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J Thorac Cardiovasc Surg 2003;125:1268-1275
© 2003 The American Association for Thoracic Surgery
Surgery for Congenital Heart Disease |
From the Departments of Surgery,a Pediatrics,b Biochemistry,c and Internal Medicine,d the University of Iowa College of Medicine, Iowa City, Iowa.
Supported in part by a Scientist Development Grant from the American Heart Association (C.A.C.), a grant from the National Institutes of Health (RO1 HO64770; C.A.C., T.D.S., and J.L.S.), a Grant In Aid from the American Heart Association (W.G.L.), and a grant from the Children's Miracle Network (C.A.C.).
Received for publication June 16, 2002. Revisions requested July 23, 2002; revisions received Aug 12, 2002. Accepted for publication Sept 13, 2002. Address for reprints: Christopher A. Caldarone, MD, Division of Cardiovascular Surgery, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada, (E-mail: christopher.caldarone{at}sickkids.ca).
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1-5 Apoptosis is distinct from necrosis in that the cell is dismantled by a family of specific proteases, the caspases, and endonucleases. Early in apoptosis, myocardial cells become dysfunctional because of mitochondrial changes and derangement of structural and functional proteins. 6 They might remain in this dysfunctional state for a variable period or proceed to cleavage of genomic DNA and segmentation of the cell into apoptotic bodies, which are endocytosed by neighboring cells.
Although myocardial apoptosis has been thought of as the result of pathologic processes, it has also been shown to be a normal component of fetal cardiac development and early postnatal maturation. 7,8 Within the neonatal period, expression of proapoptotic signals is increased in the myocardium, 9 suggesting a greater susceptibility to apoptosis. It has recently been suggested that apoptosis is initiated during cardioplegic arrest in diseased adult hearts and might play a role in postoperative myocardial stunning. 10 We therefore hypothesize that in a clinically relevant model of neonatal cardiac surgery, a brief period of hypothermic cardioplegic arrest will result in increased apoptotic cell death and activation of apoptotic signaling pathways.
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General anesthesia was maintained for 6 hours after reperfusion, during which time arterial blood gases, electrolytes, and hematocrit levels were maintained in a physiologic range, and maternal blood or crystalloid solution was infused as necessary to maintain arterial pressure. Inotropic agents were not administered. After 6 hours, the heart was quickly excised and perfused through the coronary arteries with 240 mL of ice-cold phosphate-buffered saline solution. Full-thickness, 1-cm2 myocardial samples for histologic study were taken from the center of the left ventricular free wall, frozen in OCT (Sakui, Torrance, Calif) on Dry Ice, and then stored at -80°C until sectioning or stored in 10% neutral (phosphate)-buffered formalin at room temperature. The remainder of the left ventricular free wall was minced and snap-frozen in liquid nitrogen. Nonoperated control lambs (n = 5) were anesthetized in a similar manner, and the hearts were immediately excised and processed as above but without a 6-hour delay. Cardiopulmonary bypass-only control lambs (n = 5) were started on cardiopulmonary bypass in the empty beating state without cardioplegic arrest. After 90 minutes, they were weaned from bypass, decannulated, and maintained for 6 hours before the harvest of myocardial tissue.
All procedures were performed within the regulations of the Animal Welfare Act and the "National Institutes of Health Guide for the Care and Use of Laboratory Animals" and were approved by the University of Iowa Animal Care and Use Committee.
In situ TdT-mediated dUTP-digoxigenin nick end-labeling
In situ TdT-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was performed with a modification of the technique described by Olivetti and colleagues, 2 using the ApopTag In Situ kit (Intergen, Norcross, Ga). In brief, 5-µm cryostat sections were fixed with freshly prepared 1% paraformaldehyde in phosphate-buffered saline solution at room temperature for 10 minutes, preincubated with equilibration buffer for 5 minutes, and subsequently incubated with deoxyribonucleotidyl transferase in the presence of digoxigenin-conjugated dUTP for 1 hour at 37°C. The reaction was terminated by incubating the samples in stopping buffer and then with the fluorescein-labeled anti-digoxigenin antibody (yellow-green). Cell nuclei were counterstained with 4',6-Diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, Calif). Ten random high-power fields representing approximately 4000 cells were counted to enumerate TUNEL-positive cells. Formalin-fixed and paraffin-embedded 5-µm sections were stained with mouse monoclonal immunoglobulin M (IgM) anti-
-sarcomeric actin antibody (Sigma, St Louis, Mo) and Alexa Fluor 594-conjugated goat anti-mouse IgM (Molecular Probes, Eugene, Ore) and then TUNEL stained as above to identify cell type. Adjacent sections were stained with hematoxylin and eosin and examined by means of light microscopy for evidence of necrosis.
