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Ann Thorac Surg 2002;74:830-837
© 2002 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
b Division of Cardiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
* Address reprint requests to Dr. Pearl, Division of Cardiothoracic Surgery, Department of Surgery, Children’s Hospital Medical Center OSB-3, 3333 Burnet Ave, OSB 3, Cincinnati, OH, USA 45229
e-mail: pearj0{at}chmcc.org
Presented at the Thirty-eighth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28–30, 2002.
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Abstract |
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Methods. Neonatal piglets were cooled with CPB followed by 2 hours of circulatory arrest. Animals were rewarmed, removed from CPB, and allowed to recover for 2 hours. Methylprednisolone (60 mg/kg) was administered in the CPB priming solution to one group (intraoperative glucocorticoids). In another group (preoperative glucocorticoids), 30 mg/kg methylprednisolone was administered 6 hours before CPB in addition to the intraoperative dose (30 mg/kg). Control animals received no glucocorticoids.
Results. Apoptotic myocardial cells measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and caspase-3 activity were reduced in animals administered glucocorticoids compared with controls (p < 0.05). Animals receiving either intraoperative or preoperative glucocorticoids had 0.10 ± 0.07 and 0.13 ± 0.05 apoptotic cells per high-power field, respectively, whereas 0.33 ± 0.15 apoptotic cells were detected with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling in control animals. Glucocorticoid administration reduced myocardial intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 mRNA expression compared with control piglets. Maximum rate of increase of left ventricular pressure was 62% ± 9% of baseline in control animals at 120 minutes of recovery compared with 96% ± 6% and 95% ± 10% of baseline in animals receiving intraoperative and preoperative glucocorticoids, respectively (p < 0.05).
Conclusions. The reduction of neutrophil adhesion and activation proteins in neonatal myocardium was associated with less apoptotic cell death after glucocorticoid administration. The blunting of apoptosis in glucocorticoid-treated animals was also associated with improved recovery of left ventricular systolic function in neonatal animals after CPB and circulatory arrest. Glucocorticoid attenuation of myocardial apoptosis might have important implications for maintaining long-term ventricular function after ischemia and reperfusion.
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The presence of apoptosis has been detected in myocardium after reperfusion along with cellular necrosis [3, 4]. Although the specific roles of apoptosis and necrosis remain controversial, studies have demonstrated that apoptosis significantly contributes to the loss of cardiomyocytes with reperfusion of ischemic myocardium [5]. Apoptosis may be initiated by generation of reactive oxygen species and mediated through activation of the caspase cascade. Neutrophil invasion of tissue involves adhesion to the microvasculature, neutrophil activation, and transmigration into the surrounding tissues. Expression of intercellular adhesion molecule-1 (ICAM-1) by endothelial cells and cardiomyocytes particularly after stimulation by proinflammatory cytokines, as occurs after CPB, can induce migration of neutrophils into the myocardium [6]. Neutrophils adhered to cardiomyocytes can release oxygen-derived free radicals [7], induce apoptosis [8], and reduce the contractile response of cardiomyocytes [9]. Intercellular adhesion molecule-1—induced neutrophil adhesion to cardiomyocytes may thus be an early step in initiating oxidative damage. Monocyte chemoattractant protein-1 (MCP-1), produced in nearly all cells, has been associated with development of reperfusion injury [10, 11]. In addition, MCP-1 stimulates ICAM-1 expression and neutrophil adhesion to neonatal cardiomyocytes [12].
Elevated caspase-3 protein levels and activity are associated with cardiomyocyte apoptosis [13], and overexpression of caspase-3 in mice depresses cardiac function [14]. Caspase-3 inhibitors can also reduce cardiomyocyte reperfusion injury [15].
