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Ann Thorac Surg 2004;78:948-955
© 2004 The Society of Thoracic Surgeons
a Division of Cardiovascular Surgery, University of Iowa College of Medicine, Iowa City, Iowa, USA
c Department of Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa, USA
b Division of Cardiothoracic Surgery, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
Accepted for publication April 5, 2004.
* Address reprint requests to Dr Caldarone, Division of Cardiovascular Surgery, The Hospital for Sick Children, 555 University Ave, Ste 1525, Toronto, Ontario, Canada, M5G 1X8, Canada
christopher.caldarone{at}sickkids.ca
Presented at the Poster Session of the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.
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1% of myocytes). Because apoptosis is preceded by events associated with mitochondrial dysfunction, the fraction of myocytes with preapoptotic mitochondrial changes has important clinical implications (eg, postoperative myocardial dysfunction). My colleagues and I therefore hypothesized that postoperative apoptotic myocytes represent a tip of the iceberg, with more myocytes upstream with apoptosis-related mitochondrial dysfunction (ARMD). METHODS: Neonatal lambs underwent cardiopulmonary bypass, 60 minutes of cardioplegic arrest, and 6 hours of recovery (cardiopulmonary bypass with cardioplegic arrest [CPB+CP]; n = 5) and were compared with nonbypass controls (non-CPB; n = 5). Myocardium (left ventricle [LV] and right ventricle [RV]) was examined by using terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, electron microscopy, immunohistochemistry, Western blot, and isolated mitochondrial oxygen consumption measurement.
RESULTS: TUNEL-positive nuclei and electron microscopy–confirmed mitochondrial structural changes were more common in CPB+CP than non-CPB myocardium and were more common in the LV than RV (p = 0.0016). Bax (a proapoptotic mediator) translocated from the cytosol to the mitochondria (LV > RV; p < 0.05). Immunohistochemistry demonstrated diffuse mitochondrial loss of cytochrome c that was consistent with outer mitochondrial membrane permeabilization (LV > RV > non-CPB). Permeabilization was further demonstrated by augmentation of oxygen consumption in isolated mitochondria after administration of exogenous cytochrome c. The mitochondrial oxygen consumption boost was 57% for CPB+CP:LV; 23% for CPB+CP:RV; and 18% and 17% for non-CPB:LV and non-CPB:RV, respectively (p < 0.01, CPB+CP:LV vs other groups).
CONCLUSIONS: ARMD is much greater than the prevalence of TUNEL-positive myocytes in postoperative neonatal myocardium. Greater LV vulnerability may represent a relationship between increased afterload and ARMD. These changes are consistent with the early postoperative myocardial dysfunction commonly reported after neonatal cardiac operations.
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We have previously reported that completed apoptosis is present within 6 hours after cardioplegic arrest in the neonatal lamb [5] and that neonatal myocardium is more vulnerable to postoperative apoptosis than mature myocardium [6]. These reports, however, have focused on completed apoptosis (eg, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling [TUNEL]-positive nuclei), which is limited to approximately 1% of the myocytes and, consequently, is unlikely to have an appreciable effect on postoperative dysfunction because of the rarity of the involved myocytes.
Because the preliminary stages of apoptosis are intimately associated with alterations in mitochondrial structure and function, the distribution of preapoptotic mitochondrial changes throughout the myocardium may have important implications in terms of clinical myocardial performance. If these changes are diffuse, then they could have a marked effect on clinical myocardial performance; if they are limited to the sparse number of TUNEL-positive myocytes, then they are unlikely to be of clinical significance. In this study, we examined apoptosis-related alterations in mitochondrial structure and function in the early postoperative neonatal myocardium. We use the term apoptosis-related mitochondrial dysfunction (ARMD) to describe the cluster of functional and structural preapoptotic mitochondrial changes in the early postoperative period.
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For the CPB+CP protocol, cardiopulmonary bypass was initiated with a blood prime and passive cooling to 28° to 30°C. The aorta was cross-clamped, and cold antegrade crystalloid cardioplegia (Plegisol; Abbott Laboratories, North Chicago, IL) was administered (20 mL/kg body weight), followed by repeat doses (15 mL/kg) at 20-minute intervals. Topical cooling was applied, and the pulmonary artery was vented. After 60 minutes, the cross-clamp was removed, and the animal was rewarmed. After 10 to 20 minutes bypass was terminated, protamine was administered, and the animal was decannulated.