DNA electrophoresis
Snap-frozen myocardial samples were digested in lysis buffer (urea, 4 mol/L; Tris HCl, 200 mmol/L; NaCl, 20 mmol/L; and ethylenediamine tetraacetic acid [EDTA], 200 mmol/L [pH 7.4]) with proteinase K (3.3 mg/mL) purified with Miniprep columns (Roche, Indianapolis, Ind), according to the manufacturer's instructions. Equal masses of DNA were then digested with DNAse-free RNAse (Roche), radiolabeled with phosphorous 32-labeled deoxycytidine triphosphate (Amersham, Sunneyvale, Calif) and Klenow (New England Biolabs, Beverly, Mass), and purified over Sephadex G-50 (Amersham). Electrophoresis was performed in 1.8% agarose with EtBr at 5 V/cm for 2 hours. The gels were photographed with UV light to confirm equal loading of DNA and then dried and imaged with X-omat film (Eastman Kodak, Rochester, NY). DNA was extracted from adult sheep heart as a negative control and from peri-infarct lamb myocardium as a positive control, as previously described. 11
mRNA microarray
Total cellular RNA was prepared from frozen myocardial samples by using the RNEasy Protect Midi kit (Qiagen, Valencia, Calif) and analyzed by using the Human Apoptosis-2 GEArray kit (SuperArray, Bethesda, Md), according to the manufacturers' instructions. Transcript abundance was evaluated in comparison with ß-actin and reduced glyceraldehyde-phosphate dehydrogenase by using a Storm 860 phosphor imager (Amersham Biosciences [Molecular Dynamics], Sunnyvale, Calif).
Caspase activity assay
A fluorimetric caspase activity assay was performed, as previously described. 12 Snap-frozen myocardial samples were homogenized in cold detergent buffer (Tris-HCl, 10 mmol/L [pH 8.0]; Triton X-100, 1%; sucrose, 0.32 mol/L; and EDTA, 5 mmol/L) with proteinase inhibitors (phenylmethylsulfonyl fluoride, 1 mmol/L; dithiothreitol, 2 mmol/L; leupeptin, 10 µg/mL; and aprotinin, 10 µg/mL) and cellular debris peleted at 13,000g for 10 minutes at 4°C. Supernatant protein concentration was standardized on the basis of a chromogenic assay (Bio-Rad, Hercules, Calif). Samples were diluted in assay buffer (sucrose, 10%; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 100 mmol/L [pH 7.5]; NaCl, 100 mmol/L; and CHAPS, 0.1%) with proteinase inhibitors as above. Tetrapeptide substrates of caspase 3 (Ac-DEVD-AFC), caspase 8 (CBZ-IETD-AFC), and caspase 9 (Ac-LEHD-AFC; all from Sigma, St Louis, Mo) were added (50 µmol/L), and the fluorescence before and after 1 hour of incubation at 37°C were measured with a PerkinElmer LM50B fluorimeter (EG&G, Wellesley, Mass) and compared with a curve of known AFC concentrations diluted in each sample.
Western immunoblotting
Protein was purified from frozen left ventricular myocardial samples by means of homogenization in nondetergent lysis buffer (Tris, 50 mmol/L; NaCl, 150 mmol/L; EDTA, 10 mmol/L [pH 7.5] with leupeptin, 10 µg/mL; 2-mercaptoethanol, 2 mmol/L; aprotinin, 10 µg/mL; and phenylmethylsulfonyl fluoride, 1 mmol/L) and centrifugation at 13,000g for 10 minutes at 4°C and then quantified by means of chromogenic assay (Bio-Rad). Equal amounts of protein were separated by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Equal protein loading was confirmed by means of Ponceau staining. Bcl-2 was detected with hamster monoclonal anti-Bcl-2 IgG (BD Biosciences, Franklin Lakes, NJ). Bax was detected with hamster monoclonal anti-Bax IgG2b (Santa Cruz Biotechnology, Santa Cruz, Calif). Bcl-xL was detected with mouse monoclonal or rabbit polyclonal antibody (both BD Biosciences). Antigen-antibody complexes were labeled with appropriate horseradish peroxidase-conjugated secondary antibodies and revealed with the SuperSignal enhanced chemiluminescence kit (Pierce, Rockford, Ill). Chemiluminograms were digitized and quantified by using the National Institutes of Health Image program (rsbweb.nih.gov).