Glucocorticoids have been the basis of therapy aimed at decreasing inflammation after CPB. However, the mechanisms by which glucocorticoids improve myocardial function have not been elucidated. Furthermore, the effectiveness of steroids in neonates and the proper timing of administration to take full advantage of the benefits of glucocorticoids have not been fully addressed. Therefore, this study was undertaken to determine whether glucocorticoids could improve hemodynamics and myocardial function after CPB and deep hypothermic circulatory arrest (DHCA), at least in part, by suppressing neutrophil infiltration, monocyte activation, and apoptosis that results in cardiomyocyte loss. In addition, we wanted to determine whether glucocorticoid administration before and during operation could be more efficacious than intraoperative administration alone.
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Material and methods |
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Animals were administered heparin (300 U/kg) and placed on CPB with cannulation of the carotid artery and right atrium. The CPB prime consisted of 800 mL of direct-drawn whole porcine blood stabilized with sodium citrate (Animal Biotech Industries, Danboro, PA). A single dose of methylprednisolone (60 mg/kg) was administered in the pump priming solution in the intraoperative glucocorticoid group (n = 8), whereas a dose of 30 mg/kg was given 6 hours before CPB in addition to the intraoperative dose of 30 mg/kg in the preoperative glucocorticoid group (n = 9). Control piglets (n = 9) received no glucocorticoids. Two separate groups of animals were included to analyze baseline data. Baseline animals (n = 6) were sacrificed and had ventricular tissue collected immediately after sternotomy. Baseline glucocorticoid-treated animals (n = 6) were administered methylprednisolone (30 mg/kg) 6 hours before sternotomy, then sacrificed, and tissues were collected immediately after sternotomy. Comparisons of data before and after CPB and DHCA were made among groups. Animals undergoing CPB were cooled to a rectal temperature of 18°C for more than 40 minutes. Hematocrit on CPB was maintained at 25% to 30% and calcium at 0.6 mmol/L with a flow rate of 100 mL/kg per minute. The oxygen tension of the CPB circuit was maintained at greater than 250-mm Hg. The bypass circuit was then turned off and the head packed in ice. The heart was protected with topical cold saline and ice. Deep hypothermic circulatory arrest was maintained for 120 minutes after which CPB was resumed, and the animals were warmed to 38°C for more than 40 minutes. Ultrafiltration during warming returned the hematocrit to 30% to 35%. The piglets were removed from CPB and maintained for 2 hours. Dopamine at 5 µg/kg per minute was administered during weaning from CPB, then discontinued after 1 hour. Cardiopulmonary function was monitored continuously during the period before and after CPB. Transmural LV and RV tissues from the same area of the outer ventricular walls in each animal were obtained at the terminal time.
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animals Resources and published by the National Institutes of Health (National Institutes of Health publication 86-23, revised 1985). The Animal Care and Use Committee at Children’s Hospital Research Foundation also approved the protocol.
Measurement of mrna expression
Ribonuclease protection assays were used for determination of levels of specific mRNA in LV and RV samples frozen in liquid nitrogen at the point of collection. A custom probe set containing DNA templates was used for T7 RNA polymerase-directed synthesis of antisense probes (PharMingen, San Diego, CA). The set includes templates for probes that hybridize with porcine ICAM-1, MCP-1, and the housekeeping genes, L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The [
32P]uridine triphosphate-labeled probes were generated by in vitro transcription, then hybridized in excess to total mRNA (10 µg) isolated from porcine tissues. Free probe and single-stranded RNA were digested with ribonuclease A and T1. Ribonuclease-protected probes were purified by phenol-chloroform extraction, resolved on a 5% denaturing polyacrylamide gel, and imaged by autoradiography. The identity of each mRNA species in the original samples was determined on the basis of the appropriate size of the protected probe fragment. Phosphor imaging of the gel allowed for the relative quantification of each of the mRNAs (Molecular Dynamics, Sunnyvale, CA). Housekeeping genes (L-32 and GAPDH) in the template allowed RNA levels to be normalized within and between gels. Messenger RNA expression is reported as a ratio of target mRNA to GAPDH mRNA to correct for background effects.