General anesthesia was maintained for 6 hours after reperfusion. Arterial blood gases, electrolytes, and hematocrit were maintained in a physiologic range. Maternal blood or crystalloid was infused as necessary. Inotropic agents were not administered. After 6 hours, the heart was quickly excised, and the coronary arteries were flushed with ice-cold phosphate-buffered saline. Full-thickness myocardium was minced, snap-frozen in liquid nitrogen, and stored at –80°C. Other tissues were fixed in 10% formaldehyde and embedded in paraffin. For the non-CPB protocol, a thoracotomy was performed, and cardiectomy was performed with identical tissue harvest. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Association for Assessment and Accreditation of Laboratory Animal Care (March 1999).
Mitochondrial fractionation for biochemical analysis
Frozen ventricular myocardium samples were mechanically homogenized in ice-cold isotonic buffer. Unlysed cells and nuclei were removed by centrifugation at 750g for 5 minutes at 4°C. The supernatant was spun at 10,000g for 25 minutes at 4°C, and the resulting mitochondrial pellets were then resuspended in the buffer and frozen at –80°C.
In situ TUNEL staining
TUNEL staining was performed with a modification of the technique described by Olivetti and colleagues [7] by using the ApopTag In Situ kit (Intergen, Norcross, GA) as previously reported [5, 6]. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). To identify cell type, formalin-fixed and paraffin-embedded 5-µm sections were stained with mouse monoclonal immunoglobulin (Ig)M anti–
-sarcomeric actin antibody (Sigma, St. Louis, MO) and Alexa Fluor 594–conjugated goat anti-mouse IgM (Molecular Probes, Eugene, OR) before TUNEL staining.
To enumerate TUNEL-positive cells, 10 random high-power fields (HPF) representing approximately 4,000 cells were counted by a blinded observer. Adjacent sections were stained with hematoxylin and eosin and examined by light microscopy for evidence of necrosis. Necrosis was not detected in any of the specimens (data not shown).
Western blotting for Bax and cytochrome c oxidase subunit IV
Homogenized samples standardized for protein content were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 15% linear gradient gels and subsequently transferred to nitrocellulose membranes. The membrane was probed for Bax or cytochrome c oxidase subunit IV (COX IV) with anti-mouse IgG monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies were coupled to horseradish peroxidase (anti-mouse IgG/horseradish peroxidase: Upstate Biotechnology, Lake Placid, NY; anti-rabbit IgG/horseradish peroxidase: Santa Cruz Biotechnology). Autoradiographs were scanned and quantified with a densitometer (NIH Image software).
Buffers
Mitochondrial isolation buffer (MIB) was made from the following reagents: 5 mmol/L 3-(N-morpholino)propanesulfonic acid (1.05 g/L), 2 mmol/L ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.76 g/L), 70 mmol/L sucrose (23.8 g/L), 220 mmol/L mannitol (40.04 g/L; pH 7.2), and 5 N KOH. Mitochondrial respiration buffer was made from the following reagents: KCl 130 mmol/L (0.97 g/100 mL), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 20 mmol/L (0.478 g/100 mL), MgCl2 2.5 mmol/L (0.051 g/100 mL), and ethylenediaminetetraacetic acid (EDTA) 0.5 mmol/L (0.0146 g/100 mL), titrated to pH 7.2 with KOH. Respiration stock solution was made from KH2PO4 (250 mmol/L), glutamate (250 mmol/L), and malate (250 mmol/L). Phosphate/EDTA buffer was made from KH2PO4 (50 mmol/L) and EDTA (1 mmol/L) and was titrated to pH 7.2 with KOH. For mitochondrial oxygen consumption measurements, mitochondrial respiration buffer (9.8 mL) was mixed with respiration stock solution (200 µL) daily.
Mitochondrial isolation for oxygen consumption measurements
After lambs were killed, the left (LV) and right ventricular (RV) myocardial free wall segments ( 1 g) were harvested and immediately placed on ice in 10 mL of MIB with 0.1% bovine serum albumin and finely minced. Tissue was homogenized on ice by using a blade homogenizer and pelleted by centrifugation (600g for 5 minutes x 3). The pellet was resuspended in 10 mL of MIB and centrifuged (8,500g for 10 minutes x 2). The final pellet was resuspended in 1 mL of MIB. Protein concentration was determined by following the Bio-Rad (Hercules, CA) protein assay protocol.