Data are expressed as means ± SEM. Statistical comparisons were performed by means of analysis of variance, followed by the Student t test.
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13 Only recently, however, has evidence of apoptosis been sought after what is believed to be the protected ischemia of brief, hypothermic, depolarized cardioplegic arrest. 10
The data that most significantly support the presence of apoptosis after cardioplegic arrest in the neonatal lamb come from TUNEL staining. This method has been very widely applied in the study of myocardial apoptosis; however, its specificity has been questioned. 14 To increase our confidence in the specificity of TUNEL in our model, we have combined TUNEL with immunofluorescent labeling of cardiomyocytes through -actin. Although at earlier time points after injury other investigators have identified more prominent apoptosis among endothelial cells, 15 the majority of TUNEL-positive nuclei in our samples were seen in actin-positive cells. More important, the specificity of TUNEL in this study is supported by the finding of ladder patterns in DNA from postcardioplegia hearts. Because laddering is the result of internucleosomal cleavage of DNA by endonucleases, it has previously been seen as a virtual sine qua non of apoptosis. Although in experimentally produced necrosis false-positive laddering caused by endogenous, Ca2+- and Mg2+-dependent endonucleases has been reported, 16 our technique included immediate freezing, thawing in the presence of high EDTA concentrations to inhibit endonucleases, and early proteinase K digestion of nuclear proteins, after which DNA is susceptible only to random length degradation.
The data from mRNA microarrays showed no increase in most of the target transcripts; however, there was a highly significant increase in signal for Bad, a proapoptotic effector, and for Bcl-x. The function of Bcl-x is to antagonize the mitochondrial pathway of apoptosis induction. The mitochondrion releases cytochrome c in response to ischemic injury and oxidative stress, forming a complex with cytosolic Apaf and procaspase 9 to yield active caspase 9 and subsequently active caspase 3. 17,18 Bcl-xL opposes this process. Because TUNEL positivity was greater after cardioplegia than after cardiopulmonary bypass alone and because the addition of cardioplegia imposes ischemia and oxidative stress, the finding of early induction of Bcl-x transcription corroborates our TUNEL findings.
The caspases are a family of highly specific proteases that participate both in the activation of apoptosis and in its execution. Caspase 3 is a central mediator: its activation is associated with significant impairment of cardiac contractility and its inhibition restores hemodynamic performance in certain models. 19 Our finding of a lack of increase in caspase 3 activity is in contrast with reports from rodent models and from cultured cardiomyocytes 20 and situations involving more severe cardiac injuries. 12 Human myocardial apoptosis has been reported without caspase 3 activation. 21-23
Caspases 8 and 9 are more upstream mediators in apoptosis signaling. Caspase 8 is involved in transmitting cell-surface apoptotic signals, such as from tumor necrosis factor and Fas. Although cardiopulmonary bypass is known to increase tumor necrosis factor and Fas levels, 24-26 caspase 8 activity was not significantly different between groups. Caspase 9 is activated by the mitochondrial pathway, as described above. After cardioplegia, there was a more suggestive but still nonsignificant trend (P = .16) to higher caspase 9 activity than after bypass alone. The lack of increased caspase activity despite TUNEL evidence of completed apoptosis might indicate that the fraction of cells undergoing caspase activation was too small to detect above intragroup variability. It is also possible that caspase activity was opposed through apoptosis-regulatory mechanisms. Alternatively, species variability in enzyme specificity for the tetrapeptide substrates used might be responsible.
The lack of change in prevalence of Bcl-2 and Bax protein levels neither supports nor refutes the occurrence of apoptosis in this model. Bcl-family proteins are known to exert many of their functions in the short term by altering their subcellular localization or through homodimerization and heterodimerization, rather than solely by means of de novo expression. 27-29 Unchanged bcl-xL protein abundance, despite increased mRNA transcript abundance, might be due to the acute nature of the stimulus.
