubated with the oxidant scavenger 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol, Sigma Aldrich) at 2 or 10 mM at 37°C for 30 minutes, washed, and then loaded with DCFDA and exposed to H2O2.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

I thank David Leifer, Michael Payne, and Dr. Hongbin Liu for technical assistance with these studies.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES