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Ann Thorac Surg 2006;81:2055-2062
© 2006 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery and the Carlyle Fraser Heart Center, Emory University School of Medicine, Atlanta, Georgia
Accepted for publication January 4, 2006.
* Address correspondence to Dr Vinten-Johansen, Cardiothoracic Research Lab, 550 Peachtree St NE, Atlanta, GA 30308 (Email: jvinten{at}emory.edu).
Presented at the Forty-first Annual Meeting of The Society of Thoracic Surgeons, Tampa, FL, Jan 24–26, 2005.
| The Thoracic Surgery Directors Association (TSDA) Resident Research Award was established in 1990 to encourage resident research in cardiothoracic surgery. Abstracts submitted to The Society of Thoracic Surgeons (STS) Program Committee representing research performed by residents were forwarded to the TSDA to be considered for this award. The abstracts were selected by the TSDA Executive Committee consisting of Douglas Mathisen, MD, President, Jeffrey Gold, MD, President-Elect, John Calhoon, MD, Secretary/Treasurer, Edward Verrier, MD, Immediate Past President, George Hicks, MD, Councillor-at-Large, and Bartley Griffith, MD, Councillor-at-Large. The TSDA Resident Research Award was given to Faraz Kerendi, MD, a resident at Emory University School of Medicine. He received a monetary award of $500.00 and an engraved desktop award. The TSDA, with support by Medtronic, Inc, makes this award annually, using the above selection procedure. The resident author of the selected study is recognized at the STS meeting.
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Abstract |
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METHODS: Anesthetized neonatal piglets were randomly assigned to four groups (n = 6 in a group): control (NaCl injection); hypoxia (pO2 of 30 to 40 mm Hg for 3 hours); DFX injection; or CoCl2 injection. Twenty-four hours later, the animals underwent cardiopulmonary bypass (CPB) and 110 minutes of DHCA. One week after CPB, percentage of apoptotic myocytes (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling [TUNEL] assay) and expression of the pAKT, Bcl-2, and Bax were assessed by Western blot.
RESULTS: Although preconditioning with hypoxia and DFX failed to show a protective benefit, CoCl2 pretreatment significantly attenuated myocardial apoptosis (9.3% ± 4.1%) versus controls (33.8% ± 9.7%, p = 0.042). That was associated with increased myocardial pAkt expression (0.19 ± 0.006 in CoCl2 versus 0.12 ± 0.008 in control, p < 0.001). The expression of Bcl-2 was also significantly higher in the CoCl2 group (0.15 ± 0.02) versus control (0.11 ± 0.01, p = 0.007), whereas Bax expression was lower (0.34 ± 0.04 versus 0.54 ± 0.03 for control, p < 0.001).
CONCLUSIONS: Preconditioning with CoCl2 before prolonged DHCA in neonatal piglets attenuates myocardial apoptosis by mechanisms involving phosphorylation of Akt, upregulation of the antiapoptotic protein Bcl-2, and decreased expression of the proapoptotic protein Bax.
Deep hypothermic circulatory arrest (DHCA), while useful for the repair of complex congenital cardiac defects and acquired aortic arch abnormalities, is associated with a significant level of end-organ compromise. Variable levels of injury, consisting of cellular necrosis or apoptosis, or both, have been described after DHCA in the heart [1], brain, kidney, and gastrointestinal mucosa [2]. Whereas neurologic injury is often clinically manifested by seizures or developmental abnormalities, injury to the myocardium may not be as readily evident. Nevertheless, detrimental effects attributed to cardiomyocyte apoptosis have been described, including impaired cardiac contractility [3] and delayed postoperative recovery of function [1, 4]. However, while there has been an abundance of investigation focused on preventing neuronal injury and apoptosis, relatively little research exists on minimizing myocardial apoptosis specifically after DHCA [1].