Glutathione peroxidase activity
Glutathione peroxidase activity was measured with a commercial kit (R&D Systems, Minneapolis, MN). Ventricular tissue homogenates were diluted in 0.05 mol/L Tris-HCl and 5 mmol/L ethylenediaminetetraacetic acid and mixed with reduced nicotinamide-adenine dinucleotide phosphate reagent. tert-Butyl hydroperoxide (0.007%) was added in the spectrophotometer, and the change in absorbency at 340 nm was recorded every 30 seconds for 3 minutes. Protein concentrations of samples were measured with the Bio-Rad protein assay (Hercules, CA). Activity is reported in milliunits per milligram of protein.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay
Tissues from the piglets were also fixed overnight in 10% neutral-buffered formalin. Apoptotic cells were identified by the incorporation of biotin into DNA fragments using the CardioTACS kit according to manufacturer’s directions (R&D Systems). The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay generated a blue precipitate in the presence of DNA fragmentation with a streptavidin-conjugated horseradish peroxidase substrate. The samples were stained with red counterstain C, then examined under a light microscope. Incubating tissue sections with a nuclease generated positive controls, and negative controls were incubated in buffer without terminal deoxynucleotidyl transferase. The number of positive-staining cells in each sample was counted in a grid pattern containing 20 fields of view (x40) and covering most of the section. The apoptotic index was the mean number of positive cells in each field of view from two separate tissue sections collected from each animal. Analyses of apoptotic cells were conducted by researchers blinded to the treatment group.
Caspase-3 activity
Myocardial tissue samples were collected from the same two areas in the outer wall of the LV and RV after CPB-DHCA or immediately after sternotomy. Tissues were homogenized in MOPS buffer and assayed for caspase-3 activity with a commercially available kit (BioMol Research Laboratories, Plymouth Meeting, PA) using a fluorogenic substrate to measure DEVDase activity. Fluorescence generated by substrate cleavage was recorded every 10 minutes for 90 minutes. Addition of purified caspase-3 or DEVD-CHO, an inhibitor of DEVDase activity, to some samples was used as a control. Caspase-3 activity was compared in tissues collected before and after CPB-DHCA.
Active caspase-3 immunoblot analyses
Ventricular myocardium was flash frozen in liquid nitrogen at the point of collection. Tissue was homogenized in 10 mmol/L MOPS buffer. Homogenates were centrifuged and the supernatant retained. Protein concentration was determined by the Bio-Rad protein assay, and the samples were stored at -80°C until used. Western blots were performed with the standard protocol with 30 mg of total proteins separated on 4% to 12% acrylamide bis-tris gels (Invitrogen, Carlsbad, CA) by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. Antibodies for immunoblotting were anti-human caspase-3 (0.2 µg/mL, PharMingen) that recognized only the activated 17-kDa fragment of caspase-3. Immunoblots were also probed with antibodies for GAPDH (0.05 µg/mL, Chemicon International, Temecula, CA). Secondary antibodies were alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG. Proteins were visualized with the Western Breeze chemiluminescent detection system according to the manufacturer’s instructions (Invitrogen). Protein levels are reported as a ratio of target protein to GAPDH levels on the same immunoblot to correct for background effects.
Statistical analysis
Repeated-measures analysis of variance was used to analyze serial data as a function of time, and post hoc comparisons made by Fisher’s protected least significant difference test were used when appropriate to evaluate differences between individual times. Comparisons between treatments were made by analysis of variance with a probability value less than or equal to 0.05 considered significant. Personnel conducted analyses using StatView 4.01 software (Abacus Concepts, Inc, Berkeley, CA). Data are presented as the mean ± the standard deviation.
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Comment |
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Cardiomyocyte apoptosis was measured directly, by TUNEL assay, and by inference from caspase-3 activation. Although the specificity of TUNEL assays for detecting DNA fragmentation in apoptosis versus necrosis has been questioned, only very low levels of TUNEL-positive cells were detected in baseline hearts, and glucocorticoid administration reduced TUNEL staining to baseline levels in animals undergoing CPB-DHCA. Caspase-3 activity was measured by cleavage of a fluorogenic DEVD substrate, and active caspase-3 protein was detected by immunoblot analysis. Inasmuch as caspase-3 and caspase-7 share the substrate DEVD, it is possible that the fluorogenic assay is not specific for caspase-3. However, the entire caspase cascade is thought to flow through the activation of caspase-3 and the subsequent cleavage of cellular proteins [5]. Caspase-3 immunoblots detected the same pattern of activity as the DEVD substrate assay. The use of a combination of markers for apoptosis in the current study all defined a similar pattern consistent with cell death after reperfusion of ischemic neonatal myocardium.
The finding that reperfusion injury is associated with activation of caspase-3 and the development of apoptosis is consistent with other studies [8, 16]. Although apoptosis is likely to have both short-term and long-term adverse effects on ventricular function, the initiating events are likely to happen in close temporal approximation to the injury. Apoptosis has been detected within 30 minutes of reperfusion in isolated perfused rat hearts [8], and caspase-2, caspase-3, and caspase-7 are activated during ischemia and reperfusion of myocardium [15]. In the present study, ventricular function was evaluated at 90 and 120 minutes after reperfusion. It is possible that some of the cardiac dysfunction noted in these experiments is a consequence of true myocardial stunning and is therefore reversible. Nevertheless, the increase in apoptotic cells in untreated hearts suggests a permanent component of cardiomyocyte injury.
The present data suggest glucocorticoids may reduce oxidative stress within the myocardium after CPB-DHCA, which may in turn be one mechanism by which steroids prevent apoptosis [8]. Reactive oxygen species including hydrogen peroxidase, superoxide, and hydroxyl radicals increase with reperfusion of ischemic myocardium [17] and can initiate apoptosis in cardiomyocytes [18]. Glutathione peroxidase is activated by oxidative stress and detoxifies hydrogen peroxide. In addition, glutathione peroxidase can reduce free radical injury associated with ischemia and reperfusion [19]. In the current study glucocorticoid treatment was associated with lower glutathione peroxidase activity, indicating higher oxidative stress in untreated animals.
A further link between glucocorticoid treatment, prevention of oxidative stress, and decreased apoptosis is provided by the data regarding ICAM-1 and MCP-1. Although ICAM-1 levels are not direct measures of neutrophil infiltration into myocardium, previous studies have directly correlated ICAM-1 mRNA with neutrophil adherence and cellular injury after myocardial infarction [20, 21]. Lu and colleagues [22] found that neutrophils adhered to cardiomyocytes and released oxidants such as superoxide and hydrogen peroxide that cause intracellular oxidative stress and cell death. Furthermore, anti-ICAM-1 antibodies reduce infarct size in reperfused heart [23], and ICAM-1 null mice are less susceptible to reperfusion injury [24, 25], indicating an important role for ICAM-1 in reperfusion injury. In the current study glucocorticoids prevented the increase in ICAM-1 mRNA that was detected after CPB-DHCA. Elevated MCP-1 mRNA in the current study indicates potential monocyte infiltration after CPB-DHCA. Inflammatory cytokines induce MCP-1 expression by cardiomyocytes and lead to attraction and activation of monocytes [26]. Like ICAM-1 antibodies, MCP-1 antibodies reduce infarct size in rats after ischemia and reperfusion [10]. Interestingly, MCP-1 antibody has also been shown to markedly decrease ICAM-1 mRNA expression [10]. Expression of ICAM-1 mRNA is regulated by MCP-1 in rat cardiomyocytes, and MCP-1 stimulation of ICAM-1 leads to neutrophil infiltration [12]. Peak levels of MCP-1 mRNA induction occur as soon as 3 hours after reperfusion of ischemic myocardium in rat heart, demonstrating a time course similar to the current study.