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Dr Hammel and associates from the University of Iowa Children's Hospital have performed a comprehensive evaluation of the relationship between myocardial apoptosis, apoptosis-related mediators, and cardiopulmonary bypass with cardioplegic arrest in a neonatal lamb model. They have demonstrated rather convincingly on the basis of the 5-fold increase in TUNEL-positive nuclear myocytes that there is an increase in apoptosis in the fetal myocardium after aortic crossclamping.
This is not something generally on the mind of most congenital heart surgeons when they are performing an arterial switch procedure, but perhaps it should be. The clinical implications of this study might be quite significant. As the authors have speculated, this might contribute to the frequently seen postoperative sag in cardiac output 6 to 12 hours after neonatal cardiac procedures. We might also speculate that it could be a cause of later ventricular dysfunction in certain lesions.
I have several questions for the authors.
Can you speculate on potential clinical methods that would be used to prevent this apoptosis and perhaps modulate the effect seen in your model? Specifically, do you believe there is any contribution of anti-inflammatory-type measures, such as Decadron administration, aprotinin, or leukocyte depletion to decrease apoptosis?
You raised the question yourself regarding the lack of corroborating evidence with regard to the modest increase of caspase 3. Do you think the TUNEL positivity might actually be a false-positive result?
Finally, why did you select 6 hours for your end point? With my limited understanding, I am not sure that we are sure that this apoptosis is actually going to become cell death.
I congratulate you on a well-designed, well-executed, and nicely reported experiment. You have greatly heightened the awareness and possible implications of apoptosis in neonatal cardiac surgery.
Dr Hammel. Thank you, Dr Backer, for your kind comments.
To answer your first question regarding potential clinical techniques that could be used to prevent apoptosis and modulate this effect, current techniques for myocardial protection were developed, with myocardial stunning and myocardial necrosis protection as end points. Just the knowledge that apoptosis is a potentially important end point suggests investigation of techniques that prevent apoptosis. There are ways to inhibit apoptosis, for example, through caspase inhibitors, which are being tested in other models of cardiac apoptosis, or through use of alternative cardioplegic agents, such as potassium channel openers, which stabilize the mitochondrial membrane potential during ischemia and prevent mitochondrially induced apoptosis.
With regard to anti-inflammatory measures to decrease apoptosis, we were suspicious that inflammatory mediators, which are known to be increased during cardiopulmonary bypass, might potentially be the cause of this activation. We did not find evidence of that because we would expect to see an activation of caspase 8, which transmits the signal from those inflammatory mediators at the cell surface into the downstream mediators. We did not see that activity, but I would not say that we have ruled that possibility out.
Furthermore, when inflammatory cells are recruited into the myocardium after reperfusion, those cells increase the oxidative stress to which the mitochondrion is exposed and favor the induction of apoptosis. Therefore, there is a potential role for anti-inflammatory agents, such as steroids or complement inhibitors, during cardioplegia.
With regard to caspase 3 activity, it is a bit troubling not to see activation. Of course it is possible that this TUNEL stain is a false-positive result. But because this is the foundation that we are going to be basing a lot of work on, we went to some lengths to confirm it. The DNA laddering is strong corroborative evidence because DNA laddering is difficult to observe in tissue DNA extracts. When it is present, it has historically been a sine qua non of apoptotic DNA degradation. Furthermore, we repeated the TUNEL assays in a second separate laboratory, using different reagents from a different manufacturer and using both formalin-fixed and cryostat sections, and we came to the same conclusions, although the numbers were slightly different.
The lack of caspase 3 activation has actually been observed in human models and other large-animal models. It is more the rodent models and cell culture systems in which large increases in caspase activity have been reported. Figuring out why caspase 3 activity is not increased might end up teaching us something about the sequelae of cardioplegia in this model.
Why the 6-hour time point? Dr Caldarone's interest in this subject came from the observation that postoperative myocardial dysfunction seemed to actually increase as neonates approached that 6-hour time point. Therefore, we picked a time point at which apoptosis might be contributing to that phenomenon. We have looked at other time points, and we see TUNEL positivity in fact increasing beyond 6 hours, but we do not quite have that data ready to present.
Dr Bradley S. Allen (Oak Lawn, Ill). I have 3 questions. First, on your bypass controls, did you cool to the same temperature? This is important because apoptosis can be caused by temperature changes.
Second, your first statement was that the neonate is in a proapoptotic state, and this is the reason for the increased rate of apoptosis. However, apoptosis has been shown to occur in adult hearts subjected to cardioplegic arrest. How do you know there is more apoptosis in the neonate compared with that in the adult? Do you have a comparison group of mature hearts subjected to the same ischemic stress to prove this statement?