Fluorescent immunohistochemistry
Tissue sections were deparaffinized and rehydrated, and antigen retrieval was performed by microwaving in sodium citrate buffer 10 mmol/L, pH 6.0, for 20 minutes. The sections were blocked with 3% bovine serum albumin and phosphate-buffered saline for 60 minutes. Before immunostaining, slides were treated with 0.2% phosphomolybdic acid solution for 2 minutes to reduce cytoplasmic background fluorescence. Mouse monoclonal anti–cytochrome c (1:200 dilution; BD Biosciences Pharmingen, San Diego, CA) was used as the primary antibody for cytochrome c staining. Sections were incubated with secondary antibody (fluorescein isothiocyanate–conjugated goat anti-mouse IgG, 1:200; Sigma). Mouse monoclonal anti–COX IV (1:200; BD Biosciences Clontech, Palo Alto, CA) was used as the primary antibody for COX IV staining. Sections were incubated with secondary antibody (rhodamine-conjugated goat anti-mouse IgG, 1:200 dilution; Sigma). Four 5-µm sections from each sample, cut in 20-µm intervals, were used for staining. The images from 20 sections in each group were analyzed by a blinded observer, who used a Leica (Deerfield, IL) fluorescent microscope with OpenLab software (Improvision, Lexington, MA). All images were acquired with identical gain and offset settings.
Mitochondrial oxygen consumption
All measurements were made by using a Clark-type oxygen electrode at 25°C. Mitochondrial respiration buffer with respiration stock solution (700 µL) was added to the chamber, and 200 µg of mitochondria was then added to the chamber. After thermal equilibration, 5 µL of adenosine diphosphate 1 mol/L was added. For exogenous cytochrome c assays, 30 µL of 2 mmol/L equine cytochrome c (made in phosphate/EDTA solution) was added to the chamber before the addition of mitochondria and adenosine diphosphate.
Statistical analysis
Data are described as frequencies, medians with ranges, or means with standard deviations, as appropriate. Statistica 5.5 (StatSoft Inc, Tulsa, OK) statistical software, with the default settings, was used for statistical analyses. A p value less than 0.05 was set as the level of statistical significance. Kruskal-Wallis analysis of variance was used for nonparametric analysis of multiple groups, and the Mann-Whitney U test was used for individual comparisons, with correction for multiple comparisons. Student's t test was used to compare groups within a single Western immunoblot. The Tukey test was used to make post hoc comparisons between multiple groups with normally distributed data.
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) [12–14]. Aebert and colleagues [15] demonstrated that the serum after cardiopulmonary bypass contained factors capable of inducing apoptosis in cultured endothelial cells. Third, ischemia-reperfusion injury is a well-described stimulus capable of initiating apoptosis. Multiple investigators have described the initiation of apoptosis after cardioplegia-protected ischemia-reperfusion in animal models [5, 16] and human studies [17].
The functional consequences of postoperative apoptosis, however, are not well defined. In terms of early postoperative myocardial function, it is unlikely that the loss of 3 myocytes per HPF reported in this study ( 1% of myocytes) could have any appreciable effect on global function. An important question, therefore, is whether the sparse number of TUNEL-positive cells represents the entire population of myocytes undergoing apoptosis (suggesting minimal physiologic effect) or whether the TUNEL-positive myocytes represent a small minority of apoptosis-stimulated myocytes that have escaped apoptosis regulatory mechanisms. The latter interpretation, the "tip of the iceberg" concept, suggests the potential for greater physiologic effect because of the intimate relationship between the early stages of apoptosis and alterations in mitochondrial structure and function.
Arguments opposing the tip of the iceberg concept center on abundant evidence that release of cytochrome c from the mitochondria is an irrevocable commitment to "rapid, complete and kinetically invariant" cell death in cell culture models [18, 19]. If these data are applicable to the in vivo postcardioplegic neonatal heart, then in light of the diffuse extent of cytochrome c release noted in this study, massive apoptosis would be predicted. Because postoperative neonatal myocardial function is well preserved in clinical studies [1–3], however, it seems improbable that cytochrome c release inevitably leads to cell death in our model. This challenges the applicability of the in vitro data to the postoperative neonate.
In contrast, supporting the tip of the iceberg concept, Martinou and colleagues [20] have demonstrated that under certain conditions cytochrome c release from the mitochondria can be a recoverable event. In addition, widespread cleavage of caspase-3 and cytochrome c release have been reported in the setting of chronic human cardiomyopathy, with completed apoptosis limited to a small fraction of myocytes [21, 22]. Narula and associates [23] hypothesized that apoptosis-related release of cytochrome c from mitochondria in chronic heart failure leads to temporary functional impairment but that, because nuclear fragmentation is suppressed, the dysfunctional myocytes continue to exist and, in unspecified favorable conditions, the apoptotic process subsides. Although determination of the ultimate fate of myocytes with mitochondrial permeabilization and cytochrome c release is beyond the scope of this study, we speculated that ARMD represents a temporally brief episode of this process in the early postoperative neonatal myocardium.
Cytochrome c is released by several mechanisms. The convoluted shape of the inner mitochondrial membrane has a large surface area in comparison to the outer mitochondrial membrane. Consequently, osmotic swelling of the inner mitochondrial membrane after reperfusion can result in rupture of the outer mitochondrial membrane and nonspecific (eg, not related to apoptosis) release of the contents of the outer mitochondrial space into the cytosol [24]. In contrast, Bcl-2 family proteins such as Bax can translocate from the cytosol to the outer mitochondrial membrane and form multimers with selective pore-forming capability, through which cytochrome c can egress from the outer mitochondrial space [25]. In this study, we demonstrated an increase in Bax concentration in the mitochondrial fraction in association with release of cytochrome c into the cytosol, and this suggests that Bax may participate in outer mitochondrial membrane permeabilization.
Because apoptosis is an energy-requiring process, the inner mitochondrial membrane potential must be maintained in the early stages of apoptosis, and electron transport continues, although with some functional impairment [26, 27]. In the absence of adequate energy production, the mode of cell death can shift to necrosis. The energetic consequences of permeabilization of the outer mitochondrial membrane include loss of electron transport capability due to the lack of available cytochrome c to shuttle electrons from complex III to complex IV [24]. In addition, the accumulation of reducing equivalents can induce increased production of reactive oxygen species through direct transfer of electrons to oxygen [28]. Finally, permeabilization of the outer mitochondrial membrane can allow access for cleaved caspase-3 to directly inhibit the activity of complexes I and II in the electron transport chain [29]. We and others [6, 16, 30] have demonstrated cleavage of caspase-3 after cardioplegia-protected myocardial ischemia; consequently, permeabilization of the outer mitochondrial membrane may permit inhibition of electron transport by cleaved caspase-3.
The finding that apoptosis-related changes predominated in the LV myocardium is consistent with data reported by Pearl and associates [30] in a piglet model of cardiopulmonary bypass and circulatory arrest. Apoptosis is commonly observed as a part of normal postnatal remodeling, although differing reports assign a greater propensity for postnatal apoptosis to the LV [31] or the RV [10]. The faint cytochrome c staining in the non-CPB:LV myocardium may reflect some normal postnatal remodeling. The predisposition of the postoperative LV myocardium in this study may also be related to a greater afterload in the LV (relative to the RV). Stretching of neonatal rat myocytes promotes elaboration of reactive oxygen species and a dose-response relationship with TUNEL positivity, suggesting a relationship between afterload and apoptosis [32]. In addition, reactive oxygen species are detectable in human LV myocardium after cardiopulmonary bypass and cardioplegic arrest [33], and there is a close association between elaboration of reactive oxygen species and apoptosis [34]. Consequently, further studies to evaluate the effect of pharmacologic afterload reduction, a common postoperative neonatal management strategy, on ARMD in the early postoperative period would be of interest.
Important limitations of this study include the acquisition of data at a single time point in the postoperative period. Consequently, as noted previously, the ultimate fate of myocytes with ARMD was not directly evaluated. A second important limitation is the absence of a determination of the relative contributions of more traditional forms of myocardial stunning (eg, calcium overload) in comparison to ARMD. The Bax translocation data, however, suggest some degree of apoptosis-related specificity in the mechanism of mitochondrial permeabilization; nevertheless, both processes are likely to be present. Finally, necrotic cell death may be present at a level below our ability to detect with hematoxylin and eosin staining of sections adjacent to the TUNEL-staining myocardial sections, or it may be in its early stages but not yet detectable by these staining methods.
In conclusion, ARMD (manifested by translocation of Bax to the mitochondria, permeabilization of the outer mitochondrial membrane, and mitochondrial release of cytochrome c) is present in the early postoperative period after cardioplegic arrest in the neonatal lamb. Subjectively, the release of cytochrome c is out of proportion to the limited number of myocytes that are TUNEL positive, and these mitochondrial changes are consistent with the early postoperative myocardial dysfunction commonly reported after neonatal cardiac operations.
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= CPB+CP; 
= non-CPB. (CPB+CP = cardiopulmonary bypass with cardioplegic arrest; LV = left ventricle; non-CPB = nonbypass controls; RV = right ventricle.)