Ischemic preconditioning, a potent cardioprotective strategy against myocardial ischemic injury, has been associated with attenuation of myocyte apoptosis after ischemia and reperfusion [1, 5]. Similarly, hypoxic preconditioning, through induction of hypoxia-inducible factor (HIF-1
), has been shown to have a protective effect against ischemia-reperfusion injury in rodent myocardium and neuronal tissue [6, 7]. Furthermore, the administration of cobalt chloride (CoCl2 [a transitional metal]) and deferoxamine (DFX [an iron chelator]) before an acute ischemic event pharmacologically mimics hypoxic preconditioning by providing similar protective benefits [7, 8]. Although the protective mechanisms of CoCl2 and DFX preconditioning are not fully understood, it is thought that these agents prevent the degradation of HIF-1, leading to increased expression of its target gene products, including vascular endothelial growth factor and erythropoietin [9–12].
Although these effects have been well established in the brain, the effect of preconditioning by hypoxia or hypoxia-mimetic agents has not previously been assessed with respect to myocardial injury resulting from DHCA. The aim of this study, therefore, was to determine if preconditioning with hypoxia, DFX, or CoCl2 24 hours before a period of DHCA in neonatal piglets would limit apoptosis related to the injury resulting from the inherent global ischemia of circulatory arrest.
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Material and Methods |
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Each animal underwent two operative procedures: preconditioning and cardiopulmonary bypass (CPB)/DHCA. Twenty-four 2- to 3-week-old Yorkshire cross piglets were preanesthetized with an intramuscular cocktail of ketamine (22 mg/kg), acepromazine (1.1 mg/kg), and atropine (0.05 mg/kg). The animals were orotracheally intubated and ventilated (Model 2000 pediatric ventilator; Hallowell, Pittsfield, Massachusetts) to maintain arterial blood pH at a goal of 7.35 to 7.45, pCO2 at a range of 35 to 45 mm Hg, and pO2 at 100 to 110 mm Hg. Deep anesthesia was maintained with 1% to 1.5% inhaled isofluorane. A marginal ear vein was cannulated for administration of drugs and intravenous (IV) fluids, and the superficial femoral artery was cannulated with a fluid-filled catheter for monitoring of blood pressure, heart rate and arterial blood gases.
The animals were then randomly assigned to one of four groups: (1) control (ventilated with room air supplemented with oxygen to maintain an arterial blood pO2 of 95 to 125 mm Hg for 3 hours, and received an IV and intraperitoneal [IP] injection of 0.225% NaCl as a vehicle); (2) hypoxia (ventilated with a hypoxic gas mixture to maintain an arterial blood pO2 of 30 to 40 mm Hg, and received an IV and IP injection of 0.225% NaCl); (3) DFX (maintained arterial blood pO2 of 95 to 125 mm Hg, and received an IV [100 mg/kg] and IP [100 mg/kg] injection of deferoxamine); and (4) CoCl2 (maintained arterial blood pO2 of 95 to 125 mm Hg, and received an IV injection of 0.225% NaCl and IP [20 mg/kg] injection of cobalt chloride).
The animals were then allowed to recover for 24 hours, after which they were reanesthetized in a manner similar to that described above (although studies using different species have showed beneficial results from acute hypoxic preconditioning [13], this 24-hour interval was chosen based on our previous study demonstrating that the greatest level of HIF-1 and erythropoietin upregulation occur at 24 hours after the preconditioning stimuli [14]). Cefazolin (25 mg/kg IV) was administered before the procedure on each day and every 8 hours thereafter for a period of 24 hours. Deep anesthesia was maintained with 1% to 1.5% inhaled isofluorane, except during the period of DHCA. Once again, the superficial femoral artery was cannulated for monitoring of blood pressure, heart rate, and arterial blood gases. After systemic heparinization (300 U/kg), the contralateral common femoral artery was cannulated with an 8F arterial cannula (Bio-Medicus, Minneapolis, Minnesota), and the right atrial appendage was cannulated with an 18F venous cannula (Baxter RMI, Deerfield, IL) through a right anterolateral thoracotomy.