In conclusion, CPB-DHCA in neonatal pigs was associated with upregulation of neutrophil adhesion proteins, increased oxidative stress, caspase-3 activation, and cardiomyocyte apoptosis. All of these alterations were prevented to some degree by glucocorticoids either before or during operation, and glucocorticoids were associated with preservation of LV function after CPB-DHCA and reperfusion. Glucocorticoids are a promising therapy to reduce reperfusion injury and potentially prevent long-term sequelae of repair of congenital heart disease.
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Discussion |
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DR PEARL: Yes. We have very similar slides for protein expression. We initially had a little bit of trouble getting our intercellular adhesion molecule-1 assays to work in the pig model. We had to create our own antibody, but now have slides that would pretty much parallel the mRNA expression. We have also measured myeloperoxidase activity, which tends to be elevated as well in the nontreated animals and then decreased in the steroid-treated animals. We have yet to perform any electron microscopy or other histologic examination.
DR DAVID C. DRINKWATER (Nashville, TN): I very much enjoyed your presentation. Can you tell us how you arrived at the dose of steroids? Were there any dose-related studies, and is there a response curve below our therapeutic clinically used dosage?
DR PEARL: The steroid dose that we have been using clinically is 30 mg/kg in the bypass circuit. And I do not know of any real dose-response curves. I think we kind of blast them pretty hard with fairly high dosages, when you might be able to get by with less, but we have not seen any downside to giving one dose or two doses to the patient, so that is what we have used clinically.
You can look at the type of steroids used and the pharmacology, and there is a lot of suggestion that giving them preoperatively would be beneficial. We have done both clinical and animal studies now, and the trend is toward improvement with preoperative steroids, but it is not as dramatic as we initially thought it might be. But there are a lot of reasons that giving them ahead of time might be more advantageous than having them put in the pump basically at the time the insult starts.
DR ANTON MORITZ (Frankfurt, Germany): Are these data only true for the immature myocardium? Corticoids were tested for general adult heart operations, and the results were not that clear. Do you have any information on this, or do you just specify for the pediatric subgroup?
DR PEARL: The immature myocardium is more susceptible in some ways and in different ways to injury than the adult myocardium. Since we are congenital heart repair surgeons, that is what we are most interested in and that is why we have kept with the neonatal model. Earlier studies looking at steroids or any of the other modalities used have primarily been in adult models, and so we believed it was important to look at an immature myocardium because of the different response.
What we have seen with the steroids also is a significant effect on some of the sarcolemma proteins, which we are currently investigating, which has not been described in immature hearts but has been briefly in adults, such as effects on calpain, calpastatin, and troponin-I.
DR CHRISTOPHER A. CALDARONE (Iowa City, IA): Could you speculate about what you think the mechanism is for the contractile dysfunction we see postoperatively in a neonate. Clearly it seems like 6 or 8 hours after the operation we have a lot more dysfunction than you might attribute to the 2% or 3% of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nickend labeling (TUNEL)-positive cells you are detecting. I suspect there is a lot more mitochondrial dysfunction and that you are seeing only the tip of the iceberg.
DR PEARL: The purpose of looking at TUNEL assays and at other markers of apoptosis was really to get a sense of whether this transient myocardial dysfunction that magically gets better with aggressive support continues in some patients.
I think there is, obviously, as you mentioned, a lot of factors that contribute to myocardial dysfunction postoperatively, especially in neonates, such as mitochondrial issues, calcium-related issues, factors that adult myocardium tend to be a little less sensitive to. As I alluded to, looking at troponin-I degradation, we are finding significant changes in the patients as well as the animals, in troponin-I, related to the ischemia-reperfusion process. Some of those theoretically should be reversible, and maybe what we are seeing is that with time the sarcolemma proteins regenerate. We have thought about using steroids postoperatively, too, extending this out for the first 24 hours to see if we can avoid that 8-hour slump.