Last, did you perform any hemodynamic measurements? I would expect them to be normal after only a 70-minute crossclamp interval with good myocardial protection. If that is the case, the importance of this study is that it shows damage is occurring even with good myocardial protection. In contrast, if the hemodynamics were poor, then this means the method of protection was inadequate, and that poor protection was the cause of the increased rate of apoptosis.
Dr Hammel. Those are 3 very interesting questions.
The first question was about the temperature. Although for the bypass-only animals the prime was warmed a bit more before we went on so that they would not have rhythm disturbances when they were first started on bypass, they were all passively cooled, and all the lambs pretty quickly achieved a temperature of about 28°C during the bypass runs. Therefore yes, they were all cooled to approximately the same temperature.
The issue of the maturity of the subjects is also interesting. In fact, we have performed this experiment comparing with 6-week-old lambs who are significantly larger—about 4 times the body weight—and we see some apoptosis, but we see much less. We have also seen that as compared with those animals after surgical intervention, caspase activity is much higher in these neonates than it is in those 6-week animals, and we will be presenting that information at a later date as well.
With regard to hemodynamics, I think that is a very important next step. We need to find out whether hemodynamic changes occur in our model system and whether they are causally related to apoptosis. We are working on it.
Dr Pedro L. del Nido (Boston, Mass). Did you use catecholamines after pumping on any of these animals?
Dr Hammel. No, none of the animals received any inotropic agents. They all got transfusion and fluids as necessary to support their pressure.
Dr del Nido. Are they then allowed to regulate their own blood pressure?
Dr Hammel. Yes, they were all allowed to control their own pressure. They received some interventions as needed. Some of the animals were defibrillated, and some were given lidocaine, but not as a pattern.
Dr del Nido. It is one of the things that at some point you are going to have to need to control for because endogenous catecholamines, exogenous catecholamines (eg, dopamine), or both, are widely known to augment apoptosis. Therefore, it is something for which you will need to control.
My other question is this: Did you take your samples from the right ventricle or the left ventricle?
Dr Hammel. The samples for histologic studies were all taken from the middle of the left ventricular free wall. The samples for the rest of the studies were taken from the remainder of the left ventricular free wall.
Dr del Nido. Just a word of caution. A lot of the apoptotic signal, in fact, is driven by mitochondria, and subsarcolemmal mitochondria are not as involved as the interfibrillar mitochondria. There is a difference between hypertrophied versus nonhypertrophied ventricles, and physiologic hypertrophy, such as that seen in the left ventricle postnatally, in fact grows a lot of mitochondria very quickly but does not have a lot in the beginning. The right ventricle early on, because it is an important ventricle in the neonate, has a lot more mitochondria, which it loses in the first few days after birth. Again, you are going to have to look at both ventricles. It would be very interesting to repeat your exact study and look at the right ventricle. In fact, you might find that this phenomenon is even more dramatic in the right ventricle.
Dr Hammel. In fact, earlier in the study we looked at the right ventricle. I did not have a complete data set regarding that tissue, and therefore I did not present it. But TUNEL staining and laddering were also positive, and the rate of TUNEL-positive nuclei was, in fact, just about the same in the right and the left ventricle.
Dr Frank L. Hanley (Stanford, Calif). I am assuming, because you did not comment on it, that the spatial distribution throughout the volume of myocardium demonstrated a random distribution of apoptotic cells.
Dr Hammel. That is correct.
Dr Hanley. If that is correct, why did those particular cells become apoptotic? Could you speculate on the possibility that these were already cells that were on their way to cell death, and you just stimulated them along? Do you have any other explanation for why the individual cells that did become apoptotic in this setting did so?
Dr Hammel. That is a very interesting and provocative idea. Why these nuclei? They were evenly distributed as far as we could see. The other cells are all exposed to the same stimulus, and they must be affected as well. I think that you are probably right, these cells probably were cells that were just about to undergo apoptosis and were pushed over. However, they might be the tip-of-the-iceberg evidence of a phenomenon in which early and more subtle changes of apoptosis, such as cytochrome c release and mitochondrial dysfunction, might be occurring in a larger population of cells. That larger population might account for hemodynamic changes, whereas the 1% of cells that are TUNEL positive is not a significant loss on its own.
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