The CPB circuit was prepared at this time, and consisted of a nonpulsatile roller pump (Cobe Cardiovascular, Arvada, Colorado), sterile tubing, a pediatric membrane oxygenator (Lilliput 2; Cobe Cardiovascular), a venous reservoir (Lilliput 1 twin reservoir; Cobe Cardiovascular), and a 40 µm arterial filter (Cobe Cardiovascular). The circuit was primed with 500 cc whole porcine blood (Lampire Biological Labs, Pipersville, Pennsylvania), 1,000 units heparin, 30 mg/kg calcium chloride, 15 mEq sodium bicarbonate, 1 g/kg mannitol, 5 mg/kg dexamethasone, and 0.1 mg/kg pancuronium bromide.
The animals were placed on CPB and perfusion-cooled to a nasopharyngeal temperature of 18°C over a period of 20 minutes. Perfusion was initiated at a flow rate of 150 mL/kg and adjusted to maintain a perfusion pressure greater that 30 mm Hg. During the cooling and rewarming periods, hematocrit was maintained at or near 30%, and arterial blood gases were managed according to "alpha-stat" strategy, maintaining a pCO2 of 35 to 45 mm Hg, uncorrected for temperature. Once the animals reached a core temperature of 18°C, they underwent a 110-minute period of circulatory arrest. The chest was packed in ice, and the heart was arrested by topical cooling without the use of cardioplegia. During the circulatory arrest period, the venous cannula was unclamped periodically to completely drain the venous system of any accumulated blood. After reinstitution of CPB, the animals were rewarmed and weaned from CPB once they reached a temperature of 34°C. The femoral artery and right atrium were then decannulated, a thoracostomy tube was placed, and all incisions were closed in multiple layers with absorbable suture.
Postoperative Care
During recovery, oxygen saturation and heart rate were continuously monitored with a pulse oximeter (Novametrix Medical Systems, Wallington, Connecticut). When the animals were able to breathe spontaneously, mechanical ventilation was discontinued. The endotracheal tube was removed when it was no longer tolerated, and the animals were moved to a temperature-controlled recovery kennel. The thoracostomy tube was clamped and aspirated periodically, and removed when there was minimal blood drainage. The ear vein cannula was maintained patent for a minimum of 24 hours after the procedure or until the animals were able to eat and drink independently. Animals incapable of eating or drinking were supported with nasogastric feedings. Buprenorphine (0.1 mg/kg IV) was used for pain management in the immediate postoperative period.
After a 6-day survival period, the animals were sedated with an intramuscular cocktail of ketamine (22 mg/kg) and acepromazine (1.1 mg/kg). A marginal ear vein was cannulated for administration of systemic heparin (300 U/kg) and euthanasia solution (sodium pentobarbital, 100 mg/kg). The right chest was opened, and the heart was excised and immediately placed in 0.9% saline at 4°C.
Assessment of Myocyte Apoptosis—TUNEL Assay
Myocyte apoptosis in the subendocardium of the left ventricular free wall was quantified using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. Only subendocardial tissue was assessed as there was no significant injury detected in the epicardial segments on preliminary examinations. In preparation for the TUNEL assay, myocardial tissue was embedded in optimal cutting temperature compound (OCT; Sakura Finetek, Torrance, California). Cryosections from frozen tissue were obtained using a Hacker-Bright cryostat and thaw-mounted onto Fisher-Plus slides (Fisher Scientific, Pittsburgh, PA). Determination of apoptosis was performed using an in-situ cell death detection kit (Roche Applied Science, Indianapolis, Indiana). The DNA strand breaks were labeled with fluorescein-dUTP, followed by the addition of a secondary, antifluorescein antibody conjugated with alkaline phosphatase, a reporter molecule that generates a red color from Vector Red substrate. After counterstaining with hematoxylin and dehydration in graded alcohols, the number of total nuclei and apoptotic nuclei (as indicated by red staining) were quantified at x20 magnification using a computer-automated counting system and ImagePro 4.0 image analysis software. The percentage of apoptotic nuclei averaged from a minimum of 10 random fields is presented. While it is true that other cell types may be stained by the TUNEL assay, it is clear from high-power evaluation of the tissue sections that the majority of cells stained are cardiomyocytes. Nevertheless, a small percentage of cells identified by this technique may be endothelial cells or apoptotic inflammatory leukocytes.
Assessment of Myocyte Apoptosis—DNA Ladder
Appearance of DNA laddering was used as a confirmatory tool of apoptosis. The DNA ladders were detected using agarose gel electrophoresis as described previously [15]. Briefly, frozen tissue samples from transmural myocardium were minced in lysis buffer and were quickly homogenized with a microfuge tube pestle. The tissue was digested with proteinase K and incubated with RNase A. Supernatants containing DNA were precipitated with isopropanol, and the resulting DNA pellets after centrifugation were washed with ethanol and dissolved in DNA hydration solution. Electrophoresis of DNA was carried out, and DNA ladders were visualized under ultraviolet light.
Western Blot Analyses of Bcl-2, Bax, and Phosphorylated Akt
Extraction of total protein from tissue samples was prepared as previously described [15]. In brief, 100 mg tissue from left ventricular free wall was placed in lysis buffer (consisting of the following in mM: 20 Tris-HCl, 150 NaCl, 1 Na2EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 ß-glycerophosphate, 1 Na2VO4, 1% Triton, 1 µg/mL leupeptin), and protein concentration was measured by the DC Protein Assay (Bio Rad, Hercules, California) and homogenized. Protein concentrations were measured by the DC protein assay (Bio Rad). Equal amounts of protein (80 to 100 µg each) were boiled in loading buffer, loaded onto 8% SDS-polyacrylamide gel electrophoresis, then transferred to nitrocellulose filters in the presence of glycine/methanol transfer buffer in a Mini Protein II transfer system (Bio Rad). The membranes were subsequently incubated with one of the following antibodies: a polyclonal anti-rat Bcl-2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal rabbit anti-rat Bax (1:400, Santa Cruz Biotechnology) and a rabbit anti-phospho-Akt (Ser473, 1:1000, Cell Signaling, Beverly, MA) at 4°C for overnight, respectively. The resulting immunocomplex was further reacted with a horseradish peroxidase-conjugated secondary goat anti-rabbit IgG at 1:10000 for Bcl-2 and Bax, and at 1:3000 for phosphorylated Akt. The Bcl-2, Bax, and phosphorylated Akt (pAkt) were detected as 28, 23, and 60 kDa bands, respectively, identified by a molecular weight marker. The intensity of each band was scanned by an image program from National Institute of Health and is presented as percent density of bands in experimental groups relative to a control that was run contemporaneously. For Bcl-2 and Bax, values were normalized to tissue beta actin expression while p-Akt values were normalized to total (unphosphorylated) Akt. Analyses of Bcl-2 and Bax were performed for the control and cobalt groups only. Since there was no protective benefit observed in the DFX and hypoxia groups, we did not measure Bcl-2 or Bax levels in those animals.
Statistical Analysis
Data are presented as mean ± SD. Data between groups were compared using a one way analysis of variance (ANOVA) or a Kruskal-Wallis ANOVA on ranks using a post-hoc Dunnet's test to compare treatment groups with the control group. Intraoperative data within groups were compared using ANOVA with Student-Newman-Keuls post-hoc analysis for all pairwise comparisons. All analyses were performed using SigmaStat for Windows (version 3.0; SSPS, Chicago, IL). Differences were considered statistically significant for p values less than 0.05.
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Results |
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demonstrate the percentage of apoptotic nuclei in the subendocardium of control animals compared with animals preconditioned with hypoxia, DFX, or CoCl2. Whereas hypoxia (26.0% ± 6.5%, p = 0.528) and DFX (22.7% ± 8.2%, p = 0.403) failed to show a significant protective benefit, CoCl2 pretreatment significantly attenuated myocardial apoptosis (9.3% ± 4.1%) as compared with controls (33.8% ± 9.7%, p = 0.042). Laddering of DNA was used as a confirmatory marker of apoptosis. A representative sample is shown in Figure 3, which demonstrates a relative absence of laddering in the CoCl2 group compared with control, hypoxia, and DFX groups.
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Comment |
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Neuronal apoptosis associated with DHCA has been the subject of several recent investigations [17–20] and may be a significant source of morbidity after these procedures. However, there have been relatively few studies aimed at preventing myocyte apoptosis resulting from DHCA. The results of the present study confirm that cardiomyocyte apoptosis also occurs after DHCA and that a significant level of myocyte apoptosis is detectable 1 week after the period of circulatory arrest. These findings corroborate the results of previous work by Zhao and colleagues [21] in which rising levels of myocyte apoptosis were seen at 72 hours after ischemia-reperfusion. Furthermore, our results indicate that whereas hypoxia and DFX showed no beneficial effects, preconditioning with cobalt chloride led to diminishes myocardial apoptosis and was associated with increased phosphorylation of Akt and upregulation of Bcl-2, two known antiapoptotic proteins.
Although the precise mechanisms of this protective effect have not been completely elucidated in this study, one possible mechanism is the upregulation of HIF-1 by CoCl2. It has been well established that HIF-1 is upregulated by hypoxia, DFX, and CoCl2 in several previous studies [7]. Furthermore, we have previously shown in this same model of CPB and DHCA that CoCl2 significantly upregulates HIF-1 expression in the brain, leading to diminished neuronal injury and apoptosis [14]. Hypoxia-inducible factor-1 is a transcription factor that mediates adaptive intracellular mechanisms during periods of hypoxic stress. By increasing the expression of its target products, including erythropoietin, vascular endothelial growth factor, glucose transporter enzymes (GLUT-1 and GLUT-4), and glycolytic enzymes, HIF-1 increases the oxygen-carrying capacity of blood, stimulates angiogenesis, and enhances anaerobic adenosine triphosphate synthesis. These adaptive responses could potentially render cardiomyocytes more tolerant of the global ischemic insult faced during DHCA.
The association between CoCl2 preconditioning and diminished apoptosis may depend on the target products of HIF1-, particularly erythropoietin and vascular endothelial growth factor. Recent studies have shown the effectiveness of erythropoietin in limiting apoptosis in rat myocytes [22–24], vascular endothelial cells [25], human erythrocyte progenitor cells [26], and vascular smooth muscle cells [27]. Furthermore, it has been shown that the protective effects of erythropoietin are dependent on the PI-3 kinase-mediated phosphorylation of Akt, [25–27] and the downstream alteration of Bad, p53, and caspase-3 activity [28]. Furthermore, studies conducted on HepG2 tumor cells have shown that hypoxic preconditioning prevents apoptosis of serum-deprived cells in a vascular endothelial growth factor–dependent manner and that this effect is associated with decreased Bax and increased Bcl-2 levels [29]. An increase in the Bcl-2/Bax ratio has been demonstrated to prevent apoptosis through stabilization of the mitochondrial membrane and inhibition of mitochondrial cytochrome c release [5]. Our results corroborate these findings in that CoCl2-mediated protection was also associated with increased levels of phosphorylated Akt and an increase in the Bcl-2/Bax ratio.