DR CALDARONE: And second, what do you think about the disparity in the extent of apoptosis between the right and left ventricles? We have seen similar results in our laboratory. In a rat model, there is a difference in terms of the proapoptotic state of the ventricles, but the right ventricle is more proapoptotic than the left.
DR PEARL: Well, one reason we have used an immature animal for these models is because they do get pulmonary hypertension. The right ventricle in the newborn animal is well able to tolerate that, so the animals survive. Whereas if we did this in an adult with some of the pulmonary vascular resistance elevations we get, they would not survive.
Sometimes people have related the right ventricle being more or less susceptible based on myocardial preservation techniques, which we are not doing in this model, other than packing the whole heart in ice, so we do not have the right ventricle exposed to the operative field with a headlight on it or not giving it adequate cardioplegia because of using retrograde cardioplegia and those things.
I think we found both in our hypoxia—reoxygenation studies as well as in these studies that there is quite a bit of difference, even on baseline samples, between the right and left ventricles in almost everything we measure. My concern is that this may explain why a lot of right ventricles that function, or are forced to function, as systemic ventricles, such as in hypoplasties, fail despite good operations. They are just not designed the same way.
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References |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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4 integrin. Circ Res 1999;84:1245-1251.4-Integrin mediates neutrophil-induced free radical injury to cardiac myocytes. J Cell Biol 2001;152:857-866.This article has been cited by other articles:
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  F. Kerendi, P. M. Kirshbom, M. E. Halkos, N.-P. Wang, H. Kin, R. Jiang, Z.-Q. Zhao, K. R. Kanter, R. A. Guyton, and J. Vinten-Johansen Cobalt Chloride Pretreatment Attenuates Myocardial Apoptosis After Hypothermic Circulatory Arrest Ann. Thorac. Surg., June 1, 2006; 81(6): 2055 - 2062. [Abstract] [Full Text] [PDF]
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 L. G. Kevin, E. Novalija, and D. F. Stowe Reactive Oxygen Species as Mediators of Cardiac Injury and Protection: The Relevance to Anesthesia Practice Anesth. Analg., November 1, 2005; 101(5): 1275 - 1287. [Abstract] [Full Text] [PDF]
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C. A. Caldarone, E. W. Barner, L. Wang, M. Karimi, C. E. Mascio, J. M. Hammel, J. L. Segar, C. Du, and T. D. Scholz Apoptosis-related mitochondrial dysfunction in the early postoperative neonatal lamb heart Ann. Thorac. Surg., September 1, 2004; 78(3): 948 - 955. [Abstract] [Full Text] [PDF]
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J. M. Pearl, S. M. Schwartz, D. P. Nelson, C. J. Wagner, J. M. Lyons, S. M. Bauer, and J. Y. Duffy Preoperative glucocorticoids decrease pulmonary hypertension in piglets after cardiopulmonary bypass and circulatory arrest Ann. Thorac. Surg., March 1, 2004; 77(3): 994 - 1000. [Abstract] [Full Text] [PDF]
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 I. Herr, E. Ucur, K. Herzer, S. Okouoyo, R. Ridder, P. H. Krammer, M. von Knebel Doeberitz, and K.-M. Debatin Glucocorticoid Cotreatment Induces Apoptosis Resistance toward Cancer Therapy in Carcinomas Cancer Res., June 15, 2003; 63(12): 3112 - 3120. [Abstract] [Full Text] [PDF]
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 V. A. Schroeder, J. M. Pearl, S. M. Schwartz, T. P. Shanley, P. B. Manning, and D. P. Nelson Combined Steroid Treatment for Congenital Heart Surgery Improves Oxygen Delivery and Reduces Postbypass Inflammatory Mediator Expression Circulation, June 10, 2003; 107(22): 2823 - 2828. [Abstract] [Full Text] [PDF]
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