Although the clinical significance of reduced myocardial apoptosis in newborns is uncertain, Karimi and associates [30] have shown that neonates may lack the necessary adaptive mechanisms to ameliorate oxidative stress in response to cardioplegic arrest and are more likely to develop myocyte apoptosis which can lead to postoperative myocardial dysfunction. Furthermore, in adults, apoptosis has been associated with reduced cardiac contractility and ejection fraction, ultimately leading to congestive heart failure. As survival after repair of congenital defects improves and these children live longer, they will be subjected to further surgical procedures, and the subtle effects of myocyte apoptosis may become additive and clinically important.
Among the limitations of this study is that while we have shown a correlation between CoCl2-mediated reduction in myocyte apoptosis and increased levels of phosphorylated Akt and Bcl-2, we have not clearly demonstrated a causal relationship between the latter and the former. To do so would have required additional animal groups with specific inhibitors of Akt phosphorylation (ie, PI-3 kinase inhibitors) or Bcl-2. Similar studies have previously been conducted in rodent models and would not be as feasible in this large-animal model. A second potential criticism is that we did not use cardioplegia to arrest the heart in this study. It is possible that doing so would have diminished the level of apoptosis that was seen. However, it was important to create a model with a reproducible level of injury that could then be treated by our preconditioning stimuli.
In summary, this study has confirmed that myocardial apoptosis occurs after CPB and DHCA, and significant levels of myocyte apoptosis are seen for as long as a week after the initial operation. Preconditioning with CoCl2 before the episode of DHCA can limit the extent of apoptosis, which may potentially be of benefit in hearts with marginal ventricular function. Future investigations should aim to determine if this preconditioning treatment can be applied clinically in neonates undergoing corrective procedures utilizing DHCA as well as to determine if additional CoCl2- and HIF-1–benefits exist, such as vascular endothelial growth factor–induced angiogenesis and enhanced myocardial contractile function. Finally, CoCl2-mediated preconditioning should be investigated in an adult model of DHCA, as these hearts are likely subjected to similar mechanisms of myocyte injury (ie, ischemia-reperfusion).
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DR KERENDI: That's a good question, and those protective strategies that you mentioned have been tested in similar models looking at neuroprotection. We had not specifically planned on looking at myocyte apoptosis with respect to those strategies, but that is something we could consider in future studies.
DR MARSHALL L. JACOBS (Philadelphia, PA): Congratulations on your beautiful piece of research and on a very appropriate award. I think that is exciting stuff.
My question relates to the historical evidence that you referred to about hypoxia and its potential for preconditioning against postischemic apoptotic changes. And I noted in the experimental protocol you used 2- to 3-week old piglets. My question is this: prior to birth and the transition from fetal to postnatal circulation, both the myocardium and the brain of most mammals are perfused at PAO2s anywhere between about 26 and 30. Is there an age-related difference in the prevalence of these pro and antiapoptotic proteins? And is the fetal circulation with its normal degree of relative hypoxia a form of chronic preconditioning, and is that lost after transition from fetal to postnatal circulation?
DR KERENDI: Thank you for your comments. That's an excellent question. In terms of preconditioning in the first few days of life related to fetal hypoxia, it may be so, but I don't know if anyone really knows the answer to that question. I suspect that if that's the case, the preconditioning benefit would be lost by 2 to 3 weeks of age. In 1-day-old rodents, previous studies have shown that there is still significant neuroprotection if they take neurons and expose them to hypoxia in cell culture. So in our model I don't know if that would be the case, but that benefit apparently is not lost in the rodent model.
DR GABRIEL AMIR (Stanford, CA): I think it's erroneous to relate your results to deep hypothermic circulatory arrest. You're talking about myocytes, so I think it would be more appropriate to relate it to cross-clamping or myocardial ischemia.
DR KERENDI: I think that's true. And part of the injury is certainly related to ischemia. We didn't cross-clamp the aortas in these animals so there was no ischemia related to that. We also didn't use cardioplegia, which I suspect is part of the reason we saw the level of injury that we did. But you're right in that the injury is more an ischemic injury and not necessarily related to hypothermic circulatory arrest, although there is some global ischemia inherently involved with circulatory arrest.
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